Potato tuber quality management in

relation to environmental and

nutritional stress

Stephen Harper QLD Department of Primary

Industries and Fisheries

Project Number: PT99052

PT99052 This report is published by Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the potato industry. The research contained in this report was funded by Horticulture Australia Ltd with the financial support of the potato industry. All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government. The Company and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests. ISBN 0 7341 0903 2 Published and distributed by: Horticultural Australia Ltd Level 1 50 Carrington Street Sydney NSW 2000 Telephone: (02) 8295 2300 Fax: (02) 8295 2399 E-Mail: horticulture@horticulture.com.au © Copyright 2004

Queensland the Smart State

Potato tuber quality management in relation to environmental and nutritional stress. Final report HAL project PT99052 April 2004

Stephen Harper Queensland Government DPI&F

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HAL project No. PT99052 Potato tuber quality management in relation to environmental and nutritional stress. Stephen Harper Queensland Government DPI&F Gatton Research Station LMB7 MS 437 Gatton 4343. 07 5466 2222 This report outlines the factors that cause and enhance symptoms of brown fleck in potato tubers. It also evaluates potential management strategies that reduce incidence of brown fleck. Date of completion April 26 2004

Any recommendations contained in this publication do not necessarily represent current Horticulture Australia policy. No person should act on the basis of the contents of this publication, whether as to matters of fact or opinion or other content, without first obtaining specific, independent professional advice in respect of the matters set out in this publication.

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Contents

Media Summary

3

Technical Summary

4

1. Introduction

7

2. Materials and Methods - General

10

3. The Effect of Temperature on the Incidence of Brown Fleck

12

4. Effects of Calcium and Boron on BF

25

5. Understanding Field Physiology of BF

32

6. Effects of Assimilate Supplementation on BF Incidence

42

7. Brown Fleck Microscopy and Phenology of Development

46

8. Technology Transfer

54

9. Recommendations

54

10. Acknowledgements

55

11. Literature Review

56

12. References

75

.

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Media Summary The causes and management strategies for the potato tuber internal disorder brown fleck (BF) were evaluated. This disorder reduces potato growing profitability in problem regions and is a major cause of potato consumer dissatisfaction. Worldwide research on causes of BF and its solutions has not been substantiative. The results of this project show incidence and development of BF symptoms appear to be 2 separate processes. Initiation of BF relates to the death of specific sugar conducting cells in the tuber and as a consequence further severe cellular disruption occurs. There is a strong relationship between crops having high yield potential and high BF incidence, hence factors that favour rapid tuber growth also favour high BF incidence. In support of this BF incidence was increased specifically by elevated night temperature (but not day temperature) and vigorous crop foliage growth. High soil temperature enhanced symptom development. In minimising incidence of BF, growers need to prevent prolific canopy development, which can largely be achieved by ensuring that nutrient application and irrigation are not excessive. Early monitoring of tubers for the first evidence of BF incidence should be conducted which can form the basis for determining whether preventative measures are required. Short term weather outlooks could be used as a basis for determining whether imminent weather conditions would be favourable for rapid tuber growth; in particular, warm nights, mild sunny days and rain events. In the event that there is early evidence of BF and conditions are favourable for its development, the management of crop foliage should be considered. This can be achieved by potentially spraying defoliants or agents that might harden the crop up including copper or calcium. The maintenance of weed or vegetative cover at crop senescence should be encouraged so as to provide shade to soil and preventing excessive soil temperatures that favour enhancement of BF symptoms. Application of calcium or boron did not significantly or reliably reduce incidence of BF and based on this is not recommended as a means of controlling BF. These nutrients should nonetheless be applied at rates appropriate to meet normal growth requirements. The cause of the initial cell death requires further research and particularly in relation to varieties that are both susceptible and resistant to BF. As well as providing an understanding of BF causes at the cellular level this would form a basis for the breeding and developing of new varieties that are resistant to BF. Also further work is required to evaluate the potential of different defoliants or retardants and their rates and timing of application.

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Technical Summary Temperature Results over the series of experiments demonstrate that elevated night time temperatures and high soil temperature dramatically increase the expression of BF. Elevated daytime temperatures did not greatly increase the expression of BF. Moderate temperature regimes (including mild days and warm nights) were most favourable to the development of BF and this was particularly evident in the 2000 experiment where plants exposed to a constant day/night temperature regime of 23/18oC exhibited BF incidence as high as that exhibited under a shorter exposure to 28/23oC. In contrast to the night temperature effect, high daytime temperature did not appear to greatly increase the incidence of BF. The effect was clearly demonstrated by comparing BF incidence under day night treatment temperature regimes of 28/23 oC and a 28/13 oC in the 2001 experiment. Incidence of BF was substantially higher at 28/23 oC compared with that in the 28/13 oC treatment and incidence was much greater under a 23/18 oC regime than under a 28/13 oC regime despite the mean treatment temperature being was the same. Tuber dry matter % (DM%) was not affected by diurnal temperature patterns but rather was related to overall heat exposure with higher temperature reducing DM%. Whereas the BF incidence was particularly related to warm night temperature DM% was more related to the heat units experienced by the plant. Albeit at low levels, BF was still observed in tubers of plants grown under a low day night temperature regime (18 /13 oC) suggesting that elevated temperature per se does not appear to be the sole critical factor in inducing the disorder. It appears that an expansion of BF symptoms occurs at higher temperature probably due to the increased activity of oxidising enzymes and greater assimilate supply to tubers. Tuber yield and BF A positive relationship existed between yield potential and incidence of BF where high yielding plants exhibited high incidence of BF. This is further confirmed by virtue of the positive correlation between plant foliage weight and incidence of BF. Plants with a greater amount of foliage exhibited higher BF incidence. This effect is likely to be indirect and related to the greater amount of foliage increasing assimilate supply to tubers. The issue of temperature and tuber growth and BF incidence is important since increased temperature not only increased BF but consistently increased tuber yield in each experiment. The microscopy studies highlight that increased carbohydrate supply and the inability of blocked phloem sieve cells to pass the extra sucrose to newly developing cells appears to be a major factor in the expansion of BF symptoms. Nutrition The presence of low incidence of BF in day night temperature treatments 18oC/13 oC suggested other factors may cause disorder. On this basis the effect of various rates of calcium 0-127 kg ha-1 and boron 0-5 kg ha-1 on BF incidence was evaluated. The effects of both Ca and B on incidence of BF were inconsistent and generally not

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statistically significant. It is possible that any effect of either B or Ca on BF incidence is indirect through an inhibition of foliage growth, which would reduce the intensity of BF symptoms through reduced assimilate supply to tubers. There is no basis for making recommendations for application of Ca and B to reduce incidence of BF. Foliage management A series of field experiments was conducted to evaluate the effects of foliage removal on the incidence of BF. Removal and retarding of foliage generally reduced incidence of BF and shows good potential as a tool in minimising incidence of BF. The evidence presented suggests that growers should monitor potato crops for the first detectable incidence of BF and this can form the basis of a canopy management strategy. In addition, growers in regions where BF is a problem should attempt to grow crops a little on the hard side so that the proliferation of a vigorous canopy is not promoted. Various mechanisms for achieving this could be applied including use of defoliants or retardants, or restricting or withholding irrigation and not applying excessive nutrient application that favours foliage growth. A major trade off with removing foliage is the potential yield loss but early removal of only 25 % of leaf foliage does not appear to affect final yield. Further research is required to evaluate forms rates and timing of potential retardants. Supplementing assimilate Given that high night temperatures and a large plant foliage mass favoured BF symptoms two explanations were proposed. Firstly this set of circumstances could result in increased photosynthesis and assimilate supply to tubers or alternatively increased foliage respiration might have occurred leaving a deficit of assimilate to growing tubers and localised tuber cell death. In the latter case, the opportunity to supplement assimilate supply to foliage via a foliar application of sugar was evaluated, offering the potential to remediate short term foliage deficits in assimilate and BF incidence. Sucrose concentrations from 0% to 4% were applied at weekly intervals to glasshouse grown potato plants. The effect on BF incidence of sucrose treatments was not significant. Though not significant there appeared to be a trend for increasing sucrose application to increase the incidence of BF. Yield of tubers was significantly affected by sucrose application with yield being highest at a sucrose concentration of 1%. Phenology of development In phenology studies there was a significant positive correlation between tuber fresh weight and incidence of BF (p<0.001). This correlation accounted for 92.3% of the variance associated with BF. There was also a significant correlation between leaf dry weight and tuber fresh weight (p=0.030) and this relationship accounted for 39.9% of the variance in tuber weight. It appeared likely that the correlation between leaf dry weight and BF incidence as noted in all studies was subsequent to the effect that the quantity of leaf matter has on tuber growth. A higher leaf dry matter results in greater assimilate partitioning to the tubers which in turn is the driving factor behind BF symptom expression. A multilinear correlation matrix for the relationship between tuber fresh weight and specific climatic variables and leaf mass showed that tuber fresh weight was

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positively correlated with; night temperature, solar radiation, and leaf dry weight but negatively correlated with day time temperature. These factors appear to be the main factors driving yield and subsequently BF development. Cytology of BF At the earliest incidence of BF a single phloem sieve cell dies. Typically this cell is in regions of active cell growth and since it is responsible for conducting sucrose to newly developed cells the new cells are devoid of contents, adopt a glassy appearance and ultimately die resulting in a brown lesion. The initial phloem cell damage occurs when the plasmalemma suffers mild damage or trauma, evidenced by its browning. The damage progresses resulting in brown occlusions developing throughout the affected cell. This occlusion precludes sucrose transport past the damaged cell and proximal phloem cells face the difficulty of metabolising a continued supply of sucrose to regions where cell death has occurred. In response starch granules develop within these phloem cells. These studies confirm that a continued and excessive supply of sucrose under good growth conditions is consistent with an enhancement of BF symptoms as determined in the field and glasshouse studies. With further rapid photosynthesis sucrose continues to be supplied to the damaged area creating considerable internal pressure and the amounts of sucrose and solute increase. This further reduces the osmotic potential resulting in increased cell turgor pressure and providing conditions favourable for further development of BF. The death of these phloem cells or any other cell results in browning reactions and the release of oxidising enzymes that are normally contained within the cellular components and results in the disorder spreading. A critical assessment of the biochemistry and cytology of phloem cells is required to understand the initial cause of phloem cell death in BF affected tubers. This would provide, a more thorough understanding of the problem, a strong basis for genetic selection against the problem and development of a better management strategy.

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1. Introduction Internal tuber disorders are a major impediment to growers producing quality fresh market potatoes in specific regions. The presence of these disorders often prevents growers achieving a retail market specification of a maximum of 2% major defects. In Australia Brown Fleck (BF) is the most significant internal problem; its symptoms include an irregular blotchy brown discolouration of the tuber medullary tissue. Since the disorder produces no external symptoms it cannot be graded against using conventional grading technology. Consumers purchasing such produce are disaffected by the presence of such unsightly disorders and hence the problem is of major concern to potato growers, marketers and consumers. There is a great deal of confusion in the terminology associated with physiological internal disorders and this has made the interpretation of literature somewhat confusing. Broadly speaking the disorders can be divided into those that produce an internal cavity and those that cause browning of internal tissue. The definitions of internal browning disorders create the most confusion in the literature. Internal browning disorders have been variously referred to as Brown Fleck (BF)(Novak et al. 1986), Internal Heat Necrosis (IHN) (Sterrett et al. 1991ab), Heat Necrosis (Stevenson et al. 2001), Rust Spot (Baruzzini et al. 1989), Internal Rust Spot (Collier 1980), Chocolate Spot (Kamal and Marroush 1971), Internal Brown Spot (IBS) (Stevenson et al. 2001) and Mahogany Browning (anon.). Stevenson et al. (2001) provide the most delineating description of the disorders. For the purposes of this report the browning associated with tuber medulla will be referred to as Brown Fleck since this is the most widely used terminology in Australia. The cause of BF and related disorders is not well defined and hence is termed either as a physiological or nutritional disorder and there is no pathogen association. The incidence of BF is generally more pronounced in large rapidly growing tubers. The cause of these problems has been variously ascribed to factors such as nutrition, heat and irrigation practices. More recently the role of nutrition has been demonstrated to at least influence the expression of the disorders. In particular, it has been related to calcium deficiency, however, there is evidence boron deficiency may also be involved. The difference between causes and symptoms of Internal Brown Spot and Heat necrosis (HN) seem well defined (Stevenson et al. 2001). Internal Brown Spot is characterised by small round or irregular reddish-brown spots or lesions usually in the medullary tissue and in extreme cases the affected cells become corky, lack starch and are suberised. The disorder is generally most prevalent at the apical end and is related to fluctuations in growing conditions, particularly those favouring rapid growth. In contrast the other similar disorder HN develops in tubers that are exposed to extremely high soil temperatures late in tuber growth while vines are alive (Stevenson et al. 2001). The incidence of HN is most prolific under the crown eyes and it is likely that this distal end of the tuber experiences higher soil temperature extremes compared with other parts of the tuber and hence has higher HN incidence.

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Extensive IBS damage generally results in the necrosis of cells and such damage is irreversible. However, Hooker (1981) notes that the incidence of mild IBS during storage can actually decrease in severity. Similarly, in field plantings Harper (unpublished) has observed incidence of low grade BF in cv Exton at tuber bulking which was not evident at harvest. In contrast Stevenson et al. (2001) note that the incidence of IBS can even increase with storage depending on maturity and storage temperature. Penna (2001 pers comm.) noted no incidence of BF prior to storing tubers cv Sebago but a high incidence after a storage period of several days at 20oC. The tubers had a high incidence of glassy patches of cells in the medulla. This evidence suggests that the damage to cells in mild IBS cases is not strictly terminal and the initial brown products of IBS are not the consequence of cell necrosis but perhaps products of impaired metabolism. It suggests the disorder is initially biochemically related particularly in such mild cases. In the early development of IBS walls of affected parenchyma cells in the medulla and pith become dark at the corners of cells (Hooker 1981). Baruzzini et al. (1989) found that severely damaged IBS cells had deformed walls and a smaller lumen than that of unaffected cells. Tubers from plants that remain green late in the crop often have more IBS then tubers from vines which senesced prematurely (Ellison and Jacob 1952). The finding is also consistent with anecdotal evidence from field observations over 5 years (Harper unpublished) that has noted green plants at maturity are more prone to the disorder than senesced plants. In contrast other authors (Novak et al. 1986, Iritani et al. 1984 and Sterrett et al. 1991a) found that the incidence of IBS/BF increased when potatoes were held in the ground after vine senescence and maturation. Temperature is often nominated as a cause for IBS (Hooker 1981) to the point where in some parts of the world the disorder is referred to as Internal Heat Necrosis, particularly in the USA. Work by various authors demonstrates a relationship between internal disorders and temperature (Van Denburgh et al. 1979, 1986, Sterrett et al. 1991a,b). However, the disorder has been observed under hot dry conditions, hot moist conditions but also under cooler conditions (Novak et al. 1986, Sinden and Webb 1980). The spring Lockyer crop of 1996 and 1997 had extremely high incidence of IBS and this occurred despite the fact that temperatures were mild. Novak et al. (1986) present clear evidence of an environmental effect on expression of BF. In the four years 1971 to 1974, Autumn (March) planted experiments at Gatton, Australia (lat 270 30' S long 1520 15' E) never exhibited BF whereas the same experiments when planted in winter (July) at the same site exhibited high incidence of BF (up to 25%). Research conducted on the affects of heat, and its interaction with water status, in inducing IBS/BF is somewhat inconsistent, but there is general agreement that high temperatures favour the expression of the disorder. Furthermore BF is always associated with crops that appear to have a higher than average yield potential. Though considerable research has demonstrated beneficial effects of Ca in reducing incidence of internal disorders the research is largely inconclusive and highly variable (eg. Collier et al. 1980, Davies and Millard 1985, Tzeng et al. 1986, and Henninger 1991, Clough 1994, Olsen et al. 1996, Palta 1996, Monk-Talbot et al. 1991). Weir

9

and Cresswell (1993) and Shorrocks (undated circa 1980-85) illustrate visual evidence of boron deficiency inducing brown fleck, however, no formal treatise of B effects on incidence of BF or other internal disorders appears in the literature.

Silva et al. (1991) evaluated the effects of irrigation on the incidence of IBS in Atlantic over 3 seasons from 1987-1989 and determined that excessive irrigation increased the incidence of IBS in 2 of 3 years over that in a scheduled irrigation regime. Novak et al. (1986) evaluated the effect of withholding irrigations prior to maturity on incidence of IBS in cultivar Sebago. By withholding irrigation from between 4-6 weeks prior to maturity the incidence of IBS was reduced from 15% to 5%. There has been no definitive evidence to demonstrate that internal browning disorders are either nutritionally or physiologically related. A more comprehensive review of the literature is presented in section 9. The present series of experiments evaluates the factors that drive incidence of BF in potato tubers and present some potential mechanisms for the field management of the disorder.

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2. Materials and Methods - General

2.1. Glasshouse experimentation Certified seed potato (Solanum tuberosum cv Sebago) was graded into 80 ± 10 g sets. The shot whole set tubers were planted in twenty-five l polythene pots each filled with 20.0 kg of air dry soil collected from the Queensland DPI&F Gatton Research Station. The soil is described as a sandy clay loam. Sets were planted at a depth of 100mm and grown in an ambient glasshouse until plants emerged; dates of emergence were recorded. Pots were watered using the self watering inverted bottle technique of Hunter (1981) which maintains a constant luxurious water capacity. Rates of basal nutrient application were consistent with field fertilizer application rates used in regions where BF is an issue. The rates (kg ha-1) included 160 N, 20 P and 180 K plus 0.8 Zn and 0.8 B. At 28 DAP an additional 2-3 kg of soil was added to each pot to ensure setting tubers were well below the soil surface. Various experimental treatments were imposed as per relevant experiments and described as appropriate in the chapters herein. Unless otherwise specified plants were grown for a period of 112 days and harvested. Tubers were collected washed and weights recorded. In the 2000 experiment each tuber greater than 50 g was cut transversely through the apical, middle and basal part of the tuber. Four thin tissue segments (1.5 mm thickness) were taken from each zone of the tuber by cutting with a stainless steel meat slicer. One section of each zone was colour scanned using a Hewlett Packard scanner. Images were digitally enhanced to highlight BF using Ulead Photo Express Ver 3.0 photographic software. Actual incidence of Brown Fleck was calculated for each section using UTHSCSA Image Tool for Windows Version 2.0. Incidence of Brown Fleck was then variously expressed as, a percentage of tuber area affected, total BF per plant and number of tubers affected by BF. The other 3 cut sections from each zone were held for dry matter determination. Fresh weights were taken and samples dried in an oven at 70oC and dry matter content was determined. In 2001 and subsequent years the experimental pot technique was modified with the placement of a sleeve of silver sided sislation paper and 10 mm polystyrene foam around each pot to reduce the effects of direct sunlight on pot temperature. The tuber sampling technique was also changed whereby thick tissue segments (about 8 mm thickness) were taken from the crown and heel zone of tubers greater than 100g and from the middle of tubers in the 50-100g range and tuber dry matter content determined. A quantitative fleck analysis was made on the sections as in previous years with the exception of 2 issues. The image enhancement of BF was conducted using the more advanced software package PhotoImpact 7.0 and the pixel counting by the updated UTHSCSA ImageTool 3.0.

