MSc thesis in the Danish-Swedish Horticulture Programme 2006:4

2006-06-15

(ISSN 1652-1579)

Possible interactions between Rhizoctonia solani and plant parasitic nematodes (PPN) in Swedish potato

fields

−A pilot study

© Lars Wiik

By

Anette Karlsson Student on the Danish-Swedish Horticultural Programme, SLU, Alnarp

MSc thesis in the Danish-Swedish Horticulture Programme 2006:4

2006-06-15

(ISSN 1652-1579)

Possible interactions between Rhizoctonia solani and plant parasitic nematodes (PPN) in Swedish potato

fields

−A pilot study

By Anette Karlsson

Student on the Danish-Swedish Horticultural Programme, SLU, Alnarp

Subject: Plant pathology Supervisors: Lars Wiik and Sanja Manduric

Dept. of Crop Science, SLU, Alnarp

Abstract

Karlsson, A. 2006. Possible interactions between Rhizoctonia solani and plant parasitic nematodes (PPN) in Swedish potato fields, −A pilot study. MSc Thesis. ISSN 1652-1579

Rhizoctonia solani is becoming an increasing problem for Swedish potato growers, causing quantitative and qualitative yield losses. The increasing problems might be due to synergistic interactions between plant parasitic nematodes (PPN) and R. solani. Synergistic interactions between PPN and various fungi have been reported in several crops.

The present study investigated potential interactions between R. solani and PPN. Soils from 41 fields in southern and western Sweden were sampled and the contents of PCN, free-living PPN, R. solani infection and nutrients were determined. Stem canker symptoms on ten plants in four different places in each field were observed and quantified. The nematode content and the coverage of R. solani mycelia were determined for the roots of 4 plants collected in each field. Micropropagated potato tubers were grown in closed containers of the soil samples to get a measure of the infection potential of R. solani and PPN in the soil. A questionnaire concerning background information was sent to commercial producers.

This preliminary study produced indications that synergistic interactions occur between PPN, especially endoparasitic nematodes, and R. solani in Swedish potato fields. Nematode populations below the threshold limit appeared to play a part in interactions with the fungus. The inoculum source of R. solani, i.e. seed or soil, was also found to be of great importance for disease development and the extent of the yield loss. Soilborne inoculum of R. solani and severe stem canker symptoms was found to cause development of black scurf on the tubers, whereas seedborne inoculum caused a greater development of stem canker. A yield reduction could be observed in fields infected by R. solani. The nutritional status of the soils included in this study was found to be of minor importance for the development of the disease.

Keywords: Rhizoctonia solani, plant parasitic nematodes, Swedish potato fields, interactions, disease complex, disease severity, inoculum source.