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2.2. Field experiments The main field experiments were conducted at the Queensland DPI&F - Gatton Research Station. Whole set certified seed potato (Solanum tuberosum cv Sebago) was planted in each trial conducted at the research station. Planting dates and details are specified in each experimental report. Rates of basal nutrient application and side dress application rates (in kg ha-1) of 160 N, 20 P, 180 K and 120-130 S were applied as CK77S (13.0:2.2:13.3:18.7 N:P:K:S). In each nutrition experiment combinations of potassium nitrate, potassium sulfate and ammonium nitrate were used to balance N and K inputs. In the 2001 experiment the trial was irrigated using solid set but in 2002 and 2003 the trials were irrigated using trickle irrigation. Unless otherwise stated in specific experiments, plots were delineated at full emergence by thinning the planting to establish plots of 6.0 m length at intervals of 1.5 m. Plots were harvested by digging the 2 middle rows in each plot as the datum. All tubers from each plot were washed and graded according to size (<80 g, 80-200g, 200-350g and >350 g). A sample of 20 tubers from the 80-200g and 200-350g grades and all tubers over 350 g were kept for evaluating the incidence of fleck. The weight of each tuber from the sample of each treatment was measured. In 2001 each tuber was cut transversely across the crown, middle and heel and rated for IBS incidence on a scale of 0-5 (0 being no IBS and 5 being severe IBS). Tissue segments (about 7-8 mm thickness) were taken from each zone of the tuber and scanned. In subsequent years (2002 and 2003) a single BF section was taken by selecting the section having the highest incidence of BF. A scanned colour image was taken of the cut sections using a Hewlett Packard scanner. The images produced were digitally enhanced to highlight BF using Ulead PhotoImpact7 photographic software. The actual incidence of IBS (square mm of affected tissue) was then calculated for each section using UTHSCSA Image Tool for Windows Version 3.0. The fresh sections were dehydrated in an oven at 70oC and dry matter determined. Various statistical analyses were used to evaluate results, including ANOVA and covariance analysis. The covariates included tuber number, tuber dry matter, yield, foliage mass, days to emergence as well as other variants as appropriate. Skewed variance distributions for BF incidence were noted in some studies and required either square root or cube root transformations to normalise the variance distribution. Linear regression was used to determine the relationship between various parameters and BF incidence.

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3. The Effect of Temperature on the Incidence of Brown Fleck.

Though an association between incidence of BF and temperature is generally recognised, research into this association has not progressed. However, in the USA research on a similar disorder Brown Centre has evaluated temperature effects. This chapter reports on a series of 4 experiments conducted during 2000-03 that evaluated the varying effects of temperature on the incidence of BF in potato (cv Sebago). Materials and methods

Temperature regime effects on incidence of BF 2000 In 2000 a controlled environment glasshouse pot experiment was conducted to determine the effects of altering temperature regimes on the incidence of BF. Whole sets were planted and plants initially grown in an ambient glasshouse and planted on 31 May 2000. At full emergence pots were shifted to the relevant controlled environment glasshouse rooms for the preliminary growth period (Period1). Eleven pots were allocated to each of 5 blocks according to their date of emergence. A total of 11 treatments were imposed. In treatments 1-9 (Table 3.1) plants were grown in a controlled environment glasshouse set at a day/night temperature of 18oC/13oC to the end of Period 1 (91 DAP). In treatments 10 and 11 plants were grown at a day/night temperature regime of 23oC/18oC to the end of Period 1. For treatments 1 to 9 a series of differential day night temperature regimes (18oC/13oC, 23oC/18oC and 28oC/23oC) were imposed in growth periods 2 (91-101 DAP) and 3 (102-112 DAP) giving all 9 possible temperature combinations (Table 3.1).

Table 3.1 Temperature treatments imposed in controlled environment experiment 2000.

Treatment Period 1 (14-91DAP)

Period 2 (91-101 DAP

Period 3 (102-112 DAP)

1 18oC/13 oC 18oC/13 oC 18oC/13 oC 2 18oC/13 oC 23oC/18 oC 18oC/13 oC 3 18oC/13 oC 28oC/23 oC 18oC/13 oC 4 18oC/13 oC 18oC/13 oC 23oC/18 oC 5 18oC/13 oC 23oC/18 oC 23oC/18 oC 6 18oC/13 oC 28oC/23 oC 23oC/18 oC 7 18oC/13 oC 18oC/13 oC 28oC/23 oC 8 18oC/13 oC 23oC/18 oC 28oC/23 oC 9 18oC/13 oC 28oC/23 oC 28oC/23 oC

10 23oC/18 oC 18oC/10 oC 23oC/18 oC 11 23oC/18 oC 23oC/18 oC 23oC/18 oC

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The treatments were imposed by interchanging pots between appropriate controlled temperature glasshouses. For treatments 10 and 11 a single temperature comparison was made as per Table 3.1. These 2 treatments were imposed to make comparisons with similar temperature treatments used overseas to induce Brown Centre and Hollow Heart. Unfortunately, plants grown in Treatment 10 suffered from a wilt disorder immediately upon transfer to the cooler glasshouse and senesced early. At 112 DAP plants were harvested. In addition to the practices outlined in the general materials and methods the soil depth to the top of each tuber was recorded and the fresh yield determined for each tuber in each pot. The dimensions of each tuber were recorded determining width and depth (cv Sebago has an ovoid shape) and length of tuber.

Effects of day and night temperature 2001 On July 3, 2001 a controlled environment experiment was planted to evaluate the effects of combinations of day and night temperatures on incidence of BF. At emergence, pots were shifted to an 18oC/13 oC day/night temperature controlled environment glasshouse. Four pots were allocated to each of 5 blocks according to their date of emergence. The treatments consisted of 4 day/night temperature regimes which were imposed at 98 DAP for a period of 14 days. These day/night temperature regime treatments included 18oC/13 oC, 23oC/18 oC, 28oC/13 oC and 28oC/23 oC. The comparison of results between treatments 28oC/13 oC and 28oC/23 oC gives a night temperature effect. The comparison of results between treatments 28oC/13 oC and 18oC/13 oC gives a day temperature effect. Whilst the comparison of results between treatments 28oC/13 oC and 23oC/18 oC gives the mean temperature effect, since the mean temperature for these 2 treatments was 20.5 oC. Plants were harvested at 112 DAP and standard measurements made as per the materials and methods section. Ambient air temperature and relative humidity were monitored for the duration of the experiment using automatic loggers. Soil temperature in a selection of pots was also logged over the duration of the experiment.

Soil and Air temperature effects on BF incidence 2002 On July 1 2002 a controlled environment experiment was planted to evaluate the effects of soil and air temperature on incidence of BF. At emergence, about 14 DAP, pots were allocated to each of 4 blocks according to their date of emergence and transferred to a temperature controlled glasshouse at 18oC/13 oC day/night temperature. The experimental design was a factorial replicated 4 times. The factors included two air temperatures and 3 soil temperatures. At 98 DAP pots were split into the 2 air temperature treatments (18oC/13 oC and 23oC/18 oC day/night) for a period of 14 days. The series of pots in each of these rooms was simultaneously split into 3 sets of 4 pots. Soil temperature treatments (nominally 18oC, 23oC and 28oC) were imposed by placing each set of pots in a temperature controlled bath maintained at the appropriate temperature.

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In previous experiments a high temperature regime of 28/23 oC has been used. In this experiment the milder high temperature regime (23/18 oC) was used so as to minimise difficulties in maintaining temperature in the soil treatment temperature baths. Changes in soil temperature in a selection of pots from each treatment were logged at 15 minute intervals over the duration of the experiment. The actual mean soil and air temperatures for the treatment period is presented in Table 3.2. Table 3.2. Measured temperature data for treatments in controlled environment experiment.

Nominal soil temperature Nominal Air temp oC

Actual mean air temp oC 18oC 23 oC 28 oC

18/13 oC 16.4 17.3 23.8 25.9 23/18 oC 20.0 18.0 24.8 27.1

Days exposure to high temperature 2003 The experiment was planted 1 July 2003 and plants initially grown in ambient glasshouse conditions. At emergence pots were transferred to a controlled environment glasshouse at 18/13 oC day/night temperature. The treatments imposed consisted of varying periods of exposure to high temperature (28/23 oC day/night temperature). Four treatments were imposed consisting of a 0, 1, 2 and 4 day exposure period. A cautionary note is that the seed in this experiment appeared to be aged. This seed broke dormancy very quickly, had rapid emergence and plants showed signs of senescence at a very early stage about (13 weeks), despite being grown under mild temperature. For this reason the treatments were imposed at 96 DAP and the experiment harvested at 100 DAP; this contrasted with previous experiments, which were harvested at about 112 DAP. Hence at the time treatments were imposed plants were not likely to have been actively growing. Results

Effects of phasic temperature regimes on BF incidence and growth parameters 2000 Phasic temperature regimes in the final 3 weeks of potato plant growth and tuber bulking had a substantial effect on the incidence and expression of BF. The mean percentage tuber area affected by BF in Treatment 1 was 4.55% compared with that in Treatment 9 which had 21.7% surface area affected. The percentage number of tubers affected by BF was 28.2% in Treatment 1 compared with 67.6% in Treatment 9. Treatments where only one phase of high temperature was imposed (Treatment 3, 7 and 8) exhibited intermediary incidence of Brown Fleck of about 8%. The maintenance of low temperature regimes particularly in the final stages of tuber bulking (Treatments 1 and 4) suppressed the development of BF (Table 3.3). Treatments 1 and 4 had a lower total area of BF per plant, lower mean tuber area

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affected with BF and a lower percentage of tubers affected with BF (P=0.05). In contrast, high temperature regimes in the final stage of tuber bulking (Treatments 6 and 9) dramatically increased the incidence of BF (Table 3.3) as reflected in all BF parameters. In all other treatments BF incidence was intermediary between these extremes and consistent with the milder temperature regimes. Of important note was the presence of very high BF incidence in treatment 11, which was grown at a continuous 23/18oC day night temperature. Table 3.3 Results of controlled environment experiment evaluating effects of phasic temperature regimes on incidence of Brown Fleck and growth components in potato cv Sebago.

Treat. No.

Tuber area affected by BF

(%) Dry Matter % Total BF area

per pot (mm2) Number tubers with BF (%)

Dry Matter Yield

(g pot-1) 1 4.5 ab 19.9 h 94.1 a 28.2 a 150 d 2 8.0 abc 17.7 ef 166.7 a 29.0 ab 110 ab 3 8.4 abc 15.7 ab 195.3 ab 40.6 abcd 103 ab 4 3.1 a 18.8 g 76.5 a 40.0 abc 148 cd 5 10.1 bc 17.1 cdef 212.9 ab 41.5 abcd 119 abcd 6 17.6 de 15.2 a 431.4 cd 57.8 cd 118 abc 7 9.0 abc 17.9 fg 216.0 ab 55.5 bcd 132 bcd 8 8.2 abc 16.3 abc 186.6 ab 54.7 abcd 132 bcd 9 21.7 e 16.7 bcde 510.6 d 67.6 d 124 bcd

10 4.1 ab 16.6 bcd 99.5 a 32.4 abc 89 a 11 14.7 cd 17.6 def 341.0 bc 67.2 d 141 cd

LSD = 6.645 LSD = 1.085 LSD = 159.7 LSD = 27.13 LSD = 31.16 1Treatments with the same letter are not significantly different at P < 0.05.

Treatment No.

1 2 3 4 5 6 7 8 9 10 11

Tube

r Fre

sh Y

ield

(g)

500

600

700

800

900

1000

Fig 3.1 Tuber Fresh yield response to temperature treatments in potato cv Sebago.

Low temperature regimes particularly in the final treatment period resulted in higher dry matter (DM%) compared with high temperature regimes (Table 3.3). In Treatments 1, 4 and 7, where the final period temperature was 18oC/13 oC, high dry

16

matter contents (19.9%, 18.8% and 17.9% respectively) were recorded whereas in Treatments 3, 6 and 9, where the phase 3 temperature was 28oC/23 oC, the dry matter content was much lower (15.7%, 15.2% and 16.7% respectively). Tuber dry matter accumulation appeared to be less sensitive to temperature in Phase 2. There was also evidence of heat necrosis in tubers in this experiment and this was largely a function of the pots not being insulated and higher soil temperatures recorded due to the plastic warming well above the air temperature despite being in a controlled environment glasshouse. The highest yield was displayed in the treatment where plants were continuously grown at 23oC/18 oC (Treatment 11) (Fig 3.1). Across all treatments this treatment also had amongst the highest incidence of BF. Yield was also elevated in treatments 1 and 4 under mild temperature exposure.

Importance of Day and night temperatures in BF expression Varying the day and night temperature regimes significantly affected the incidence of BF. In particular, a high night time temperature favoured the development of BF since the incidence of BF in the 28oC/23 oC treatment was much greater than that in the 28oC/13 oC (Table 3.4 and Fig 3.2). In comparison, the effect of day temperature though apparent was less pronounced since the incidence of BF in the 28oC/13 oC was not significantly greater than that in the 18oC/13 oC (Table 3.4 and Fig 3.2). Table 3.4 Effects of Day and night temperature regimes on the incidence of brown Fleck in potato tubers. Temperature regimeI BF incidence 18oC/13 oC 23oC/18 oC 28oC/13 oC 28oC/23 oC Total BF per pot (mm2) ** 10.9a 441.0b 92.2a 552.3b Average BF per tuber

(mm2) ** 2.3a 112.4b 21.2a 144.0b Tubers affected with BF (%) * 22.5a 58.5b 38.7ab 55.8b *denotes significantly different at 5% **denotes significantly different at 1% I denotes treatments with the same letter are not significantly different at p=0.05 The effect of the summed heat units (mean temperature) on BF is demonstrated by comparing the BF incidence in the 23oC/18 oC and 28oC/13 oC treatments, for which the mean temperature for both treatments was 20.5oC. The incidence of BF in the 23oC/18 oC was far greater than that recorded in the 28oC/13 oC. This occurred despite the fact that the daytime temperature in the 23oC/18 oC treatment was cooler than that in the 28oC/13 oC; further highlighting the importance of night temperature in the development of BF symptoms.

17

Temperature Regime (oC)

18/13 23/18 28/13 28/23

Tota

l BF

per p

ot (m

m2 )

0

100

200

300

400

500

600

Figure 3.2 Effect of temperature on the incidence of BF in potato tubers expressed as the mean affected area mm2.

Dry matter accumulation Table 3.5 Effects of day and night temperature regimes on the yield and dry matter accumulation in potato tubers averaged per pot. Mean of all tubers per pot 18oC/13 oC 23oC/18 oC 28oC/13 oC 28oC/23 oC lsd Dry matter % ** 21.6 21.2 19.5 18.9 1.6 Fresh yield (g) NS 728 814 674 695 DM yield (g) NS 159.7 171.2 136.4 132.4 *denotes significantly different at 5% **denotes significantly different at 1% The dry matter percentage (DM%), varied across treatments, with the 18oC/13 oC and 23oC/18 oC treatments recording higher dry matter than that in the other two treatments (Table 3.5 Fig 3.3).

18

Temperature Regime

18/13 23/18 28/13 28/23

Tube

r Dry

Mat

ter (

%)

16

18

20

22

24

Fig 3.3 Effect of temperature on the dry matter percentage of potato tubers cv Sebago. Figure 3.3 highlights the dry matter difference (% dry matter) for tubers across treatments. The combination of cool day and night temperatures in the 18oC/13 oC treatment gave highest dry matter. A day/night temperature regime of 23/18oC was more favourable for rapid tuber growth than the higher 28/23 oC or lower 18/13 oC. This is confirmed in the data presented in Table 3.5 where the 23oC/18 oC treatment had the highest fresh yield and dry matter yield (product of Fresh yield x DM%) of all treatments (P=0.07). This trend though not significant would suggest that higher night respiration at higher temperature was not the driving factor behind increased BF incidence since higher dry matter accumulation was recorded at higher night temperature.

Soil and Air temperature effects 2002 Air and soil temperature effects on BF incidence Irrespective of soil temperature, increasing the air temperature from 18/13 oC to 23/18 oC significantly increased the amount of BF in tubers (Table 3.6). This demonstrates and confirms that the air temperature experienced by foliage is a key factor in the development of BF symptoms. The increase in BF across this temperature differential was about 30%. The high temperature treatment in this study was only 23/18 oC compared with the higher regime (28/23 oC) used in previous studies (2000 and 2001) and more intense BF development is expected at the higher temperature. Furthermore, there was a significant positive correlation between the amount of foliage at harvest and the incidence of BF. The effect of air temperature on incidence of BF was most pronounced at a soil temperature of 18 oC and its relative importance was reduced when the soil temperature was increased (Fig 3.4). At soil temperatures of 23 and 28 oC there was only a small difference in BF incidence between the 2 air temperature treatments. The

19

effect of air temperature at the lowest soil temperature appeared to be greater than at higher soil temperatures but this was not significant (p=0.05).

Soil Temperature (oC)

18 23 28

BF

inci

denc

e (m

m2 )

0

100

200

300

400

18oC AirTemp23oC AirTemp

Fig 3.4 Effects of Air and soil temperature on the incidence of BF in cv Sebago Table 3.6. Effect of Air temperature on incidence of BF, fresh weight, Dry matter %, and dry matter yield in potato cv Sebago. Air Temperature regime

Total BF (mm2)*

Fresh Weight Dry matter (%)

Dry Matter Yield (g pot-1)

18/13 oC 217 945 20.38 190 23/18 oC 284 874 20.13 178 LSD 55.3 69.1 ns ns

*Denotes significantly different at p=0.05 An increase in soil temperature from 18 oC to 23oC to 28 oC progressively increased the incidence of BF (Table 3.7). Table 3.7. Effect of soil temperature on incidence of BF, fresh weight, Dry matter %, and dry matter yield in potato cv Sebago. Nominal Soil Temperature

Total BF (mm2)**

Fresh Weight Dry matter (%)**

Dry Matter Yield (g pot-1)

18 oC 101 893 21.56 193 23 oC 277 938 20.20 189.6 28 oC 374 897 18.99 169.6 LSD 68.1 ns 1.099 ns

**Denotes significantly different at p=0.01

20

Effect of Air and soil temperature on tuber yield parameters The fresh weight yield in the 18/13oC air temperature treatment was significantly greater than that in the 23/18oC treatment (Table 3.6). Air temperature did not significantly affect tuber dry matter %. There was no significant difference in the fresh yield and dry matter yields between all treatments at soil temperatures of 18 oC and 23 oC (figs 3.5 and 3.6), irrespective of air temperature. However, in the 28 oC soil and 18/13 oC air temperature treatment, fresh yield (1005g) was much greater than that in all other treatments. Increasing soil temperature also dramatically reduced the dry matter content of tubers (Table 3.7). At 18 oC soil temperature the dry matter content was 21.56% whereas in the 28 oC treatment dry matter content was only 18.99%. This confirms previous suggestions that tuber dry matter is most strongly influenced by soil temperature effects. In support of this the net dry matter of tubers was also reduced at high soil temperature. Air temperature did not significantly affect tuber dry matter % though the trend was for the lower air temperature to produce higher tuber dry matter (Table 3.6). The high air temperature regime in this study was mild and a greater response to air temperature might be expected at higher extremes.

Soil Temperature (oC)

18 23 28

Fres

h yi

eld

(g/p

ot)

0

200

400

600

800

1000

1200

18/13 oC 23/18 oC

Fig 3.5. Effects of day/night air temperature regimes (18/13oC and 23/18oC) and soil temperature on fresh yield of potato cv Sebago.

21

Soil Temperature (oC)

18 23 28

Dry

mat

ter y

ield

(g/p

ot)

0

50

100

150

200

250

18/13 23/18

LSD p=0.05

Period of temperature exposure Though not significant at p=0.05, the incidence of BF increased with relatively short exposure to high temperature of 28/23oC. There was a steady increase in, the area of BF per tuber, the number of BF lesions per tuber, and the average size of lesions (Figs 3.7 a,b,c). There was a small apparent increase in average fresh tuber fresh weight with increased duration of exposure to the higher temperature (Fig 3.8).

a) b) c)

Treatment period (days)

0 1 2 4

BF a

rea

(mm

2 /tube

r)

0

5

10

15

20

25

30

35

Treatment period (days)

0 1 2 4

Mea

n B

F le

sion

are

a (m

m2 )

0

2

4

6

8

10

Treatment period (Days)

0 1 2 4

BF

lesi

ons

(Cou

nts

per t

uber

)

0.0

0.5

1.0

1.5

2.0

Fig 3.7. The Effect of temperature treatment exposure (28oC/23oC day night) over varying time periods on a) Area of BF per tuber, b) average lesion size per tuber and

c) average lesion count per tuber.

Fig 3.6. Effects of day/night air temperature regimes (18/13oC and 23/18oC) and soil temperature on dry matter yield of potato cv Sebago.

22

Treatment period (days)

0 1 2 4

Ave

rage

Tub

er F

resh

Wei

ght (

g)

0

20

40

60

80

100

120

140

160

Fig 3.8. The Effect of temperature treatment exposure (28oC/23oC day night) over varying time periods on average tuber fresh weight.

Discussion Results over the series of experiments clearly demonstrate that elevated temperatures dramatically increase, the expression of BF and fresh yield, but reduce tuber DM%. In particular high nighttime temperature and high soil temperature result in increased expression of BF. Elevated daytime temperature did not greatly increase the expression of BF. Mild or high night temperature regimes were most favourable to the development of BF. In the 2000 experiment plants exposed to a constant day/night temperature regime of 23/18oC exhibited BF incidence as high as that exhibited under a shorter exposure to 28/23 oC whereas at 18/13 oC BF incidence was much lower. In 2001, there was little difference in BF incidence between treatments where exposure was 18/13 oC and 28/13 oC, which reinforces the notion that high night temperatures are critical in driving the development of BF symptoms. In contrast, the BF incidence was much greater under a 23/18 oC and a 28/23 oC regime than under the 28/13 oC regime. The highest yield tended to be obtained in the plants grown under a constant regime of 23/18oC (fig 3.1) highlighting the relationship between high yield potential and BF incidence. It is likely that the temperature regime 23oC/18 oC provides a combination of mild days favourable for increased plant photosynthesis and warm nights that favour assimilate mobilisation to tubers and BF development. This is further confirmed by virtue of the positive correlation between plant foliage weight and incidence of BF. Plants with a greater amount of foliage exhibit higher BF incidence. This effect is likely to be indirect and due to the greater amount of foliage increasing assimilate supply to tubers. The finding is in keeping with all other experiments where foliage mass is consistently correlated with incidence of BF. In contrast to the night temperature effect, high day time temperature did not appear to greatly increase the incidence of BF. This is likely to be due to a reduced net

23

assimilate production at higher temperature as a result of increased foliage respiration. Also, elevated temperatures tend to favour foliage growth through increased gibberellic acid (GA) production (Menzel 1985). In turn this would restrict the supply of assimilate to tubers thereby reducing the expansion of BF symptoms. In all experiments low incidence of BF was found in plants grown under an 18/13oC day night temperature regime, highlighting that although high temperature accentuates the symptoms of BF, temperature per se is not the sole factor determining the incidence of the disorder. This feature was most noticeable in the first experiment, which was planted in early May compared with the other 3 experiments which were planted in early July. Notwithstanding, elevated temperatures favour induction of BF since the average lesion count per tuber and tubers affected with BF both increase under higher temperature. It is apparent that an expansion of BF symptoms occurs at higher temperature probably due to the increased activity of oxidising enzymes and greater assimilate supply to tubers. The latter is consistent with the finding that elevated night temperatures increased the expression of BF since mild night temperatures favour assimilate production and tuber growth. The issue of temperature and tuber growth and BF incidence is important since increased temperature not only increased BF but consistently increased tuber yield in each experiment. The microscopy studies highlight that increased carbohydrate supply and the inability of blocked phloem sieve cells pass the extra sucrose to newly developing cells appears to be a major factor in the expansion of BF symptoms. Research in 2001 also suggested that soil temperature was perhaps not a critical factor in inducing BF. The incidence of BF in a 28/13 oC treatment was less than in a 23/18

oC treatment even though the soil temperature was about 1 oC higher in the 23/18 oC treatment. The study in 2002 subsequently highlighted that higher soil temperature does in fact also favour the development of BF symptoms. Thus combinations of high soil and high night air temperature are favourable for BF development. In this study the effect of soil temperature appeared to be greater than that of air temperature. However, the maximum air temperature imposed in the 2002 experiment was mild in comparison to the soil temperature imposed, and in comparison to air temperatures under which BF is experienced in the field. Furthermore the highest soil temperature of about 27oC is in excess of the soil temperature at which potatoes would generally experience in regions where BF is experienced. Olsen et al. (1996) grew potato plants cv. Russet Burbank in a pot experiment under two soil temperature regimes of 18oC and 32oC the plant tops were maintained at ambient day/night temperature of 21oC /16oC. Increasing the soil temperature did not increase the percentage of tubers affected by IBS, however, the severity of IBS was increased. It is likely that the main effect of increased soil temperature on incidence of BF is through an expansion of existing symptoms through increased activity of oxidising enzymes and greater tuber respiration. In a field situation both soil and air temperature could induce BF. Under full canopy cover, prior to senescence, the soil is well covered from direct sun and maintained at relatively low temperatures (about 20oC in field studies). Under this situation it is likely that air temperature will principally determine incidence of BF and particularly

24

mild to high night temperatures that favour high sucrose transfer to tubers. However, in crops where senescence is uneven, soil temperature is likely to be relatively higher due to greater soil exposure to sun and hence the incidence of BF could be higher. In a field situation a major factor that could induce this problem is physiologically young seed that results in uneven emergence, maturation and senescence. Temperature in the final 3 weeks of tuber bulking appears critical in determining the proportion of dry matter in tubers. In the 2000 experiment cool bulking conditions as in Treatment 1 resulted in the highest tuber dry matter (19.88%). This was achieved in the non-crisping cultivar Sebago and equated to an S.G. (specific gravity) of 1.079. In contrast tubers that bulked under high temperatures, Treatment 9 and 6, had the lowest dry matters 16.65 and 15.23% respectively equating to S.G.s of 1.063 and 1.057 respectively. In 2001 experiment the higher the temperature exposure the greater the reduction in DM%. A reduction in DM% was noted in the 28oC/13 oC and 28oC/23 oC compared with the 18oC/13 oC and 23oC/18 oC (Fig 3.3) indicating high day temperature, though not necessarily favourable for inducing BF, reduced the dry matter accumulated in tubers. It is likely that at high day temperature the efficiency of photosynthesis is reduced and foliage respiration is increased resulting in less dry matter production and partitioning to tubers. This result is consistent with the findings in the field phenology study where a significant negative correlation was recorded between tuber weight gain and averaged daytime temperature. Also, the DM% in the 28oC/13 oC was higher than that in 28oC/23 oC indicating the further action of high night temperature reducing the amount of dry matter in the tubers. The factor that prominently drives tuber DM% is respiratory losses from both foliage and tubers under generally elevated temperatures whereas BF incidence is particularly related to warm night temperature. In concluding there appears to be 2 distinct components relating to incidence of BF. On the one hand there appears to be an initiation of BF that can occur across a range of temperatures including cool temperatures but is more evident at mild temperatures (e.g. 23/18 oC day/night). This is the process whereby an initial phloem sieve cell dies. Then there is a further direct effect of temperature whereby warm nights favour symptom development likely to be related to increased sucrose loading. Further research is required to elucidate the effects of temperature on the initiation of BF and growth and development of symptoms.

25

4. Effects of Calcium and Boron on BF Background In controlled environment studies plants grown continuously at 18oC/13 oC have exhibited low-level incidence of BF. This suggests that factors other than just high temperatures alone influence the expression of the disorder. Considerable overseas research has been conducted on the effects of Ca on the incidence of BF but boron has received little or no attention. This is despite the fact that scant reference to boron deficiency inducing internal browning in potatoes is presented by Weir and Cresswell (1993) and Shorrocks (circa 1985). In the Lockyer Valley region of Queensland, where BF is an issue in potato production, soils contain unusually high levels of soil Ca and responses to B are more likely. By way of a series of glasshouse and field experiments the effects of boron and calcium on BF incidence and yield in potatoes was evaluated. Methodology

Field nutrition trial 2001 The first field nutrition experiment was conducted at the Queensland DPI&F - Gatton Research Station and planted on July 10 2001. The field plot size consisted of 62 rows (at 0.75 m spacing) each of 90 m length. The experiment was conducted as a split plot design. The main plots were Ca rates (0, 50 and 100 kg ha-1) and sub-plots were four Boron rates (0, 1, 2 and 3 kg ha-1), replicated 4 times. Individual plot size was 3 m (4 rows at 0.75m spacing) x 5m long. In the 1, 2 and 3 kg ha-1 B treatments, 1 kg ha-1 equivalent was applied as Sodium tetra-borate at 21 DAP. In the 2 and 3 kg ha-1 treatments the remainder was applied at 63 DAP. Calcium was supplied as broadcast calcium nitrate (CaNO3) at 71 DAP when the king tuber was about 3-5 cm and irrigated in using solid set spray irrigation. The boron was applied as dissolved aliquots applied over the hills of each plot and similarly irrigated in. Rates of basal nutrient application and side dress application rates (in kg ha-1) of 160 N, 20 P, 180 K and 120-130 S were applied as CK77S and a combination of potassium nitrate and ammonium nitrate as per the general materials and methods. The extra nitrogen applied in the Ca treatments was balanced by reducing the ammonium nitrate application at side dressing. At 84 DAP a leaf sample (youngest expanded blade) was taken from each treatment and held for nutrient analysis as appropriate. Plots were harvested by digging the 2 middle rows in each plot as the datum. Tubers were graded and evaluated as per the general materials and methods.

26

The growing season for this field experiment was marked by continuous strong westerly wind influences, which reduce the yield capacity of the crop and are generally considered much less favourable for inducing BF. Furthermore, the tuber filling period late October to November experienced unusually cool nights (data not presented), which as determined in the glasshouse studies do not favour the development of BF.

Glasshouse nutrition study 2002 On July 1 2002 well shot whole set tubers were planted in each of 65 pots. In contrast to other glasshouse temperature experiments plants were grown under ambient glasshouse conditions for the duration of the experiment. At emergence, about 14 days after planting (DAP), pots were allocated to each of 4 blocks according to their date of emergence. Ambient air temperature and relative humidity were monitored for the duration of the experiment using automatic loggers. Soil temperature in a selection of pots was also logged over the duration of the experiment. Nutrition Treatments The calcium and boron treatments consisted of 3 Ca rates and 4 Boron rates in factorial combination with four replications. These rates were: Calcium 0, 50, 100 kg ha-1 equivalent Boron 0, 1, 2, 4 kg ha-1 equivalent In the 1, 2 and 4 kg ha-1 B treatments, 1 kg ha-1 equivalent B was applied as Sodium tetra-borate at 21 DAP. A further 1 kg ha-1 equivalent was added to the 2 and 4 kg ha-

1 treatments 49 DAP and a final B application of 2 kg ha-1 equivalent was made to the 4 kg ha-1 B treatment at 70 DAP. Calcium was supplied as calcium nitrate in 2 split applications applied at 63 and 70 DAP. The extra N applied as CaNO3 in the highest Ca treatment was balance in the other Ca treatments using appropriate rates of ammonium nitrate. The total rate of basal nutrient application and side dress application was 160 N, 180 K and 90 S (in kg ha-1) equivalent. All other experimental details were as per the general materials and methods.

Field nutrition experiment 2002 This experiment was conducted as a field trial at the Queensland DPI&F - Gatton Research Station. Whole set certified seed potato (Solanum tuberosum cv Sebago) was planted on July 8 2002. The experiment was conducted as a factorial in a randomised complete block design. Factors included four Boron rates (0, 1.3, 2.7 and 5.0 kg ha-1) by three Ca rates (0, 67 and 127 kg ha-1) replicated 4 times. Individual plot size was 2.25 m (3 rows at 0.75m spacing) x 6 m long. Boron was applied to the B treatments as Sodium tetra-borate at hilling (about 28 DAP) and via trickle irrigation. Calcium was supplied as Calcium nitrate at 63 DAP via trickle irrigation. Rates of basal nutrient application and side dress application rates (in kg ha-1) of 160 N, 180 K and 120-130 S was applied as CK77S at planting and a combination of potassium nitrate and ammonium nitrate through trickle irrigation. The extra nitrogen

27

applied in the Ca treatments was balanced by reducing the side dressing ammonium nitrate application. The trial was harvested at 112 DAP.

Grower nutrition study This experiment was conducted on a grower property at Mulgowie via Laidley Queensland. Cut certified seed potato (Solanum tuberosum cv Sebago) was planted on July 17 2002. Individual plot size was 3.2 m (4 rows at 0.80 m spacing) x 10 m long. Four Boron rates (0, 0.5, 1 and 2 kg ha-1) were imposed and replicated 4 times. Boron was applied as Sodium tetra-on 12 September as aliquots sprayed onto hills, incorporated at hilling and irrigated in using overhead spray irrigation. On 28 November (126 DAP), the trial was harvested by digging the 2 middle datum rows in each plot. The net weight of tubers was determined and a sample of the 20 largest tubers collected for evaluating the incidence of brown fleck.

Results and discussion

Field nutrition trial 2001 There was a significant interaction between the effects of Boron and Calcium on the incidence of BF. Application of boron at 2 and 3 kg ha-1 significantly reduced the incidence of BF at a Ca rate of 0 kg ha-1. Similarly, Ca at 100 kg ha-1 tended to reduce the incidence of BF particularly at a B application rate of 0 kg ha-1. Given a square root transformation on the data was required Table 4.1 presents data for the incidence of BF as the square root of the actual total area of BF in a 20 tuber sub-sample.

Table 4.1. Effect of Boron and calcium application on mean total BF per plot sample a* in field grown potatoes 2002.

Ca Rate B rate (kg ha-1) kg ha-1 0 1 2 3

0 24.1 15.1 6.8 8.5 50 18.0 14.8 14.5 19.6 100 8.3 15.6 18.0 14.3

*denotes significantly different at 5% (lsd = 11.01) **denotes significantly different at 1%

There was a strong negative interaction between Ca and B on the incidence of BF. That is, combinations of high rates of Ca (100 kg ha-1) and B (2-3 kg ha-1) were no more effective at reducing BF incidence, than was either the Ca or B rate alone. This interaction is difficult to explain on the basis of nutritional effects per se and highlights the complexity of interactions between Ca and B. Furthermore it provides scant support to the validity of overseas reports that indicate Ca can alleviate the disorders symptoms.

28

Table 4.2. Effect of Boron and calcium application on total yield in field grown potatoes 2002

Ca Rate B rate (kg ha-1) kg ha-1 0 1 2 3

0 36.4 41.3 39.3 39.1 50 40.9 36.4 37.8 36.5

100 37.0 33.0 35.3 38.1 Not significant at p = 0.05 There was no significant difference in yield between treatments however there was a trend for B treatments at 1 and 2 kg ha-1 at 0 kg ha-1 Ca to out yield all other treatments (Table 4.2). The underground irrigation water in this trial does contain significant concentrations of Ca, which may have impacted on results, but nonetheless application by this source would have been consistent across the trial site. Increasing the Ca rate resulted in lower dry matter yield (Yield per ha multiplied by tuber dry matter %) (P=0.05). The dry matter yields at 0, 40 and 80 kg ha-1 Ca, were 6.93, 6.58 and 6.63 tonne ha-1 respectively. Given the relationship between yield and incidence of BF it is possible that the reduction in yield with Ca application contributed to the reduced BF incidence.

Glasshouse nutrition study 2002 In the glasshouse experiment there was no significant effect of Ca and B on the incidence of BF, tuber yield, dry matter content or total dry matter yield per pot, nor were there any apparent trends in the effects of Ca and B (Table 4.3). Boron at progressively higher rates in combination with Ca at 0 and 100 kg ha-1 appeared to reduce the incidence of BF but not in the 50 kg ha-1 Ca treatment (Fig 4.1).

Boron rate (kg ha-1)

0 1 2 4

BF

inci

denc

e (m

m2 /p

lant

)

0

200

400

600

800

1000

1200

1400

1600

1800

Ca 0 kg ha-1

Ca 50 kg ha-1

Ca 100 kg ha-1

Fig 4.1 Interaction between Ca and B rate on the incidence of BF in field grown potato tubers 2002.

29

The trends in BF incidence for the 0 and 100 kg ha-1 Ca treatments over B rates were the opposite of that for the 50 kg ha-1 Ca treatment. The F value probability for this relationship was 0.07 indicating a reasonable probability the relationship exists. This anomaly cannot be explained by the Ca and B rates alone. More likely it could be related to the different ratios of ammonium and nitrate nitrogen imposed in the 3 Ca treatments. The Ca treatments were imposed using highly soluble CaNO3 and to balance the accompanying N, ammonium nitrate was added to the 0 and 50 kg ha-1. The difference in ratios of NH4

+:NO3- across Ca treatments could have resulted in greater

growth at the intermediary Ca rate resulting in higher BF incidence. Cao and Tibbitts (1993) have demonstrated that the optimal ratio of NH4

+:NO3- for potato growth

appears to be between about 20:80. Given the unusual interaction in the first field experiment between Ca and B it is possible the effect of differing NH4

+:NO3- ratios

was also influencing BF expression. This unanticipated apparent effect is unfortunately confounded with the Ca treatment itself and hence cannot be meaningfully interpreted. Table 4.3. Effect of Calcium (0, 50 and 100 kg ha-1) and Boron (0, 1, 2 and 4 kg ha-1) on a) brown fleck incidence (mm2) per pot, b) mean tuber fresh weight (g) per pot, c) mean tuber dry matter percentage and d) Mean tuber dry matter yield per pot (g) in a glasshouse experiment 2003.

a)

BF incidence (mm2) ns

b)

Tuber weight (g pot-1) ns

Ca Rate Ca Rate B Rate 0 50 100 B Rate 0 50 100

0 1026 271 1372 0 1012 1101 1053 1 1153 985 1031 1 1013 1102 984 2 905 1371 790 2 1122 954 1036 4 753 1745 745 4 1099 1197 1068

c)

Tuber Dry matter (%) ns

d)

Tuber dry matter yield (g pot-1) ns

Ca Rate Ca Rate B Rate 0 50 100 B Rate 0 50 100

0 19.8 18.9 19.9 0 201 208 210 1 18.5 19.3 19.1 1 193 214 186 2 19.2 18.4 19.1 2 216 176 198 4 19.0 19.6 19.4 4 210 234 208

ns denotes not significant at p = 0.05 In the field nutrition experiment 2003 there was no significant difference between nutrition treatments for BF incidence, Total fresh yield and Dry matter yield (Table 4.4). Furthermore, there were no obvious trends that would indicate benefits of

30

adding Ca and B to reduce incidence of BF or increase yield. The application of B at the highest rate appeared to increase tuber dry matter % (Table 4.4), though this is not likely to be a real result as other trials have shown an opposite effect. Table 4.4.Effect of Calcium (0, 65 and 127 kg ha-1) and Boron (0, 1.3, 2.7 and 5.0 kg ha-1) on a) mean tuber brown fleck incidence (mm2) b) Tuber fresh yield (tonne ha-1), c) mean tuber dry matter (%) and d) Tuber dry matter yield (tonne ha-1) in a field experiment at Gatton 2003.

a)

Mean tuber BF incidence (mm2) ns

b)

Total tuber yield (tonne ha-1) ns

Ca Rate Ca Rate B Rate 0 65 127 B Rate 0 65 127

0 133 147 153 0 61.2 56.1 54.51.3 100 161 95 1.3 59.7 64.8 59.02.7 150 148 177 2.7 60.9 58.3 62.65.0 116 110 92 5.0 61.0 63.1 57.3

c)

Tuber Dry matter (%) ns

d)

Tuber dry matter yield (tonne ha-1) ns

Ca Rate Ca Rate B Rate 0 65 127 B Rate 0 65 127

0 17.3 16.9 17.5 0 10.6 9.5 9.51.3 17.4 17.6 18.1 1.3 10.3 11.4 10.72.7 17.2 17.5 17.3 2.7 10.5 10.2 10.95.0 17.8 18.1 18.2 5.0 10.8 11.5 10.4

ns denotes not significant at p = 0.05 The grower trial at Mulgowie evaluated the effects of B (0, 0.5, 1 and 2 kg ha-1 ) on BF incidence. There was no significant effect of B on BF incidence though there appeared to be a trend for the higher rate of B to reduce BF incidence (Table 4.5). Similarly, progressive increases in B rate appeared to reduce tuber dry matter, however this effect was also not significant. This apparent reduction in dry matter % at the high rate of B appears contrary to that observed in the GRS trial where the highest rate of B increased tuber dry matter %. The effects of both Ca and B on incidence of BF are inconsistent and generally not statistically significant. The application of B at high rates of about 4 kg ha-1 at times appears to reduce BF but not in a reliable manner. It is possible that any effect of either B or Ca on BF incidence is indirect through an inhibition of foliage growth which would reduce the intensity of BF symptoms. Also, Dekock et al. (1975) claim that in tissues containing adequate Ca, the Ca chelates phenolic compounds. The apparent effect of Ca supplementation in reducing BF incidence could be simply through the complexing of brown compounds and hence a masking of BF symptom development. There is no basis for making recommendations for application of Ca

31

and B to reduce incidence of BF. The issue of the form of N application as ammonium or nitrate and relationship to yield and BF warrants further investigation. Table 4.5. Effect of Boron (0, 0.5, 1 and 2 kg ha-1) on mean brown fleck incidence (mm2) per tuber, mean tuber fresh yield (Tonne ha-1) and mean tuber dry matter percentage in trial conducted on grower property Mulgowie. Boron rate 0 0.5 1.0 2.0 Mean BF per tuber ns 83.7 63.8 44.0 41.6 Fresh Yield (Tonne ha-1) ns 49.1 48.2 46.2 49.6 Dry matter (%) ns 18.42 17.36 17.08 16.86 ns denotes not significant at p = 0.05

32

5. Understanding Field Physiology of BF Background Previous experimentation has shown that high night temperatures combined with vigorous plant foliage favours BF incidence. Two explanations are put forward to explain this. Firstly, excessive cellular respiration in the plant tops could result in a night time deficit in assimilate resulting in cell death and hence BF. Alternatively, excessive assimilate supply under conditions favourable to rapid growth might cause major disruption to cells in the tuber. Both are consistent with field observations where in unevenly senesced crops green plants exhibit BF but senesced plants do not. The management of assimilate supply to tubers or foliage respiration would appear to be useful mechanisms for managing the incidence of BF. The experiments in this chapter evaluated the effects of various physiological effects on incidence of BF in the field. Three main effects were evaluated and included, removal of green tops, night time plastic covering and shading. The rationale behind these treatments was that:

I. Removal of green tops would reduce the extent of foliage respiration or assimilate supply and potentially reduce BF incidence.

II. Plastic covering at night would elevate night temperature and potentially increase BF incidence.

III. Shading would reduce both the amount of solar energy intercepted by the foliage as well as the heat experienced by foliage and this would potentially affect incidence of BF.

The aim of the experiment was to gain a more thorough understanding of the factors that underpin development of BF and evaluate opportunities to manage BF, in a field situation. The experiment thus attempts to simulate conditions typically associated with high BF incidence to give a better understanding of the factors that drive BF. These conditions include hot overcast days (somewhat achieved through shading) and warm nights (covering with plastic). The removal of green tops prior to maturity could provide a management tool to reduce BF incidence particularly when conditions might be favourable for the disorder. Materials and Methods Field trial 2002 This experiment was conducted as a field trial at the Queensland DPI&F - Gatton Research Station. Whole set certified seed potato (Solanum tuberosum cv Sebago) were planted on July 8 2002. The experiment was conducted as a factorial in a split plot design. The main plots included three plant topping regimes (Nil, tipped (25% top removed) and topped (50% top removal)). Two shading regimes (0% and 50%) (NilSH and SH) and 2 night time heat treatments (with and without plastic + Pl) were also imposed and replicated 4 times.

33

All treatments were imposed at 98 DAP. The topping treatments were imposed by removing 0% and approximately 25 and 50% of foliage from relevant plots. The shading treatments were imposed by permanently placing 50% shade cloth over relevant plots for the duration of the treatment period. The night temperature treatment was imposed by covering relevant plots with clear plastic prior to sunset and removing it after sunrise (about 7:00 AM) for a period of 10 days. Both the shading and plastic treatments were imposed for a period of 10 days. Grower physiology trial A further grower field trial was conducted which evaluated the effects of 0, 50% and 100% top removal and shading on incidence of BF. This experiment was conducted on a grower property at Mulgowie via Laidley Queensland. The treatments were imposed at 102 DAP. On 28 November (126 DAP), the trial was harvested by digging the 2 middle datum rows in each plot. The net weight of tubers was determined and a sample of the 20 largest tubers collected for evaluating the incidence of brown fleck. Analysis for Brown fleck was as previously described in the general materials and methods. Field top removal experiment 2003 The experiment was conducted as a field trial at the Queensland DPI&F - Gatton Research Station and planted on July 7 2003. This experiment was conducted as a randomised complete block and replicated 4 times. Treatments included

• Nil foliage removal • 25% foliage removal at T1 (Time 1) • 50% foliage removal at T1 • 100% foliage removal at T1 • 25% foliage removal at T2 (Time 2) • 50% foliage removal at T2 • 100% foliage removal at T2 • Total top removal at T2

The T1 treatments were imposed at 91 DAP and the T2 treatments imposed at 102 DAP. The topping treatments were imposed by removing 0, 25, 50 and 100% of leaf foliage from relevant plots. The plant material removed from each plot was immediately weighed and dried and the dry weight recorded. A severe hail storm defoliated the entire trial 2 days prior to harvest which precluded the final harvest of the plant tops. Agronomic practices, tuber grading and sampling were as per the general materials and methods.

34

Results and Discussion Top removal shading and plastic effects 2002 Green top removal, shading and plastic covering strongly affected the incidence of BF. There were significant interactions between, shading and top removal, and, plastic covering at night and top removal, and the incidence of BF. Furthermore, there was a significant 3 way interaction between shading, plastic and top removal and its affect on incidence of BF. Effects of top removal on BF incidence Across the shading and plastic treatments the incidence of BF (average per tuber) was not significantly affected by levels of green top removal (Table 5.1), though there was a trend for BF to be higher in the Nil treatment reducing in the tipped treatment but again increasing in the topped treatment. Top removal also dramatically reduced tuber yield (Table 5.1). In this experiment the yield reduction was in the order of 6 tonne ha-1 marketable yield under the topped treatment and in the tipped treatment about 2.5 tonne ha-1 compared with the Nil treatment. Similarly, both top removal treatments significantly reduced tuber dry matter %, through a reduced capacity for assimilate production. There was no significant main effect of plastic covering on the incidence of BF. The objective of applying plastic covering at night was to increase the night-time air temperature and though this was achieved to a minor extent (Table 5.2) no significant effect on BF incidence was recorded. Table 5.1. Marketable and total yield and dry matter percentage in cv Sebago under removal of green tops at Gatton Research Station 2002. Topping was conducted 11 days prior to harvest (16 week crop).

TreatmentI Nil Tipped Topped

BF (mm2/tuber) ns 32.9 23.1 30.0 Marketable yield*** 47.0a 44.4b 41.4c Total Yield*** 56.1a 52.6b 49.1c Dry matter %*** 16.77a 16.12b 15.78c ***denotes significantly different at p=0.001 I Values that have the same letter are not significantly different at p=0.05 NS denotes not significantly different p=0.05 Interactions between shading and plastic and top removal Plastic covering and top removal effects on BF There was a significant negative interaction between plastic covering and top removal on the incidence of BF on both a per tuber and treatment basis (Fig 5.1 a,b). Under the Nil top removal treatment covering with plastic resulted in lower BF than the comparable nil topping treatment without plastic covering. With progressive increases in top removal, plastic covering increased the incidence of BF whereas in the absence of plastic covering BF incidence was reduced with top removal. The presence of low BF under the Nil top removal with plastic is unusual since the

35

elevated temperature under plastic, combined with a full plant canopy and near ideal growth conditions, should provide ideal conditions for BF development. The most likely explanation for this anomalous result is that there was a reduction in supply of assimilate to the tubers, as related to the canopy structure and independent of a temperature effect; particularly since the air temperature variance across plastic and shaded treatments was not great. It is likely that the continual covering and uncovering of plots with plastic damaged plants or flattened the canopy, particularly in the Nil top removal treatment. Flattening the canopy may have restricted or altered light interception and hence reduced assimilate production thereby reducing BF incidence. Alternatively, the continual covering and uncovering of plots with plastic may have damaged the canopy or stems of plants thereby restricting the movement of assimilate to tubers. Either way, such a reduction in assimilate production or transfer would reduce BF incidence in accordance with the findings presented in other chapters; particularly the microscopy and phenology studies. Under plastic covering and elevated temperature it is expected that plant growth and yield would be increased, this did not occur. The yield of plants in the Nil top removal with plastic was marginally lower than in the comparable without plastic treatment (not significant p=0.05)(yields were 55.4 and 57.3 tonne ha-1 respectively). Under the Tipped and Topped treatments with plastic the level of canopy disruption was likely to be less than that for the Nil top removal and increased BF incidence was observed. The increase in BF over the Nil top removal treatment was dramatic. It is possible the combination of top removal and plastic provided conditions favourable for rapid tuber growth. The heavy top removal may have exposed lower leaves to greater light thereby increasing the foliage assimilate: respiration ratio. This would result in greater assimilate availability for tubers resulting in a rapid increase in tuber growth, consistent with conditions giving rise to BF. a) b)

Top Removal Treatment

Nil Tipped Topped lsd (5%)

BF in

cide

nce

(mm

2 /tube

r)

15

20

25

30

35

40

45

Plastic CoveredUncovered

Top Removal Treatment

Nil Tipped Topped lsd (5%)

BF

inci

denc

e (c

m2 /tr

eatm

ent)

10

15

20

25

30

35

40

Plastic Covereduncovered

Fig 5.1. Effect of top removal and covering with plastic on the incidence of BF on a) a per tuber basis and b) a treatment basis. Furthermore, the removal of tops exposes the soil to more direct sunlight, which in turn under plastic covering may have elevated night temperatures and relative humidity to an optimum for tuber growth. The temperature increases under the Nil

36

top removal tend to support this notion (Table 5.2). In contrast in the tipped and Topped treatments without plastic reductions in BF occurred due to removal of plant top and it is likely that assimilate production and mobilisation to tubers was reduced. Shading and top removal effects on BF a) b)

Top Removal Treatment

Nil Tipped Topped lsd (5%)

BF in

cide

nce

(mm

2 /tube

r)

15

20

25

30

35

40

45

UnshadedShaded

Top Removal Treatment

Nil Tipped Topped lsd (5%)B

F in

cide

nce

(cm

2 /trea

tmen

t)10

15

20

25

30

35

40

UnshadedShaded

Fig 5.2. Effect of top removal and shading on the incidence of BF on a) a per tuber basis and b) a treatment basis The ANOVA indicated a strong negative interaction between the effects of shading and top removal on incidence of BF on both a per tuber and total BF per treatment basis. However, this interaction was dependent on the very low incidence of BF in the Nil top removal treatment with plastic but not shaded (Fig 5.3). The data was partitioned to exclude the plastic covered treatments and revealed that the 2 way interaction for shading by top removal (Figs 5.2) was in fact not significant. On this basis there does not appear to be any interaction between shading and top removal on incidence of BF. Top removal however reduced the incidence of BF. It is likely the effect of shading in reducing light interception and hence assimilate production is offset by the effect of shading in reducing leaf temperature and hence respiration. The net effect is that the assimilate mobilisation to tubers is the same irrespective of shading.

LSD p=0.05

Top removal treatment

Nil Tipped Topped

Bro

wn

Flec

k (m

m2 /tu

ber)

0

10

20

30

40

50

NilSH +Pl NilSH -Pl SH +PL SH -PL

Fig 5. Effects of green top removal and night covering with and without plastic (+ PL) and shading (NILSH and SH) on the incidence of BF in potato tubers cv Sebago.

37

38

Tabl

e 5.

2 A

ir te

mpe

ratu

re, s

oil t

empe

ratu

re a

t 10

cm, a

nd re

lativ

e hu

mid

ity d

ata

for u

ntop

ped

treat

men

ts in

a fi

eld

phys

iolo

gy tr

ial a

t Gat

ton

Res

earc

h St

atio

n 20

02 e

valu

atin

g sh

adin

g (S

H),

nigh

t tim

e pl

astic

cov

erin

g (P

L) a

nd g

reen

top

rem

oval

on

pota

to c

v Se

bago

.

Perc

enta

ge ti

me

in e

ach

tem

pera

ture

zon

e Te

mpe

ratu

re

Nig

ht ti

me

D

ay ti

me

rang

e (o C

) N

il S

H +

Pl

Nil

SH

-Pl

SH +

PL

SH

-PL

N

il S

H +

Pl

Nil

SH

-Pl

SH +

PL

SH

-PL

<10

2.2

9.3

8.0

9.4

0.

6 1.

8 2.

2 1.

8

10-1

3 11

.3

3.7

5.9

3.5

2.

0 2.

8 3.

4 2.

8 13

-18

34.3

37

.8

36.9

37

.6

7.

3 8.

3 7.

3 9.

7

18-2

3 43

.7

41.7

41

.1

42.0

13.0

18

.3

13.0

17

.9

23-2

8 8.

5 7.

6 8.

1 7.

4

26.4

24

.9

24.9

24

.9

>28

- -

- -

51

.3

45.8

51

.5

44.8

Soi

l Tem

p 22

.3

22.4

21

.9

21.8

22.8

21

.8

21.1

20

.8

Mea

n R

H

78.8

80

.7

78.0

79

.4

48

.9

65.3

50

.0

57.2

39

Grower top removal and shading trial A trial on a grower’s property evaluated the effects of 0%, 50% and 100% top removal and shading on BF incidence, yield and dry matter %. Top removal dramatically reduced BF incidence (11.7 mm2 per tuber at 100% top removal), however, it also dramatically reduced total yield (Table 5.3). Furthermore, in the absence of shading, tuber dry matter was negatively affected by top removal but shading combined with top removal resulted in minimal effect on dry matter (Fig 5.4). It is likely that shading plots that had tops removed resulted in lower soil temperature and hence higher tuber dry matter due to reduced respiration in tubers. This trial confirms that green top removal prior to crop senescence has the potential to reduce BF incidence. Table 5.3. Tuber BF incidence and total yield in cv Sebago under removal of green tops at grower trial 2002. 0% Topping 50% Topping 100% topping Mean tuber BF*** 54.3a* 20.8b 11.7b Total Yield*** 50.6a* 43.6b 38.36c *Values with different letters are significantly different at p=0.05. *** denotes treatments were significantly different at P=0.1%

Fig 5.4. Effect of shading and green top removal on tuber DM% cv sebago.

Topping Treatment

Nil 50% 100%

Dry

Mat

ter (

%)

14

15

16

17

18

Nil shadeShaded LSD (p=0.05) = 0.64

40

Top removal experiment 2003 The removal of plant foliage at various times and proportions significantly reduced the intensity of BF expressed in both the total and marketable tuber ranges (Table 5.4). Removal of foliage at 50% and 100% at T1 significantly reduced the overall treatment incidence of BF by about 40% compared with the Nil removal treatment as well as the amount of BF per tuber. Removal of only 25% foliage at T1 appeared to reduce BF incidence compared with the Nil treatment, though the effect was not significant at p=0.05. The removal of any amount of plant foliage at T2 had no significant effect on incidence of BF. Interestingly, the 25% foliage removal treatment at T1 had amongst the highest yield but its BF incidence was relatively lower than other treatments that had comparable yield. This might suggest that small amounts of top removal early could result in lower BF incidence without a major sacrifice in yield. The first incidence of BF, as determined in the phenology trial, occurred on 25 Sept 2003 (80 DAP). In contrast the T1 treatments in this experiment were imposed on 6 Oct 2003 (90 DAP) and by this time a significant amount of BF was present in tubers sampled from the phenology trial. Table 5.4 The effect of foliage removal at 0%, 25%, 50%, and 100% at two different times (T1 and T2) on incidence of BF.

Treatment BF per treatment

(Marketable Tubers in cm2) 1

BF per treatment (all

tubers in cm2) 1

BF per marketable tuber (mm2) 1

Nil 20.6 c 24.0 c 120 b T1 Top 25% 17.8 c 19.8 c 99 b T1 Top 50% 14.6 ab 14.6 ab 73 ab T1 Top 100% 11.9 a 11.9 a 59 a T2 Top 25% 20.9 c 24.0 c 120 b T2 Top 50% 19.5 bc 21.4 bc 107 b T2 Top 100% 20.9 c 24.9 c 125 b T2 Whole Top 17.0 abc 18.1 abc 90 ab ** ** * 1Treatments with the same letter are not significantly different at P < 0.05. * denotes significant effect p=0.05 ** denotes significant effect p=0.01 The effect of foliage removal on marketable or total fresh yields was not significant at 5% though there was a trend for the 25% top removal treatments to have the highest yield (Table 5.5) and treatments where 100% foliage or whole tops were removed had the lowest yields. The marketable yield for the T1 100% top removal treatment was about 90% of that for the Nil top removal treatment but the BF incidence was about 60% of that in the Nil. The effects of top removal are confirmed in the dry matter yield data, calculated by multiplying fresh yield by dry matter %. The 25% top removal treatments had the highest DM yield of all treatments (p=0.05) and the 100% foliage removal and whole top removal had the lowest. The DM% was directly related to the amount of foliage removed with the highest dry matter recorded in the Nil and 25% topped treatments at both T1 and T2 (Table 5.5). The removal of only 25% foliage at T1 resulted in the same total DM% as recorded in the Nil indicating that the short term stress of removing tops had no long term effect

41

on tuber DM%. Irrespective of timing, both the marketable and total tuber DM%s were reduced in treatments where 100% foliage or whole top were removed. This was particularly evident in the 100% foliage removal treatment at T1 where the marketable and total DM% was only 15.56%. This effect was likely to be related to high soil temperatures increasing tuber respiration and a reduction in assimilate production with crop foliage removal. Table 5.5 The effect of foliage removal at 0%, 25%, 50%, and 100% at two different times (T1 and T2) on Marketable (Mkt) and total yield, dry matter % (DM%) and dry matter yield DMYld .

Treatment Mkt Yield

Mkt DM%

Mkt DMYld Tot Yld TotDM%

TotDM

Yld Nil 40.1 16.68 6.78 45.0 16.65 6.94

T1 Top 25% 42.4 16.55 7.05 45.7 16.54 7.21

T1 Top 50% 41.3 16.11 6.65 45.2 16.11 6.65

T1 Top 100% 36.4 15.56 5.69 40.3 15.56 5.69

T2 Top 25% 44.5 16.70 7.42 48.6 16.75 7.60

T2 Top 50% 42.4 16.19 6.88 46.4 16.17 6.98

T2 Top 100% 40.6 16.07 6.57 44.5 16.06 6.74

T2 Whole Top 38.2 16.04 6.12 42.0 16.06 6.23

lsd 0.60 0.96 0.59 0.954

NS ** * NS ** * 1Treatments with the same letter are not significantly different at P < 0.05. * denotes significant effect p=0.05 ** denotes significant effect p=0.01 Potato crop canopy management could be a useful tool in minimising the incidence of BF. The evidence presented suggests that growers should monitor potato crops for the first detectable incidence of BF and this can form the basis of a canopy management strategy. In addition, growers in regions where BF is a problem should attempt to grow crops a little on the hard side so that the proliferation of a vigorous canopy is not promoted. Various mechanisms for achieving this could be applied including use of defoliants or retardants, or restricting or withholding irrigation and not applying excessive nutrient application that favours foliage growth. Further research is required to evaluate forms rates and timing of agents that remove, inhibit or harden crop foliage.

42

6. Effects of Assimilate Supplementation on BF Incidence. Background The controlled environment and field experiments (2000, 2001 and 2002) demonstrate that under conditions that favour rapid tuber growth, development of BF symptoms is enhanced. This is particularly so given the earlier mentioned positive correlations between quantity of foliage and incidence of BF and the fact it is most prevalent at high night temperature. Two explanations for this are proposed. Firstly this set of circumstances could result in excessive photosynthesis and assimilate supply to tubers or alternatively it could result in increased foliage respiration leaving a deficit of assimilate to growing tubers and localised tuber cell death. It is possible the initiating cause of BF is low assimilate availability or supply under high demand conditions. Given that BF appears to specifically result in the death of single phloem vessels at the distal end of the tuber relatively small deficits in assimilate supply might be required to initiate BF. In this event the opportunity to supplement foliage assimilate requirements via a foliar application of a sugar may be sufficient to meet any short term deficit in respiratory requirements and potentially averting problems with BF. The presence of a positive effect would not only reinforce the above evidence but also provide a practical mechanism for reducing the incidence of BF in field grown potatoes. Sugar (sucrose) is a low cost input and its ability to correct the BF problem could potentially be a cost effective solution. The first experiment reports the effect of various foliar application rates of sugars (sucrose and molasses) on incidence of BF in glasshouse grown potatoes. In previous experiments top removal has significantly reduced BF incidence and there is both empirical and anecdotal evidence in glasshouse studies of an association between foliage damage and low incidence of BF. Positive correlations are recorded between foliage mass and incidence of BF. In 2001 foliage damage occurred due to application of a spray on one set of glasshouse plants. Despite conditions having been favourable for BF no BF was observed in the damaged plants. Furthermore, severe mite infestation was recorded on just 2 plants in a glasshouse study in 2002 resulting in foliage damage. Interestingly these 2 plants were the only ones in the entire study not to have BF. A second field trial is reported that evaluated various means of retarding plant top growth, supplementing assimilate requirements and crop nutrition management. This would provide a useful tool in the field management of BF. Methodology Glasshouse Experiment on sucrose and molasses The experiment was planted on June 30 2003 as per previous glasshouse experimentation. In contrast with other glasshouse temperature experiments this experiment was conducted under ambient glasshouse conditions. At emergence, about 14 days after planting (DAP), pots were allocated to each of 4 blocks according to their date of emergence. The experimental design was a randomised complete block replicated 4 times.

43

The treatments included

• Foliar sucrose application at 0.0%, 0.125%, 0.25%, 0.5%, 1%, 2% and 4% solution concentration

• Foliar application of molasses of about 0.164% and 0.656% (molasses calculated on a dry weight equivalent to 10 and 40 kg ha-1).

A measured aliquot of about 25 ml of each relevant solution was sprayed onto each replicates of each treatment and the application volume recorded. Single weekly applications were made during weeks 8-15 giving a total of 8 applications. Rates of basal nutrient application and side dress application were as per previous studies and sufficient to meet plant requirements. At 112 DAP, plants and tubers were harvested. The green tops were also harvested weighed and dried in a dehydrator at 70oC; dry weight was determined. Tuber measurements were as per general materials and methods. Field amendment experiment The field spray and amendment experiment was planted at the Queensland DPI&F - Gatton Research Station on July 7 2002. The field plot size consisted of 15 rows (at 0.75 m spacing) each of about 98 m length. The replicate plot size was of width 2.25 m (3 rows) by 6.5 m length. This experiment was conducted as a randomised complete block and included the following treatments;

• Nil treatment (no amendment) • Wetting agent application (1% at 98DAP) • Sucrose application 2 rates (1% and 2%) • Molasses application (40 kg ha-1) • Copper application (5 kg ha-1 as Copper Sulfate pentahydrate at 98 DAP) • Boron 3 kg ha-1 (additional, as basal application) • Zinc 3 kg ha-1 (additional, as basal application) • Mo at (250 kg ha-1 Na molybdate as basal application)

The sucrose and molasses were applied as foliar applications at 89, 96, 103 and 110 DAP. Rates of basal nutrient application and side dress application rates (in kg ha-1) of 160 N, 180 K and 120-130 S were applied as CK77S at planting and a combination of potassium nitrate and ammonium nitrate through trickle irrigation. As a basal application boron and zinc were each applied at 1kg ha-1 as Sodium tetra-borate and zinc sulfate heptahydrate at hilling (about 28 DAP) and via trickle irrigation. The Boron and Zinc treatments were applied as a balance of element in the same form as a sprayed band over the hill prior to hilling.

44

Severe hail defoliated the trial at 110 DAP and this precluded measuring the plot whole tops. The trial was harvested at about 114 DAP by mechanically digging the middle datum row in each plot. Results and discussion Glasshouse Sucrose and molasses amendments The effect of sucrose or molasses treatment on either total plant BF or mean tuber BF was not significant at p=0.05 (Fig 6.1a,b). The highest recording of BF on a per tuber basis was in the Nil treatment, but the highest overall BF per treatment was recorded in the 2% Sucrose treatment. The latter result was consistent with the fact that in this sucrose treatment yield was amongst the highest. Though not significant there appeared to be a trend for increasing sucrose application to increase the incidence of BF. This is consistent with the yield data (Fig 6.2 a,b), which demonstrated a significant increase in yield at a sucrose concentration of 1%. Similarly, the total dry matter and whole plant dry matter were significantly increased in the 1% sucrose treatment over that in the Nil. Sucrose application at 0.125% significantly reduced the tuber fresh weight and whole plant dry matter compared with the Nil. The lowest concentration of sucrose appeared to induce a very mild leaf fungal mildew infection; the level of infection progressively decreased with higher sucrose application. Interestingly the 0.125% treatment also had the overall lowest incidence of BF (Figs 6.1 a,b) consistent with previous findings that that BF incidence is directly related to foliage quantity and vigour. a) b)

Treatment

Treat 0 0.125 0.25 0.5 1 2 4 M10 M40

Mea

n Tu

ber B

F (m

m2 )

0

10

20

30

40

Treatment

0 0.125 0.25 0.5 1 2 4 M10 M40

Tota

l Bro

wn

Flec

k (c

ub rt

)

0

1

2

3

4

5

6

Fig 6.1. The effect of sucrose (0-4%) and molasses (10 and 40 kg ha-1 equivalent) on a) incidence of BF per tuber and b) Total BF per pot in a glasshouse experiment. A regression analysis of the relationship between BF incidence and tuber fresh weight was highly significant (p=0.001) and accounted for 36% of the variance in BF incidence. There was a significant correlation between plant top dry matter and BF (p=0.004) and this accounted for 21% of the variance in BF. The higher the tuber yield potential and quantity of plant tops the higher was the incidence of BF. Apart from temperature effects these factors play a critical role in the expression of BF. The application of sucrose significantly increased yield of potatoes but did not significantly affect BF incidence though there appeared to be a trend for increased application of sucrose to increase BF incidence.

45

The initial aim of this experiment was to determine whether sucrose application could alleviate the BF disorder by supplementing potential short term deficits in carbohydrate in the event that BF is a result of excessive foliage respiration. There is no evidence that this is occurring. Sucrose application did however increase tuber yield. a) b)

Treatment

0 0.125 0.25 0.5 1 2 4 M10 M40

Tube

r Fre

sh W

t (g)

600

700

800

900

1000

1100

1200

lsd p=0.05

Treatment

0 0.125 0.25 0.5 1 2 4 M10 M40

Who

le P

lant

Dry

Mat

ter Y

ield

(g)

100

120

140

160

180

200

220

240

lsd p=0.05

Fig 6.2. The effect of sucrose (0-4%) and molasses (10 and 40 kg ha-1 equivalent) on a) Tuber fresh weight b) Whole plant dry matter in a glasshouse experiment. Field amendments experiment There was no significant effect of any of the amendments on any yield components or BF on a plot or tuber basis. The results are highly variable. One point of interest is that of all treatments Boron at 4 kg ha-1 at least appeared to reduce the incidence of BF. It is likely that the mechanism for reducing BF is related to a reduced photosynthetic capacity evidenced by an apparent reduction in both tuber dry matter % and a reduced marketable yield (Fig 6.3). Application of B at this rate is well in excess of crop B requirements at a yield of 50 tonne ha-1 and is not recommended given the high unreliability and ineffectiveness of B application in reducing BF incidence.

a) b) c)

Treatment

Boron 4

Coppe

rMo

Molass

es Nil

Sugar

1%

Sugar

2%

Wett

ing Age

ntZinc

Plo

t BF

Inci

denc

e (m

m2 )

0

50

100

150

200

250

Treatment

Boron 4

Coppe

rMo

Molass

es Nil

Sugar

1%

Sugar

2%

Wett

ing Age

ntZinc

Mar

keta

ble

Tube

r Yie

ld (T

onne

ha-1

)

25

30

35

40

45

50

55

Treatment

Boron 4

Coppe

rMo

Molass

es Nil

Sugar

1%

Sugar

2%

Wett

ing Age

ntZinc

Tube

r Dry

Mat

ter (

%)

16.0

16.5

17.0

17.5

18.0

18.5

Fig 6.3. The effect of various canopy management strategies on a) BF incidence b) Marketable tuber yield and c) Dry matter %, in a field experiment.

46

7. Brown Fleck Microscopy and Phenology of Development Studies on disorders similar to BF highlight gross disruptions in cellular structure and function (Baruzzini et al. 1989). A detailed study of BF cellular symptoms from affected tubers grown under Australian conditions has not been conducted and compared with symptom descriptions developed overseas. Studies of affected tubers has focussed on cases where damage is relatively advanced. The phenology of development of BF is a critical part of understanding the problem. This study reports on the phenology of development of BF symptoms and associated cellular changes. Methodology This study monitored the development of BF and its incidence in field grown potatoes over time. The trial was conducted at the Queensland DPI&F - Gatton Research Station. Whole set certified seed potato (Solanum tuberosum cv Sebago) was planted on July 7 2003. The field plot size was 5 rows (at 0.75 m spacing) each of about 100 m length and divided into sampling plots each of 10 m length. All agronomic practices were as per the field trials in the general materials and methods. At 77 DAP one plant from each of the 10 plots was randomly selected and the plants dug and separated into foliage, stems and tubers. The foliage and stem fresh weights were determined for each plant and the samples dried and dry weights recorded. This sampling procedure was conducted twice weekly generally on Mondays and Thursdays until 112 DAP. A final sampling was conducted at 124 DAP. The study suffered severe hail damage at 110 DAP which precluded further sampling of foliage after this date. A weather station was positioned at either end of the trial and logged the following data at 10 min intervals

• Relative humidity • Air temperature • Soil temperature • Solar radiation

The laboratory measurements of BF and yield and dry matter were conducted as per the general materials and methods. A microscopic examination of tuber tissue at the different stages was conducted. Fine tissue segments were cut using a Leica microtome and either stained or left unstained as appropriate to the clarity of the sectioned material. The stains used were malachite green for cell wall components and iodine for starch. By means of regression the incidence and time of expression of BF was related to weather data and plant physiological traits. The BF data had a logarithmic growth rate and transformations on it and yield were conducted to make a linear relationship.

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Results and discussion Phenology of development There was a significant positive correlation between tuber fresh weight (Log fresh weight) and incidence of BF (Log Total BF)(p<0.001). This correlation accounted for 92.3% of the variance associated with BF. There was also a significant positive linear correlation between the amount of leaf dry matter and the incidence of BF (Log total BF) (p=0.035), which accounted for about 37.6% of the variance in BF incidence. In contrast there was no significant correlation between either total plant top dry matter or stem dry matter and BF incidence. The absence of a significant relationship for these components would suggest that excessive foliage respiration is not a critical factor in expression of BF since only the leaf dry matter was significantly correlated with BF incidence. There was also no significant correlation between tuber no and BF incidence. Importantly there was also a significant correlation between leaf dry weight and tuber fresh weight (p=0.030) and this relationship accounted for 39.9% of the variance in tuber weight which was a very similar relationship to that determined between leaf dry weight and BF incidence. It appears likely that the correlation between leaf dry weight and BF incidence (also noted in glasshouse experiments) is subsequent to the effect that the quantity of leaf matter has on tuber growth. Consistent with the findings of the other studies within this project it is likely that a higher leaf dry matter results in greater assimilate partitioning to the tubers which in turn is the driving factor behind BF symptom expression. Conditions favourable for rapid tuber growth are also likely to favour the development of BF. A multilinear correlation matrix for the relationship between tuber fresh weight and specific climatic variables and leaf mass is presented in table 7.1. The correlation between these parameters and Tuber fresh weight accounted for 62.3% of the variance in Tuber fresh weight (p<0.001). Tuber fresh weight was positively correlated with; night temperature, solar radiation, and leaf dry weight. That is the higher the, solar radiation, night temperature and leaf dry weight the higher the tuber fresh weight gain. In contrast, tuber fresh weight was negatively correlated with day time temperature indicating that as day temperature increases tuber fresh weight is decreased, probably because the efficiency of photosynthesis is reduced due to greater photorespiration or latent foliage respiration. These factors appear to be the main factors driving yield and subsequently BF development. In a field situation, conditions of relatively high night temperatures, mild day temperature and high solar radiation are conditions favourable for rapid tuber growth and likely to be the driving factors behind BF incidence. The presence of a large active plant canopy further promotes assimilate production and transfer to tubers thus resulting in higher BF incidence.

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A graph of night temperature against the incidence of BF (fig 7.1 ) suggests a relationship exists between incidence of BF and the night temperature at about 5-7 days prior to the first incidence of BF. There was a steep climb in night temperature from 70-80 DAP and the first incidence of BF was recorded at 80 DAP after which a similar rapid increase in BF was recorded. As such the incidence of BF was regressed against mean temperature parameters for three time periods. These include the means for the immediate 3 days prior to sampling, 4-7 days prior to sampling and 7-10 days prior to sampling. The best correlation existed for the temperature period at 5-7 days prior to first incidence. This indicates an approximate 3 day lag between the BF increase compared with the temperature increase. Table 7.1 Correlation components for relationship between Tuber Fresh Weight and named parameters Parameter Estimate Significance Solar Radiation 2.00 *** Night Temperature 238 *** Day Temperature -173.3 *** Leaf Dry Weight 13.01 *** Constant term -1046 ** *** denotes significant at p=0.001 ** denotes significant at p=0.01 A similar response for tuber fresh weight with night temperature was recorded with the exception that the tuber growth response to temperature was more immediate than was that for BF (about 1-3 days and 5-7 days respectively) (Fig 7.2). This too suggests that the increased incidence of BF is more related to increased tuber growth under favourable conditions.

Days after planting

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Fig 7.1 The relationship between mean night time temperature and incidence of BF over time in field grown potatoes cv Sebago.

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Days after planting

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Microscopy At the earliest incidence of BF a single cell dies. Exhaustive sampling notes this cell is typically a phloem sieve cell responsible for conducting sugars to newly developing tuber storage parenchyma cells. Plates 7.1a,b. depict longitudinal and cross sections of healthy and conducting cells. The very first evidence of cell damage due to BF is shown in the longitudinal section of an affected phloem sieve cell (Plate 7.2). Note that at the point where the plasmalemma is named, and in the inset phot, a browning reaction within the plasmalemma has commenced indicating the membrane is damaged. The plasmalemma is an internal cell membrane that contains and protects the cell contents. This damage has occurred prior to cell death and at this point there is no cell wall damage. The phloem cells are elongate and smaller than the predominant cells of the tuber, which are the parenchyma cells that store starch (see plates 7.1b and 7.2). As the damage progresses the initial browning reaction in the phloem cell plasmalemma advances and brown occlusions (blockages) develop adjacent to the plasmodesmata (Plate 7.3a). The plasmodesmata is a pore like channel that bridges the contents of cells allowing the movement of solutes (in particular sugars) between cells. There is also further occlusion development on the sieve elements at the ends of the phloem sieve cells (Plate 7.3b). The development of this occlusion within the sieve phloem cells precludes sucrose transport past this point. In response to the phloem damage starch plastids are released off the plasmalemma and develop in both the damaged phloem sieve cell as well as adjoining phloem sieve cells proximal to sucrose supply source (Plate 7.4a,b). The tuber is faced with the difficulty of metabolising a continued supply of sucrose to regions where cell death has occurred. It appears that the development of starch granules occurs within phloem cells in response to this continued supply. Van Bel and Knoblauch (2000) report on specific development of plastids in phloem sieve cells in response to phloem sieve cell damage. The microscopy studies confirm the notion that a continued and

Fig 7.2 The relationship between mean night time temperature and tuber fresh yield over time in field grown potatoes cv Sebago.

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excessive supply of sucrose under good growth conditions is consistent with an enhancement of BF symptoms. In addition with further rapid photosynthesis sucrose continues to be supplied to the damaged area creating considerable internal pressure and the amounts of sucrose and solute increase. This subsequently reduces the osmotic potential resulting in increased cell turgor pressure and providing conditions favourable for further development of BF. It is analogous to the gate valve on an irrigation line being turned off while a pump is under full pressure. There is not only a direct pressure build up due to this solute movement. The increased amounts of solutes (particularly sucrose) creates a positive osmotic potential and with increasing water supply pressure increases. This increased pressure is likely to further disrupt phloem vessels and other cells resulting in rapidly increasing development of BF symptoms. This is consistent with field observations that rainfall events at or near maturity can dramatically increase BF incidence. The continued supply of sucrose appears to further enhance symptom development and gross cellular changes result. The death of these phloem cells or any other cell results in browning reactions and the release of oxidising enzymes that are normally contained within the cellular components and this results in a spreading of the disorder. This is shown graphically in plates (7.5 a,b,c). The browning reaction and cell death not only affect adjacent storage cells but the browning can extend throughout the phloem vessel continuum as shown in plate 7.5b and ultimately healthy storage cells that are well filled with starch can also become brown and ultimately necrotic (plate 7.5c). In and around the phloem vessels new parenchyma cells are continually produced. With the initial death of a single phloem cell these surrounding juvenile undeveloped parenchyma cells appear to no longer receive a supply of sucrose essential for their further development and these cells too ultimately die. This is evidenced in plate 7.5d, which shows the necrotic phloem conducting vessels surrounded by clear undeveloped or immature parenchyma storage cells. It is for this reason that in the very early stages of development of BF affected tubers often contain glassy patches (about 2 mm diameter) with a single dead spot (about 0.5 mm), representing the dead phloem sieve cell. This is consistent with observations of commercial crops (Penna pers comm.) and in glasshouse studies within this series of experiments. The physiology of phloem cells has long been a perplexing subject due to the difficulties in studying them. Phloem cells invoke an immediate and rapid physiological response to any form of damage, the response time frame is less than 4 minutes. The response results in the formation of brown occluded blockages particularly at the sieve plate due to the formation of brown phloem protein bodies or “sludge” accumulate on the sieve plate. The issue of phloem sieve cell responses is well reviewed by Van Bel and Knoblauch (2000). They note that in collective experimentation disruption of turgor is the main activator in the development of the brown proteinaceous occlusions and plastid release in phloem sieve elements. Turgor in tuber cells is a function of sucrose supply to tubers and water status. A critical assessment of the biochemistry and cytology of phloem cells is required to understand the initial cause of phloem cell death in BF affected tubers. The subsequent symptom development appears to be related to conditions that favour

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rapid tuber growth. Van Bel and Knoblauch (2000) raise the question as to why the proteinaceous bodies that line the plasmalemma of the phloem sieve cells are not dragged along with the turbulent and rapid sucrose movement in these cells. They site evidence that minute attachments bind these to the plasmalemma, however this research is still in its infancy. It is possible that under conditions favourable for rapid growth and sucrose loading in tubers that protein forming organelles are damaged resulting in the catalyst for cell death. An evaluation of cell wall, plasmalemma and biochemical traits across sensitive and resistant varieties could form a good basis for determining breeding strategies to develop varieties that are resistant to BF as well as understanding the precise cause of phloem sieve cell death.

a) b)

Plates 7.1 a)Longitudinal section of healthy tuber tissue b)cross section of healthy tuber tissue

a) b)

Plates 7.2 a)Longitudinal section of early BF damage in phloem sieve cells note the

browning of plasmalemma in inset b)cross section of BF affected phloem sieve cells.

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a) b)

Plate 7.3 a) Longitudinal section of phloem sieve element showing protein occlusion at plasmodesmata b) cross section of sieve plate at the junction of 2 phloem sieve cells.

a) b)

Plate 7.4 a) Longitudinal section of phloem sieve element with starch granules in adjacent phloem sieve elements.b) cross section of phloem sieve cells with starch granules.

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a) b)

c) d)

Plates 7.5 a) the spread of BF into adjacent parenchyma cells, b) throughout a series of conjoined phloem sieve cells c) to healthy parenchyma cells filled with starch d) a BF lesion in the tuber medulla with

surrounding glassy cells that fail to grow and enlarge.

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8. Technology Transfer Technology transfer presentations have been made to grower groups in Gatton, Atherton and Bundaberg as well as to the Queensland and NSW crisping growers Inc. In addition various interim reports have been made available to industry representatives and growers (2000, 2001 and 2003). Update articles have been published in Potato Australia years 2001, 2002 and 2003. A poster presentation of the results was made at the University off Queensland Gatton Horticulture Expo field day 2002. A radio presentation on the significance of the disorder was made in 2002. A farm note will now be prepared out of the findings and made available on the Queensland Government DPI&F website. 9. Recommendations The incidence and development of BF symptoms appear to be 2 separate processes. The initiation of BF occurs in tubers with the death of single phloem sieve cells that are responsible for conducting sugars to new and existing cells. The death of theses cells results in further mass cellular disruption in the tuber medulla. The initiation of this cell death requires further research and particularly in relation to varieties that are both susceptible and resistant to BF. As well as providing an understanding of BF causes at the cellular level this would form a basis for the breeding and developing of new varieties that are resistant to BF. There is a consistent and strong correlation between tuber yield and incidence of BF where high yielding crops have higher incidence of BF. Yield was also directly correlated with high/mild night temperatures, high solar radiation and a large plant foliage mass. High soil temperature further acted to enhance symptom development probably through increased tuber respiration. High day time temperatures were not strongly conducive to BF development possibly because photosynthesis is retarded at such temperatures. Indeed the field phenology study highlighted a negative correlation between tuber growth and day time temperature which would suggest high day time temperatures would not be favourable for BF development. Thus in minimising incidence of BF, growers need to adopt strategies that prevent prolific canopy development and exposure of tubers to high temperatures at senescence. This in essence will restrict the development of BF. All factors that favour a prolific crop canopy will favour BF. Ensuring that excessive nutrient application and irrigation are not made will assist in reducing BF incidence. Evaluating seed age prior to planting and plant growth during crop development will provide indicators for crop vigour. Though not evaluated in this study it is likely that seed optimally aged, for high yield potential, would have greater likelihood of producing a crop with BF. The effect of uneven emergence and subsequent uneven maturation in relation to BF incidence requires evaluation. Hardening up crop foliage could be used to reduce BF incidence. This could be achieved by the application of registered retardants. Only a cursory evaluation of this was conducted in this project and a more thorough evaluation should achieve significant outcomes in the management of BF.

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At the same time early monitoring of tubers in commercial crops for the first evidence of BF incidence should be conducted. This could then form the basis for determining whether preventative measures need to be conducted. Growers could also use short term weather outlooks as a basis as to whether conditions will be favourable for rapid tuber growth. In particular, weather conditions that include warm nights and mild days or rain events would be favourable for BF development. In the event that conditions are favourable the management of crop foliage should be considered. This can be achieved by potentially spraying defoliants or agents that might harden the crop up including copper or calcium. A further detailed evaluation of the effects of forms, rates and timing of such retardants on incidence of BF is required. The maintenance of vegetative cover at the senescence phase of the crop should be encouraged so as to provide shade to soil and preventing excessive soil temperatures that favour the enhancement of BF symptoms as well as heat necrosis. This could also be achieved by oversowing the potato crop with an annual grass crop species suitable to the time of planting. This management aspect also warrants further research. Application of calcium or boron did not significantly or reliably reduce incidence of BF and based on this is not recommended as a means of controlling BF. Nonetheless these nutrients should be applied at levels sufficient to meet crop requirements. 10. Acknowledgements The assistance of the following people is gratefully acknowledged. Bill O’Donnell for his assistance in the running of experiments and harvest and processing of samples and particularly for times when weekend glasshouse checking was required. Peter Scholl, Peter Case and Brendan Nolan in the running of experiments and harvest and processing of samples and also EricColeman for assisting with harvests early in the project. Winston Bean and Janette Mercer The University of Queensland St Lucia for their assistance and provision of glasshouse facilities. Dave Schofield –Queensland DPI&F - Gatton research station manager and the farm staff (Vince Scheiwe, Chris McManus, Stephen Soderquist, Ken Quade and Joe Luck) for their assistance in the planting, managing and harvesting of field trials. To Shane Litzow for his assistance in growing one of the field trials. To Assoc. Prof. Pax Blamey, Assoc. Prof. David Edwards and Dr Ken Jackson for their advice. Mr Ian Rickuss as the then Queensland potato industry representative for championing the project in its infancy. My family (Karen, Jessie, Sophia and Bridget) for considerable time I spent on weekends ensuring glasshouse and field experiments were watered and ran smoothly.

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11. Literature review

11.1. Internal tuber disorders in potato

11.1.1. General causes of internal tuber disorders Potato tubers are affected by a large range of disorders, with causal mechanisms broadly relating to issues of pathogenic, nutritional and physiological influence. For pathogenic related internal disorders the diagnosis and description is generally quite clear cut since the isolation of the pathogen and the general prevalence of rots associated with these make diagnosis relatively easy. In addition pathogens often produce symptoms on the plant tops and on external surface of the affected tubers. Examples of such pathogens include Blackleg, Early Blight, Fusarium and pythium amongst other pathogens that are well described in Stevenson et al. (2001). The one group of pathogens that can be somewhat difficult to diagnose are some specific virus infections that produce internal browning symptoms not unlike those produced with the internal browning associated with physiological causes. Potato Leafroll virus, PVY NTN, Potato Mop-top virus and Tomato spotted wilt virus can at times produce symptoms of internal disorders. However, with these pathogens the clear visual symptoms on the plant tops and the well defined tuber symptoms make their confusion with other physiological tuber disorders unlikely. With the physiological and nutritional disorders there is no association with a pathogen. Furthermore, physiological disorders that affect potato tubers produce no obvious external symptoms, however, their incidence is generally more pronounced in large rapidly growing tubers.

11.1.2. Definition and terminology of internal tuber disorders Given there has been no definitive evidence to demonstrate that these disorders are either nutritionally or physiologically related they will henceforward be referred to as physiological disorders. There is a great deal of confusion in the terminology associated with physiological internal disorders and this has made the interpretation of literature somewhat confusing. Broadly speaking the disorders can be divided into those that affect the tuber central pith and those that cause internal browning in tissue external to the pith and have no cavity formation.

11.1.3. Disorders affecting the tuber central pith The 2 main disorders that affect the central tuber pith are Hollow Heart (HH) and Brown Centre (BC). Brown Centre is characterised by the browning of the pith section of the tuber central medulla, resultant from the death of these cells. Mogen and Nelson (1986) refer to 2 types of HH in potatoes. A physical rupturing or separation of cells, resulting in a lens shaped cavity, caused the one typically observed in their study. Tensions in cell walls associated with sudden rapid growth are likely to be the cause of such rupturing. The other form of HH was outlined by Van Denburgh

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et al. (1986) which confirmed that if the disruption of tuber pith cells due to BC is sufficiently extensive then HH can result. Davies (1998) notes that late reduction in Ca supply to tubers (tubers ca. 5 cm) resulted in IBS whereas reduction in Ca supply around the time of tuber initiation resulted in BC. It appeared the timing of Ca stress was a major influence on the expression of symptoms. The combination of these results would suggest that the underlying causes of the three disorders are at least similar. Dinkel (PhD thesis 1960 as cited in Crumbly et al. 1973) hypothesises that under stress conditions reabsorption of water, mineral nutrients and carbohydrates from small tubers by the plant tops occurs, resulting in irreparable tuber cell damage that prevents division and enlargement even when the stress is alleviated. In support of this Crumbly et al. (1973) injected 14C labelled sucrose into tubers of plant from which shoot material was removed. The subsequent presence of radio labelled sucrose in the shoots highlighted reabsorption of sucrose had occurred. Furthermore Crumbly et al. (1973) suggest that moisture stress results in the conversion of starch to sucrose and due to increased water potential rapid enlargement of the perimedullary region occurs, causing a separation in the pith that later results in a cavity.

11.1.4. Browning disorders external to the pith The definitions of browning disorders external to the pith create the most confusion in the literature. Browning disorders have been variously referred to as Brown Fleck (BF)(Novak et al. 1986), Internal Heat Necrosis (IHN) (Sterrett et al. 1991ab), Heat Necrosis (Stevenson et al. 2001), Rust Spot (Baruzzini et al. 1989), Internal Rust Spot (Collier 1980), Chocolate Spot (Kamal and Marroush 1971), Internal Brown Spot (IBS) (Stevenson et al. 2001) and Mahogany Browning (anon.). Stevenson et al. (2001) provide the most delineating description of disorders. They use the terminology Internal Brown Spot and Heat necrosis to specifically describe two different sets of symptoms. Internal Brown Spot is characterised by small round or irregular reddish-brown spots or lesions usually in the medullary tissue (Stevenson et al. 2001). In extreme cases the affected cells become corky, lack starch and are suberised. The disorder is generally most prevalent at the apical end and is related to fluctuations in growing conditions, particularly those favouring rapid growth. In contrast the other similar disorder HN develops in tubers that are exposed to extremely high soil temperatures late in tuber growth while vines are alive (Stevenson et al. 2001). They report the cause as the vines removing water and nutrients resulting in HN. In 2000 Harper (unpublished) conducted a field inspection of HN incidence in cv. Atlantic and noted prevalence of the disorder in fully senesced crops and was much less evident in crops that were at maturity and with green tops. The incidence of HN is most prolific under the crown eyes and it is likely that this distal end of the tuber experiences higher soil temperature extremes compared with other parts of the tuber and hence has higher HN incidence. Similarly, smaller tubers do not exert as close to the soil surface as large tubers and hence generally experience lower soil temperature.

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In this review terms IBS and HN will be used to refer to the fore mentioned disorders.

11.1.5. Worldwide distribution of disorders Hollow Heart, Brown Centre and Internal Brown Spot impair the internal flesh quality of the tubers of potato (Solanum tuberossum). In Australia IBS and HH are the more serious disorders, and result in downgrading and poor market perception of potatoes. Brown Fleck or IBS is observed in many parts of the world although its prevalence appears to be mostly centred in the USA. Clough (1994) describes the prevalence of IBS in potatoes grown in the Pacific North West of the USA. Other reports of IBS in the USA come from Olsen et al. (1996) Washington state and Silva et al. (1991) Michigan. Kamal and Marroush (1971) report high incidence of IBS in potatoes grown in Lebanon. Seppanen (1975) reports high incidence of IBS in Finland in 1973. Novak et al. (1986) report prevalence of IBS in Queensland, Australia. Brown Fleck (or IBS) is the most prevalent of the disorders in Australia particularly in the northern sector of the country and this review mainly focuses on it, though distinct similarities exist between the disorders.

11.2. Tuber changes associated with IBS

11.2.1. Cellular changes associated with IBS Extensive IBS damage generally results in the necrosis of cells and such damage is irreversible. However, Hooker (1981) notes that the incidence of mild brown fleck during storage can actually decrease in severity. Similarly, in field plantings Harper (unpublished) has observed incidence of low grade IBS in cv Exton at tuber bulking which was not evident at harvest. In contrast Stevenson et al. (2001) note that the incidence of IBS can increase with storage depending on maturity and storage temperature. Penna (2001 pers comm.) noted no incidence of IBS prior to storing tubers cv Sebago but a high incidence after a storage period of several days at 20oC. The tubers had a high incidence of glassy patches of cells in the medulla. This evidence suggests that the damage to cells in mild IBS cases is not terminal and the initial brown products of IBS are not the consequence of cell necrosis but merely products of impaired metabolism. It suggests the disorder is initially biochemically related particularly in such mild cases. In the early development of IBS walls of affected parenchyma cells in the medulla and pith become dark at the corners of cells (Hooker 1981). The walls of adjacent cells also darken and cellular collapse at these corners occurs. Using optical microscopy Baruzzini et al. (1989) found that severely damaged cells had deformed walls and a smaller lumen than that of cells unaffected by IBS. The degeneration and separation of the plasmalemma and cell wall occurred and cell death ultimately resulted. There was intense thickening of cell walls, which were of a rusty brown colour and consisted of corky tissue. Baruzzini et al. (1989) also found, using electron microscopy, the walls of damaged cells were deformed, initially thickened (prior to necrosis) and latterly assuming a folded lacy irregular form. With further increases in cell wall thickness gross distortion and deformation of the cell wall

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occurred probably due to the pressure of adjacent cell growth impeding normal cell wall extension. The developing wall in IBS affected tissue would sometimes fold back on itself with the abnormal thickening causing folding of the unthickened portion. The collapse of necrotic cells is probably due to pressure from healthy periderm tissue (Hooker 1981). Baruzzini et al. (1989) showed a marked delineation between unhealthy brown tissue and healthy tissue. Hooker (1981) notes the development of periderm type tissue around the IBS affected tissue to the point where it may be isolated. Compartmentalisation of the damage may be an important tuber response in preventing the further development of the disorder. The presence of suberin in cells severely affected by IBS may simply be a wound defence response mechanism. Baruzzini et al. (1989) found a substantial amount of suberin was present in the cell wall and there was a total absence of starch in the suberised zone of IBS affected cells. Dean et al. (1977) determined the structure and composition of the suberin from tissue lining hollow heart cavities in tubers of Russet Burbank. They identified octadecene-1,16 as the major component and the presence of hexadecane-1,16-diol and long chain (>C18) alcohols. This suberin lining was quite similar to that found in the periderm of external wounds and skin. Under electron microscopy the lamellar structure in the suberin of hollow heart was also similar to that in external wounds and skin. In IBS affected tissue osmiophillic granules progressively accumulated in the vacuoles and their positioning suggested they were catabolic products of a disordered metabolism (Baruzzini et al. 1989). These granules initially appeared to adhere to the tonoplast but progressively massed together to form a compact stratum on the internal surface of the vacuole until the entire lumen was occupied. In contrast, Hooker (1981) notes it is the protoplasm that becomes granular and aggregates. Evidence to explain the observed symptoms can be drawn from research conducted by Crumbly et al. (1973) on HH. They determined that HH could be induced in small tubers that were moisture stressed. This resulted in the conversion of starch into sugar and with rapid rehydration and subsequent enlargement of perimedullary cells, compared with that of pith cells, a separation in pith cells was manifested as HH. This separation occurred due to the greater pressure potential of cells containing soluble sugars. A change of this type could account for some of the changes in IBS affected tissue and the cellular changes observed by Baruzzini et al. (1989); specifically those of cell wall deformation relating to pressure from neighbouring cells as well as the total absence of starch in severely affected cells. However, it does not account for the early separation of the plasmalemma and cell wall, the browning of corners of cells nor the substantial thickening of cell walls in IBS affected tissue. Ohad et al. (1971) and Isherwood and Frederick (1976) suggest stress conditions such as low temperature cause damage to the amyloplast membrane surrounding the starch granule allowing degradative enzymes to attack starch converting it to sugars. Similarly, Iritani and Weller (1978) surmise higher temperature storage 21.1oC may similarly cause damage to the amyloplast membrane exposing starch to oxidative enzyme degradation. Premature death of the plant and exposure to higher soil temperatures could initiate membrane disintegration (Iritani and Weller 1978).

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11.2.2. Physiological changes associated with IBS Hooker (1981) notes the appearance of IBS is likely to be caused by the sub-oxidation of rapidly respiring internal tissues during active tuber growth and high temperatures. However, tubers from plants which remain green late often have more IBS then tubers from vines which senesced prematurely (Ellison and Jacob 1952). The finding is also consistent with Anecdotal evidence from field observations over 5 years (Harper unpublished) that has noted green plants at maturity are more prone to the disorder than senesced plants. In support of this, the application of the retardant Maleic Hydrazide to potato plants cv Russet Burbank at a rate of 12 l ha-1 after full bloom dramatically reduces the incidence of IBS from 33% down to 10% (Iritani et al. 1984). In contrast other authors (Novak et al. 1986, Iritani et al. 1984 and Sterrett et al. 1991a) found that the incidence of IBS increased when potatoes were held in the ground after vine senescence and maturation. Furthermore, Harper (unpublished) notes surface set green tubers appear to be rarely affected by the disorder despite being exposed to direct sun and hence extreme day time temperature. This contrasts with Hooker (1981) who notes that the disorder is most prevalent in tubers near the soil surface and less prevalent in deeper set tubers. Crumbly et al. (1973) injected 14C labelled sucrose into tubers of growing plants and pruned foliage to simulate plant stress. They determined that under this stress the plant tops were able to remobilise nutrients from the growing tuber to support plant growth. Yamaguchi et al. (1964) found that tubers exposed to the stress of high temperatures (27-29oC) had high concentrations of sugars. If stress imposed on potato plants, through moisture or temperature, is prolonged tuber starch can be converted to sugar and reabsorbed for the tops or other plant parts, to the point where tuber reserves are exhausted. Smith (1931) (as cited in Crumbly et al. (1973)) reported reabsorption of sugars in tubers as small as 50g. He reported high respiration rates in growing immature tubers but this rate was reduced with age. Crumbly et al. (1973) propose that when respiration is stimulated by high temperature or other stressful condition, cells in even small tubers can deplete starch reserves to the point where the cells cannot recover. The evidence presented on incidence of IBS suggests the disorder is most pronounced under conditions favourable for rapid growth and where vines have remained green for an extended time. The disorder clearly affects tubers in the late bulking stage of the crop and particularly large tubers. In light of the fact that IBS affected tissue is devoid of starch it is pertinent to consider the developmental processes, transport of sugars and the metabolism of starch in potato tubers. At temperatures in the order of 10-20 oC the greater part of carbohydrate in the tuber is starch at about 98% (Burton 1966).

11.3. Biochemistry of brown and dark compounds in plants The disruption of cellular tissues causes the release of phenolic compounds which can either hydrogen bond with proteins or undergo oxidation by phenol oxidase to quinones. By the action of polyphenol oxidase quinones polymerise and condense with reactive groups on proteins and amino acids to form dark pigments called

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melanin which form the basis for browning in plant tissue (Dey et al. 1997 pg 25). A more detailed review of this is provided in Dey et al. (1997).

11.4. Role of Temperature

11.4.1. Effect of Temperature on IBS Temperature is often nominated as a cause for IBS (Hooker 1981) to the point where in some parts of the world the disorder is referred to as Internal Heat Necrosis, particularly in the USA. Research conducted on the affects of heat, and its interaction with water status, in inducing IBS is somewhat inconsistent, but there is general agreement that high temperatures favour the expression of the disorder. The disorder has been observed under hot dry conditions, hot moist conditions but also under cooler conditions (Novak et al. 1986, Sinden and Webb 1980). The spring Lockyer crop of 1996 and 1997 had extremely high incidence of IBS and this occurred despite the fact that temperatures were mild. Notwithstanding, IBS is always associated with crops that appear to have a higher than average yield potential. Van Denburgh et al. (1979, 1986) present quantitative evidence of the effects of temperature on internal disorders in an experiment conducted under controlled environment conditions. They induced BC and HH in plants grown at mild temperatures of only 22-23 oC. This was achieved by imposing a cool stress of 18oC day and 10oC night temperatures for 8 days during tuber bulking. No similar studies have been conducted to elucidate temperature regime effects on incidence of IBS. Sterrett et al. (1991a,b) noted in their experiments that high temperatures, a dearth of rainfall events and delayed harvest are conditions most conducive to the onset of IBS. Sterrett et al. (1991a) found the considerable variability in the disorder was mostly related to environmental conditions though a significant but only weak correlation existed between incidence of IBS and yield components in Atlantic; even though the disorder first appeared in largest tubers. Lee et al. (1992) found significant correlations between temperature and incidence of IBS. In particular, high minimum temperatures significantly affected tuber yield and expression and development of IBS. Hooker (1981) believes IBS is caused by the sub-oxidation of rapidly respiring internal tissues during active tuber growth and high temperatures. These environmental conditions are consistent with those that favour the disorder in the Lockyer Valley Queensland. A clear environmental effect on expression of IBS in cultivar Sebago is presented by Novak et al. (1986). In the four years 1971 to 1974, Autumn (March) planted experiments at Gatton, Australia (lat 270 30' S long 1520 15' E) never exhibited IBS whereas the same experiments when planted in winter (July) at the same site exhibited high incidence of IBS (up to 25%). It is likely this differential effect in the 2 crops relates to both temperature and daylength differences at tuber bulking. Similarly, Iritani et al. (1984) found that the incidence of IBS in trials conducted at Washington State, USA was reduced when planting was delayed from March 31 to May 12 highlighting the effect environment has on IBS incidence.

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Sinden and Webb (1980) used soil mulches to reduce soil temperature; although the mulching reduced soil temperature by 5 oC the incidence of IBS was not reduced. This indicates that temperature experienced by the tubers does not cause the disorder. Olsen et al. (1996) grew potato plants cv. Russet Burbank in a pot experiment under two soil temperature regimes of 18oC and 32oC the plant tops were maintained at ambient day/night temperature of 21oC /16oC. Increasing the soil temperature did not increase the percentage of tubers affected by IBS, however, the severity of IBS was increased. The dry matter yield per plant was unaffected by increasing soil temperature which would indicate that temperature did not significantly enhance the disorder. Furthermore, as soils are a highly buffered medium major fluctuations to the point that would induce IBS are unlikely. Harper (unpublished) in 2 glasshouse pot experiments evaluating IBS has found considerable within pot variability in expression of the disorder. The presence of high within pot variability would indicate temperature per se is not the principle factor determining incidence of IBS. Iritani et al. (1984) evaluated the effects of storage temperature on the incidence of IBS in cv Russet Burbank. The IBS incidence in large tubers (>308 g) at storage temperatures 5.6oC and 8.9oC were 51% and 62% respectively. Furthermore, the percentage tubers affected with IBS in control plants increased from 22% to 34% after an 8-9 month storage period. This contrasts with Hooker (1981) who notes incidence of IBS under storage can decrease. Marinus and Bodlaender (1975) evaluated effects of mean temperature regimes of 16, 22 and 27oC on potato tuber yield across 8 cultivars. Highest dry matter yields and dry matter contents were recorded under the 16oC regime. Marsh et al. (1989) demonstrated higher growth rates in potatoes cv Norland grown at 20oC than that grown at 25oC and foliage yields dropped dramatically at the higher temperatures of 25oC and 30oC. Plants grown at 30oC were highly branched with small leaves and short internodes. At 30oC high P concentrations in the growth media increased growth over a low P media but at 20oC there was no difference in growth between the high and low P treatments. At high temperature the activity of starch synthase, the enzyme involved in the resynthesis of starch in the tuber, is reduced and consequently tuber dry matter is also reduced (Denyer et al. 1994). Increasing temperature over a threshold of about 22 oC decreases the ability of potatoes to produce starch from sucrose (Burton 1981, Mohabir and John 1988). Mohabir and John (1988) in in vitro studies on cv Desiree found a media temperature of 21.5oC was optimal for starch synthesis. Krauss and Marschner (1984) showed that a tuber temperature of 30oC starch synthesis was reduced compared with that at 20oC but the net export of sucrose to tubers did not differ at the 2 tuber temperatures. This highlighted that temperature adversely affected starch synthesis whilst the net import of sugars to tubers remained unchanged. Under conditions of high temperature and low irradiance Menzel (1985) showed that dry matter was diverted preferentially to the shoots rather than the tubers. He suggested this was likely to be due to an increase in growth substance possibly giberellin, which inhibits tuber formation.

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11.5. Nutritional effects on potato and IBS

11.5.1. Calcium

11.5.1.1. Role of Calcium A high proportion of Ca in plants is contained in cell walls where it is bound with pectins thus maintaining the structural integrity of cell walls. Furthermore, Ca is probably required for the stabilisation of newly formed membranes (Marschner 1990). Calcium is required to maintain the integrity of cell membranes and prevents the bulk movement of cellular inorganic and organic compounds. Calcium is adsorbed to negatively charged phosphate groups of cell membrane lipids and probably restricts permeability to hydrophillic solutes (Mengel and Kirkby 2001). One of the first symptoms of Ca deficiency in plants is thus a reduction of meristematic growth and the production of brown melanin compounds from polyphenol oxidation of cellular compounds. Dekock et al. (1975) claim that in tissues containing adequate Ca phenolic compounds are also chelated by Ca. The presence of this browning associated with Ca deficiency is consistent with the symptoms observed in IBS affected tissue, however, it is not consistent with the fact that under IBS affected tissue can recover to the point where symptoms are not present. The browning resultant from membrane collapse associated with Ca deficiency is clearly irreversible.

11.5.1.2. Tuber tissue Ca concentrations The periderm tissue Ca concentration is invariably much higher than that of the medulla (Table 1.1). The differential in concentrations being in the order of 4-10 fold difference. Bretzloff (1971) evaluated the distribution of Ca in tuber parts of several varieties. The pith concentrations (mg 100 g-1 fresh weight) for Russet Burbank, Red La Soda, Wauseon and BR5957-7 were 4.94, 2.41, 4.53 and 6.38 for pith and 14.57, 9.28, 15.34 and 10.21 for Cortex respectively. Simmons and Kelling (1987) evaluated effects of Ca application on yield and periderm Ca concentration in Russet Burbank. At a Ca application rate of 252 kg ha-1 periderm Ca concentration was 1.9% and this did not increase substantially when the Ca application was increased to 756 kg ha-1. Table 1.1. Concentrations of Ca determined in tuber parts as related to Ca supply in various studies. Reference Variety Rate Peel Ca (%) Medulla (%) IBS% Tzeng et al. (1986)

Russet Burbank

0 84 252

0.124 0.134 0.145

60.0 36.3 15.0

Sterrett and Henninger (1991)

Atlantic 0 900 1800

0.2 0.29 0.33

0.02 0.03 0.03

39.2 33.2 29.4

Silva et al. (1991)

Atlantic 1987 1988

0 560 0 840

0.36 0.41 0.26 0.31

14.0 6.0 21.0 11.0

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1989 0 840

0.60 0.65

8.0 4.0

Olsen et al. (1996)

Russet Burbank

-Ca +Ca

0.05-0.08 0.12-0.19

0.034-0.082 0.031-0.16

19.2-48.4 13.7-35.0

Simmons and Kelling (1987)

Russet Burbank

0 252 588

1.3 1.9 2.4

Olsen et al. (1996) evaluated the relationship between tissue incidence of IBS, Ca concentration (peel and medulla) and Ca supply at 73, 99, 127 and 146 DAP in Russet Burbank. Significant differences in peel Ca concentration between the + and – Ca treatments was only observed at 127 and 146 DAP and the –Ca treatments had higher incidence of IBS. At 99 DAP the –Ca treatment had a higher peel Ca concentration but a lower incidence of IBS. The concentration of Ca in the medulla was not different irrespective of Ca regime or stage of development. Davies and Millard (1985) determined that the concentrations of Ca in the medulla were largely water soluble whereas in the periderm a greater percentage of the Ca was bound. The medullary tissue consists of parenchyma cells with no secondary thickening. In contrast the periderm tissue consists of more structured cells of which Ca forms an integral part. These cells also have an abundance of charged binding sites that can complex Ca (Marschner 1986). Hence it is inevitable that periderm tissue will contain significantly greater concentrations of Ca than the periderm. Calcium deficiency in severe cases typically causes the degeneration of cells and it is likely for this reason considerable research has focussed on Ca effects on IBS. Furthermore, because such a strong differential exists between medullary and peel CA concentrations most research has taken the pitch of a Ca supply problem to the inner tissue of the medulla as being the likely reason for the IBS. The difference in tissue Ca concentrations between the medulla and periderm although related to difficulties of supplying Ca to symplastic tissue is also likely to be largely related to the inherent requirements of the different tissues and cells within these.

11.5.1.3. Effects of Ca on IBS incidence Considerable work has focussed on the role of Calcium (Ca) in alleviating the disorder (eg. Collier et al. 1980, Davies and Millard 1985, Tzeng et al. 1986, and Henninger 1991, Clough 1994, Olsen et al. 1996, Palta 1996, Monk-Talbot et al. 1991). Invariably studies that have focussed on Ca have demonstrated the disorder is at least partially alleviated by the application of high rates of Ca. However, results are variable and extremely inconsistent across years where the same treatments have been applied to the same variety on the same soil type. Olsen et al. (1996) evaluated the effect of Ca on IBS in Russett Burbank in a controlled environment pot experiment where a plus (+) and minus (–) Ca regime were imposed and harvested at 4 tuber developmental stages; namely tuber initiation, early bulking, late bulking and maturity. The application of Ca reduced the incidence of IBS at maturity, though nonetheless a high incidence of affected tubers was still

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observed (13.7%). Interestingly, in this study, the percentage of tubers severely affected with IBS was 5.8% at early bulking yet 0.0% at maturity. Since the effects of severe IBS are irreversible a high experimental error is assumed in this study. Plant foliage in this experiment showed no symptoms of Ca deficiency. This is not surprising as the potting media used had a high inherent Ca status. In the –Ca treatment the basal Ca contained in the silica potting mix was not properly accounted for. Based on an approximate bulk density of 1.0 kg l-1 and 60 mg of Ca kg-1, each pot was likely to have contained some 900 mg basal Ca and the estimated potato plant requirements are about 500 mg Ca. Simmons and Kelling (1987) report that despite low Ca status, symptoms of Ca deficiency are rarely observed on plant tops but Ca related disorders are likely to be due to the tubers displacement from the transpiration stream. Silva et al. (1991) in a field experiment on a sandy loam soil applied gypsum at 540 kg ha-1 in 1987 and 840 kg ha-1 in 1988 and 1989 using Atlantic as a test cultivar. They found that although Ca appeared to decrease the incidence of IBS its effect was very inconsistent from year to year and was not recommended for the purposes of alleviating IBS. The incidence of IBS in gypsum treated plots ranged between 6 and 20% from 1987 to 1989. This highlighted that factors other than Ca status were affecting IBS expression; they found a poor relationship existed between tuber peel Ca concentration and incidence of IBS (Table 1.1). Furthermore, they found both IBS incidence and peel Ca concentrations were more influenced by seasonal affects than by application of Ca. Sterrett and Henninger (1991) also evaluated the effects of both gypsum and lime rates (0, 200, 400, and 800 kg Ca ha-1 in 1976 and 0, 900 and 1800 kg Ca ha-1 in 1988) applied to a fine loam on the incidence of IBS in cv Atlantic. They found that although application of Ca reduced IBS incidence even under massive application (1800 kg Ca ha-1) high incidence was still recorded (29.4%). They also found the effect of Ca rate on peel and medullary tissue Ca concentration was inconsistent. Tzeng et al. (1986) found a strong negative correlation between tuber peel Ca concentration and IBS incidence in Russet Burbank. The application of Ca at 252 kg ha-1 reduced incidence of IBS to 15% compared with 60% in Nil Ca application treatment. Olsen et al. (1996) also evaluated the effect of temperature and Ca status on the incidence of IBS in Russet Burbank. In their pot experiment high soil temperature 32oC did not increase the incidence of IBS in either the plus or minus CA treated plants over that of plants at the lower soil temperature of 18oC. However the severity of IBS was increased in minus Ca plants at high temperature. Tubers of plants under plus Ca regime largely did not develop IBS even when the soil temperature was increased to 32oC.

11.5.1.4. Tuber Ca uptake and Supply The Lockyer Valley, Queensland is a region that has an inordinately high incidence of IBS, however, soils in this region are well supplied with Ca. Notwithstanding, responses to Ca application have been recorded in sensitive crops such as lettuce and

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tomatoes. Thus Ca disorders often relate more to uptake and availability problems as opposed to soil Ca status. Nelson et al. (1990) investigated the uptake and transport of mineral nutrients in phloem and xylem in potato cv Russet Burbank. They exposed below ground adventitious nodal roots to solutions of SrCl2 or MnSO4. Strontium (Sr) is used as a transport analogue in plant nutrition studies on Ca. After 5 hours treatment during the day they found that although elevated levels of Cl, S and Mn were found in stolon xylem and stolon phloem; Sr (the Ca analogue) was largely absent but present in stem xylem. It was likely during this daytime period xylem fluid was preferentially supplied to shoots and not tubers. Baker and Moorby (1969) showed that Sr supplied to roots can enter the tuber and that xylem flow at night was an important pathway for supplying immobile elements to tubers. Under the 19 hour treatment period Nelson et al. (1990) found substantial amounts of Sr were present in the xylem. They surmised that xylem flow was sufficient to supply Ca requirements to developing tubers provided 2 assumptions were held. Firstly, that tuber night time weight gain or volume increase was due to xylem inflow as opposed to direct tuber moisture uptake. Secondly, that Ca carried into the tuber at night remained in the tuber when leaf transpiration occurred the next day. They further surmised that if either of these conditions were compromised higher Ca concentrations in xylem would be required to maintain adequate tuber Ca concentrations. Nelson et al. (1990) also demonstrated that phloem to xylem transport of semi-mobile to mobile elements can occur in potato stolons. Kratzke and Palta (1985) found that small roots growing on stolons and tubers could transport water to developing tubers under field conditions. Kratzke and Palta (1985) used a split pot technique to evaluate differential supply of Ca to roots and tubers and the effect on tuber Ca status. They imposed 3 treatments; 100mg L-1 Ca to roots and tubers, 3000 mg l-1 Ca to Tuber and 100 mg l-1 Ca to roots, and, 100 mg l-1 Ca to Tuber and 3000 mg l-1 Ca to roots. The direct application of the highest Ca concentration to tubers dramatically increased the medullary tissue Ca concentration, 0.18% compared with 0.05%in the other treatments. Indeed the concentration was greater than that of the periderm Ca concentration of the other 2 treatments (0.09%). Incidence of IBS has been associated with calcium deficiency in tubers, due to preferential transpiratory loss to the shoots under dry conditions. However, the disorder is often associated with high humidity, which reduces moisture loss from shoots. Indeed, Ca deficiency has been induced in potato sprouts under high humidity (Hect-Buchholtz 1979), since transpiration and hence movement of Ca is restricted. Under conditions of high humidity it is expected that Ca removal from tubers via preferential transpiration to shoots would be reduced and thus less conducive to IBS. In practice this is not observed.

11.5.2. Nitrogen Effects on IBS Silva et al. (1991) determined the effect of nitrogen application on the incidence of IBS. Rates of 112, 168 and 224 kg N ha-1 were applied but incidence of IBS was unaffected. Sterrett and Henninger (1991) also evaluated the effects of N rates on

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incidence of IBS. A low rate of N increased the percentage of tubers affected by IBS at maturity (45.2 %) compared with 37.1% and 34.8% at rates of 168 and 252 kg N ha-1 respectively. McCann and Stark (1989) evaluated the effect of N supply on incidence of IBS in Russet Burbank. Three applications of N at weekly intervals after tuber initiation increased the incidence of IBS over an equivalent pre-plant application or equivalent total as smaller increments over a longer time period. Late application of N appears to favour development of IBS. Iritani and Weller (1978) found application of N at a rate of 340 kg ha-1 resulted in a longer time to senescence than in potatoes fertilised at 146 kg ha-1. Potatoes from this low fertility treatment also accumulated a greater amount of reducing sugars particularly in the basal part of tubers. Cao and Tibbitts (1993) evaluated the effects of varying ratios of ammonia and nitrate N on growth of potatoes over a 56 day period. They found that ratios 20:80 to 80:20 NH4

+:NO3- gave significantly greater dry matter yield of shoot and tuber but not roots

compared with single source N treatments of NH4+ or NO3

-. The optimal ratio of NH4

+:NO3- appeared to be between about 10:90 and 20:80. In support of this finding

the root and shoot tissue concentrations of NO3-, reduced N and Total N were greatest

in the treatments receiving a mixture of NH4+ and NO3

-. In further research Cao and Tibbitts (1994) demonstrated the sensitivity of potato to soil solution pH when grown on a single NO3

- N source. Across the range of pH values 3.5 to 7.5 a pH of 5.0 significantly increased total plant dry matter and leaf area over that at other pH values. Potato plants when grown in a 50:50 NH4

+:NO3- ratio solution exhibited a greater

tolerance range of pH values. Plant total dry weight and leaf area were similar across pH values from 4.5 to 7.0.

11.5.3. Role of Boron

11.5.3.1. Potential effects of Boron on IBS Weir and Cresswell (1993) and Shorrocks (undated circa 1980-85) illustrate visual evidence of boron deficiency inducing brown fleck. In other crops including turnip, beetroot and peanuts, boron deficiency produces symptoms which are similar to brown fleck. In addition to being well supplied with Ca, soils in the IBS sensitive region of Lockyer Valley are generally low in boron and responses are regularly recorded in many crops. Limited research has been conducted in Australia on B effects on potato growth (Pregno and Armour 1992, Sparrow et al. 1995). Sparrow et al. (1995) in field trials conducted on Russet Burbank in 1994 present evidence that suggest trace elements could play a role in the incidence of internal browning and hollow heart. Pregno and Armour (1992) demonstrated yield responses to low rates of B application on potato cv Sebago.

11.5.3.2. Interactions between B and Ca.

There are many similarities in the roles of calcium and boron in plant physiology and biochemistry (Mengel and Kirkby 2001). Calcium is responsible for maintaining cell membrane stability, as is boron, and hence a disruption of this function would result in cell death as noted by Van Denburgh et al. (1986). Dekock et al. (1975) found

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brown melanin compounds are produced in Ca deficient tissues as a result of polyphenol oxidation. Bitter pit is a disorder in apples which produces brown melanotic cells (not unlike brown fleck) that is often corrected by boron application.

In general Ca related disorders do not result from an absolute deficiency but moreover an under supply to specific organs particularly fruits and tubers (Mengel and Kirkby 2001). Calcium is a very immobile element whilst Boron at best is only partially mobile and hence they are not translocated from the plant tops to other plant parts and in particular tuberous organs such as the potato tuber. In addition, they are both carried to the growing point via the transpiration stream, hence all factors affecting water movement in a plant (viz. soil moisture, relative humidity and temperature) influence Ca and B nutrition. Calcium availability in early tuber growth is an influential factor in the development of IBS and exacerbated by high temperatures (Olsen et al. 1996).

Sterrett and Henninger (1991) found that increasing rates of Ca application increased uptake of B by tubers. Soil applications of B have been effective in improving uptake of Ca in tree crops such as apple (Dixon et al. 1973). The application of Ca at a rate of 1800 kg ha-1 increased Boron concentration from 22 mg kg-1 to 41 mg kg-1 in leaf tissue and 6.7 mg kg-1 to 7.1 mg kg-1 in tuber medullary tissue over that in the 0 kg Ca ha-1 treatment. This highlighted a direct interaction between Ca and B status.

11.5.3.3. Boron and plant growth

A large proportion of the B in plant tissues is in the form of complexed stable cis-borate esters in the cell wall (Dugger 1983), hence the maintenance of structural integrity of cell walls is an important function of B. Furthermore, B is believed to play a role in the pentose-phosphate pathway. This biochemical pathway is responsible for incorporating CO2 into sugars. One of the key intermediaries in this pathway is erythrose-4-phosphate. Erythrose-4-phosphate is a precursor for phenols. The development of IBS is associated with excess production of phenols. Mondy and Munshi (1993) found potatoes cv Ontario treated with 2 foliar applications of B at 3.36 kg Borax ha-1 reduced oxidative browning, reduced phenolic concentrations and increased the ascorbic acid concentration. Caffeic and chlorogenic acids accumulate in B deficient plant tissues (Perkins and Arnoff 1956) and these are the principal phenols in potato. Sabba and Dean (1994) found potato genotypes that readily produce brown melanin compounds have high levels of free tyrosine. The accumulation of tyrosine is observed in Boron deficient plant tissue due to the disruption of protein synthesis (Dugger 1983).

The bitter taste associated with the B related disorder bitter pit in apples is similar to that in potatoes with Hollow Heart, which results in a dramatic increase in bitter glycoalkyloids in and around the affected tissue (Jadhav et al. 1980). The relationship between IBS, HH and BC has previously been discussed. The hollowing of stems in plants, particularly Brassica Spp, is B related (Weir and Cresswell 1993). The potato tuber being a modified stem is similarly affected with HH. The browning reactions in mild BC of tubers is also consistent with an affect of B. The concentration of B in deficient potato tissues is in the order of 13-15 mg kg-1 whilst well supplied tubers had B concentrations greater than 33 mg kg-1 (Roberts and Rhee 1990).

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11.5.4. Other nutrients Jacobsen et al. (1998) found that increased phosphorus application rates increased the proportion of phosphorylated starch in potato tubers. Cao and Tibbitts (1992) evaluated the effect of varying solution Mg concentrations (0.05, 0.125, 0.25, 1, 2 and 4 mM) on dry matter accumulation and photosynthetic efficiency in cv Norland. An optimal concentration for CO2 assimilation was observed at concentrations 0.125 to 2 mM Mg. However dark respiration was more sensitive to Mg concentration where concentrations of 0.25 to 1.0 mM Mg resulted in significantly lower dark respiration than other Mg concentrations. Deficiency and excess of Mg appeared firstly to increase dark respiration and decrease leaf area subsequently resulting in reduced photosynthesis. Beringer et al. (1990) evaluated the effects of potassium as KCl and K2SO4 on tuber growth in cv Saturna. Application of KCl reduced leaf concentrations of K+ and NO3

- but increased MG2+, Ca2+ and Cl- resulting in a more negative solute potential and hence higher water content. They suggested this was the cause of higher shoot growth. In contrast K2SO4 accelerated tuber development and they suggested this was due to either a less competitive shoot sink or stimulated phloem loading and assimilate translocation due to the higher leaf concentration of K. In the presence of increased phosphate concentration the concentration of glucose-6P in potato tuber tissue is increased (Loef et al. 1999).

11.6. Irrigation and incidence of IBS

Silva et al. (1991) evaluated the effects of irrigation on the incidence of IBS in Atlantic over 3 seasons from 1987-1989. They determined that excessive irrigation increased the incidence of IBS in 2 of 3 years over a scheduled irrigation regime. The percentage of tubers affected by IBS for 1987, 1988 and 1989 were 8%, 13% and 4% respectively under irrigation scheduling and 13%, 14% and 24% respectively under excess irrigation. The oversupply of irrigation also reduced tuber specific gravity. Novak et al. (1986) evaluated the effect of withholding irrigations prior to maturity on incidence of IBS in cultivar Sebago. By withholding irrigation from between 4-6 weeks prior to maturity the incidence of IBS was reduced from 15% to 5%. McCann and Stark (1989) evaluated the effects of 3 moisture regimes on the incidence of IBS in Russet Burbank. Field experimental treatments were maintained at 80-90% available field moisture (AFM)(Constant High), 60-70% AFM (Constant Low) and between 65-100% AFM (Normal). The Constant High treatment gave the highest incidence of IBS (10%); in particular when N was supplied as 3 applications of 30 kg ha-1 at weekly intervals after tuber initiation. This effect of irrigation on IBS incidence was far less pronounced when N was applied either at planting or as 6 weekly applications of 15 kg ha-1 after tuber initiation; IBS incidence was about 2%.

One of the most dramatic effects of droughting is a rapid cessation of leaf expansion (Munns and Pearson 1974) and reduction in dry matter accumulation. Low leaf water potential reduces translocation of assimilate in potato and this reduction is proportional to the reduction in photosynthesis.

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11.7. Plant Breeding and Varietal Susceptibility

Davies (1998) notes that although there are extremes in genotypic susceptibility to IBS and BC such variation has not been exploited fully by dissecting the genetics and biochemistry of the process. The underlying environmental and biochemical causes and associated genetics of IBS are not understood and this has restricted our capacity to target the breeding of new varieties. Most of the varietal work in relation to IBS has focussed on varietal Ca status and uptake (Collier et al. 1980, Monk and Davies 1989, Sterrett and Henninger 1991, Wannamaker and Collins 1992). Monk and Davies (1989) have attributed the effect of Ca in reducing brown fleck to the greater concentrations of anti-oxidants in tubers that were well supplied with Ca. They evaluated the effect of low Ca supply on the incidence of IBS and growth of tubers of 10 IBS resistant and non-resistant potato cultivars and demonstrated a strong negative correlation between the activity of the oxidative enzyme Superoxide-Dismutase (SOD) and incidence of IBS. However, they found that although SOD activity may have contributed to varietal resistance to IBS it was not the primary resistance mechanism and could not be used for screening germplasm for IBS resistance. Sterrett and Henninger (1991) analysed the relationship between incidence of IBS and tuber Ca concentration of 4 varieties and showed that cv Atlantic had lower Ca concentration and higher susceptibility to IBS than Katahdin, Kennebec and Superior. They rated Atlantic as susceptible, Katahdin moderately resistant, and, Kennebec and Superior as moderately to highly resistant. In contrast some authors have determined there is no relationship between cultivar Ca status and incidence of IBS (Wannamaker and Collins 1992, Collier et al. 1980). Wannamaker and Collins (1992) evaluated incidence of IBS in 10 cultivars across 2 locations over 2 seasons. They determined that incidence of IBS varied dramatically depending mostly on the season and was less influenced by either location or variety. Of the varieties tested Atlantic showed the highest susceptibility to IBS. Coastal Chip was not as susceptible to IBS as Atlantic despite them being full siblings. In the high incidence year 1989, IBS in Coastal chip was 6.2-14% compared with 28.2-68.7% for Atlantic. In 1990 IBS incidence was 2.1-4.6% for Coastal Chip and 19.3-23.3% for Atlantic. This data highlights the high seasonal variability in IBS incidence within a cultivar at the same location. Collier et al. (1980) found no significant relationship between mean varietal incidence of IBS and tuber Ca concentration in an experiment evaluating effects of 3 Ca regimes over 10 varieties in potted soil culture. They concluded that although there were substantial differences in varietal susceptibility to IBS these were not accounted for by tuber Ca concentration. However, increasing the Ca concentration in the media substantially reduced the incidence of IBS. Bamberg et al. (1993) identified Solanum gourlayi as a species that could accumulate high Ca concentrations under low supply and accumulated even higher concentrations under high Ca supply. The accumulation of Ca was higher in this species than in S. Tuberosum. Further to this Bamberg et al. (1998) fine screened accessions selected within S. gourlayi genotypes for ability to accumulate Ca. They found that although

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trends among species were fairly consistent absolute mean tuber concentrations were highly variable especially on a year to year basis. Crumbly et al. (1973) notes Irish Cobbler and Norgold as sensitive to HH but Viking resistant.

11.8. Potato Botany physiology and biochemistry

11.8.1. Botany of the potato plant and tuber The potato tuber is a modified stem that has developed as a storage organ and is joined to the main stem by a stolon. The proximal end of the tuber is the ‘stem’ end whilst the distal end is the ‘bud’ end. The potato eyes are modified axillary buds and there is a condensation of these buds at the Bud end of the tuber. The small scar below these axillary buds is a rudimentary leaf scale. Kratzke and Palta (1985) identify 4 types of roots in potato plants; Basal roots, Stem-stolon junction roots, stolon roots and tuber roots.

11.8.2. Primary carbohydrates and their role in potato growth In the potato, sucrose and starch represent the principal carbohydrates required for growth and development. In order for plants to grow carbohydrate produced in the leaves as sucrose is mobilised from the leaf cells, to storage sites such as the potato tuber. The transport of carbohydrate across such a large distance is possible since sucrose is a highly soluble disaccharide consisting of a glucose and fructose moiety. Because of its high solubility and low reactivity with proteins, sucrose is readily transported via phloem to newly developing tissues (Salisbury and Ross 1985, Dey and Harbourne 1997).

11.8.3. Starch

11.8.3.1. Starch synthesis In contrast to sucrose starch is a large complex carbohydrate for which the primary purpose is to act as a storage energy reserve. Two general types of starch exist in plants both being polymers of glucose units; amylose, which contains relatively few branched structures and is of lower molecular weight and amylopectin which is highly branched and hence of greater molecular weight. Within plant tissue starch is present in both the chloroplasts of photosynthetic tissue and in amyloplasts of non-photosynthetic tissue. The amyloplasts, as found predominantly in potato tuber parenchyma cells, are specific storage organs for starch.

11.8.3.2. Starch in leaves During photosynthesis leaf chloroplasts produce triose sugars, which are exported to the cytoplasm to form sucrose. In the chloroplasts this triose sugar is converted into

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the 6 carbon (C) sugar fructose-6-P which is readily converted into Glucose; the substrate for starch formation. Plants produce starch in leaves as a short term (day-time) storage reserve but this starch cannot be transported to the tubers since it is largely insoluble in water. Plants re-convert this starch into sucrose, which is a small soluble molecule, and transport this via the phloem (sap conducting tissue) to the developing tubers where it is again re-converted into starch. Prior to tuber initiation the starch in leaf dry matter is at its highest concentration and from the outset of tuber initiation leaf starch levels decline. High concentrations of sucrose and starch occur in stems and leaves under high temperatures resulting in low starch deposition in tubers (Kolbe and Stephan-Beckman 1997). Similarly the leaf concentrations of sucrose and reducing sugars show a strict diurnal rhythm. Sugars and starch decrease during the night to a minimum at approximately 04:00. The highest reducing sugar concentrations are recorded at between 14:00 and 16:00 and sucrose between 16:00 and 18:00. Maximal uptake of CO2 occurs between 10:00 and 16:00 (Meinl 1963 in German as cited in Kolbe and Stephan-Beckman 1997).

11.8.3.3. Starch synthesis in tubers In the leaves sucrose is loaded into the phloem vessels and translocated to the tubers. Sucrose is received into the cytoplasm of tuber storage parenchyma cells where it is split into glucose and fructose. The glucose and fructose are transformed to mostly Glucose-6-P, which is transported into the amyloplast and used as a substrate for starch formation. The initiation of starch synthesis occurs with the formation of a short chain of glucose units starting with the acceptance a glucose unit by an initiating protein. This process is extremely complex and largely outside the scope of this review.

11.8.3.4. Plant maturity and starch content Starch is the predominant part of tuber dry matter accounting for between 60 and 80% of dry matter. There is a strong correlation between the percentage of starch in the dry matter and the dry matter of tubers. That is, tubers that have low dry matter also tend to have low amounts of starch in this dry matter (Burton 1966). Factors that affect the storage or accumulation of starch alter the proportion of starch in dry matter. This includes the gamut of factors that affect carbohydrate synthesis, transportation and respiration including photosynthesis, nutrition, moisture, light, temperature etc. The stage of tuber development and tuber maturity has a dramatic affect on starch granule size and starch concentration in the tuber (Christensen and Madsen 1996, Loeff et al. 1999). Christensen and Madsen (1996) note that both tuber yield and starch concentration increase dramatically up to 111 days after planting (DAP) but does not further increase from 111-139 DAP in cvs Oleva and Saturna. In the post foliage senescence period a reduction in starch content occurred but average starch granule size increased indicating that as tubers mature starch is rearranged inside the tuber.

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The inner medullary tissue of the tuber as associated with the pith is of lower dry matter than the outer medulla. Observations on the incidence of IBS highlight its association with the outer medullary tissue and particularly the medullary tissue between the pith rays at the crown of the tuber. It is this tissue, along with the cortex tissue that contains the majority of tuber dry matter as well as the greatest proportion of starch. Rapidly growing cells are surrounded by thin primary cell walls and once growth has ceased a thickened secondary cell wall is laid down. The primary cell walls consists of a cellulose, hemicellulose and pectin matrix. The predominant pectins in potato tubers are highly polymerised and in the potato tuber these cement individual thin walled parenchyma cells. The cementation of pectins occurs through the joining of carboxyl groups of adjacent pectins via Ca2+ ions. The concentration of pectin increases with tuber development and under conditions of high temperature the relative production of soluble pectins increases compared with that of insoluble pectins (Burton 1966). Burton proposes that the increase in soluble pectins under these conditions is the product of insoluble pectin decomposition.

11.8.3.5. Environmental effects on starch synthesis Considerable studies have been conducted on the effects of temperature on starch synthesis in potato (Mohabir and John 1988). The synthesis of starch in storage organs across plant species is strongly related to temperature (Mohabir and John 1988). They found that potato cv. Desire had a low optimum temperature of 21.5 oC in contrast to that of other storage organs in other species. Burton (1981) identifies an optimal temperature for carbon assimilation in potato in the presence of adequate light approximates 22 oC. At the extreme end of the temperature scale he notes the effects of high temperature are not just an increasing of foliage respiration leaving no surplus for carbohydrate export but also a hormonal association. The issue of temperature effects on starch synthesis is complicated by the fact that other temperature induced changes can occur including increases in GA levels. Though the synthesis of starch has a strong optimal temperature, Mohabir and John (1988) found that isolated potato cell amyloplasts were not as sensitive to temperature increase since starch synthesis did not decline in these when temperature increased from 21 to 31 oC. Given our limited knowledge of starch synthesis in isolated amyloplasts they tentatively suggest that assimilate transport into the amyloplast and subsequent steps in starch synthesis are not responsible for reduced starch synthesis at high temperatures. Manrique (1992) identifies temperature as the major limitation on potato production in the tropics. He notes that the leaf area index of potatoes grown at an altitude of 282 m was greater than that at 91 m and this could be ascribed to a temperature effect. Malik et al. (1992) notes a dramatic drop in dry matter of several clones between 107 DAP and 120 DAP. During the same period a dramatic reduction in foliage cover had also occurred and combined with the temperature data presented this would suggest increased respiration.

11.8.4. Potato plant physiology

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11.8.4.1. Seed age The beneficial effects of optimising the physiological age of potato seed are well recognised in terms of tuber set and yield (Bodlaender and Marinus 1987). Bodlaender and Marinus 1987 imposed cool storage treatments on seed lots of cvs Jaerla and Desiree to regulate seed age. They found that young seed produced plants that developed slowly and remained small whilst slightly aged seed developed more foliage and old seed produced little or no foliage.

11.8.4.2. Environmental effects on potato growth Cao and Tibbitts (1994b) evaluated phasic temperature regimes on potato growth and tuberization. They imposed 2 air temperature regimes (17oC and 22oC) over 3 phases with each phase being 21 days. A factorial experiment of temperature by phase was conducted using cv Norland. They found that a continuous temperature of 22oC resulted in the greatest total plant weight (243.5 g) compared with 196.7 g in the continuous 17oC treatment. However, the tuber dry weight in the continuous 17oC treatment was greater than that in the continuous 22oC treatment indicating that shoot growth is stimulated at higher temperature and tuber growth at lower temperature. Benoit et al. (1983) evaluated the effect of short term treatments of 3 days under various constant temperature regimes (10, 15, 20, 25, 30 and 35 oC) on growth of potato cv Katahdin. Maximum leaf area growth was observed at 25 oC. As the potato plant approaches full senescence a steadily lower assimilate production occurs whilst respiratory demands remain relatively constant (Kolbe and Stephan-Beckmann 1997). The often observed reduction in dry matter content and increase in crude protein (Kolbe and Stephan-Beckmann 1997) in the final stages of senescence is likely to be related to this imbalance. At the onset of tuber initiation very high respiration rates occur in the developing tubers (Kolbe and Stephan-Beckmann 1997) and at the termination of starch accumulation, when no green foliage is present, the respiration rate of the senescing tubers decreases dramatically. Under drought conditions Munns and Pearson (1974) found that a low proportion of CO2 is fixed into polysaccharide in comparison to soluble sugars. Demagante and Vander Zaag (1988) found that the cvs Desiree and Cosima grew better under shaded conditions when exposed to intense solar radiation and that cvs Red Pontiac and DTO-33 were less sensitive to intense solar radiation.

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