Author’s address: Anette Karlsson, h1karane@stud.slu.se

Swedish summary

Det är välkänt att växtsjukdomar uppstår på grund av ett komplext samspel mellan värdväxten och skadegöraren. Externa faktorer påverkar detta samspel. Basidiemyceten Rhizoctonia solani orsakar ekonomiskt betydande skördeförluster i många grödor. Svampen ger upphov till groddbränna, missbildningar och lackskorv på potatis. Potatiscystnematoderna, Globodera rostochiensis och Globodera pallida är viktiga skadegörare i potatis men även vissa frilevande nematoder har ekonomisk betydelse. Endoparasitära nematoder (som går in i rötterna) synes oftare än de ektoparasitära nematoderna (som inte går in i rötterna) vara en av skadegörarna i ett samspel med jordbundna svampar. Jordbundna svampar såsom Fusarium spp., Verticillium spp., Phytium spp., Phytophthora spp. and Rhizoctonia spp. har i ett antal studier visat sig samverka med Meloidogyne spp., Globodera spp., Heterodera spp. och Pratylenchus spp. Man tror att samspelet uppstår för att svampen får lättare att infektera potatisplantans underjordiska delar efter att nematoderna har angripit plantan. Detta beror antingen på att svampen kan infektera genom de sår som uppstår när nematoden livnär sig, eller på att nematoden inducerar förändringar i plantan som gör den mera mottaglig för svampinfektion. Målet med studien var att undersöka om det möjligen finns ett samspel mellan R. solani och växtparasitära nematoder i svenska potatisfält. Jordprover från 41 fält i södra och västra Sverige samlades in under sommaren 2005. Potatiscystnematodpopulationen, populationen av frilevande nematoder, jordburen smitta av groddbränne- och näringsinnehållet i jorden undersöktes och bestämdes. I fält graderades angreppet av groddbränna på 10 plantor på fyra ställen i varje fält. På ett prov av fyra plantor från varje fält bestämdes antalet endoparasitära nematoder och täckningen av R. solani-mycel på rötterna. Information angående tidigare angrepp av groddbränna och lackskorv, växtföljd, bevattning, m.m. besvarades av odlarna. Statistisk bearbetning (korrelations- och variansanalyser, ANOVA) gjordes i Minitab. Plantor från betat och certifierat utsäde löpte en mindre risk att bli smittade med R. solani. Lackskorv på de nybildade knölarna bildades lättare om det fanns mycket marksmitta i jorden eller om fältet uppvisade svåra symptom av groddbränna under odlingssäsongen än om marksmittan var begränsad eller om symptomen i fält var mindre framträdande. Angrepp av groddbränna i fältet orsakade både kvantitativa och kvalitativa skördeförluster. Denna preliminära studie indikerar att ett samspel mellan främst endoparasitiära nematoder och R. solani förekommer i svenska potatisfält. Populationer av växtparasitära nematoder på en nivå långt under skadetrösklarna är av betydelse, eftersom det finns tecken på att även små populationer kan ge upphov till stora skador när de är en del av ett synergistiskt samspel. Huruvida samspelet uppstår på grund av att nematoderna skapar inträdesportar eller genom modifieringar är svårt att säga. Fler och mera omfattande studier måste dock genomföras för att kunna säga något säkert.

Om det visar sig att samspel mellan R. solani och PPN är vanligt förekommande i svensk potatisodling bör hänsyn tas till detta vid utformandet av bekämpningsstrategier.

Contents

Preface, 8 Introduction, 9 Rhizoctonia solani on potato, Solanum tuberosum

Life cycle Influence of the environment Seed potato treatment

Plant parasitic nematodes (PPN) in potato Life cycle Environmental factors

Fungi-nematode interactions Synergistic interaction

Mechanisms behind the wounding theory Mechanisms behind the modification theory The role of root exudates Complexity of interaction Negative effects on nematode reproduction Species specificity Effectiveness of resistance against pathogens in host plants Effect of physical and chemical soil factors

Disease complexes involving PPN and R. solani in potato Materials and Methods, 21

Quantification of Rhizoctonia solani in the field Quantification of Rhizoctonia solani on tubers from closed containers Nematode extractions Staining of nematodes in roots Isolation of free living nematodes from plant material Questionnaire Nutritional status and soil type Statistical methods

Results, 24 Correlations

Linkage between different correlation coefficients Analysis of variance

Fungi Fungi and nematode Tuber yield Soil factors

Discussion, 36 Field studies Choice of fields Influence of cultivar Materials and Methods R. solani Nematodes Interactions Tuber yield External growth factors

Conclusions, 44 References, 45 Acknowledgements, 47 Appendix I, 48

Descriptions of the PPN extraction methods used Appendix II, 50

Explanation of factors Appendix III, 52

Questionnaire sent to producers Appendix IV, 53

Data Appendix V, 60

Geographical spread of sampling sites

8

Preface

It has long been suspected that plants are not only subjected to one potential pathogen, but many microorganisms that interact. Researchers have proven that complex interactions between plant pathogens, their host and abiotic factors that affect these interactions often lead to more serious disease outbreaks. Already in 1971, N.T. Powell pointed out that plant parasitic nematodes and plant parasitic fungi interact in disease complexes. He found that plant parasitic nematodes often played a major role in disease interactions and that soilborne fungi played a part in disease complexes. Endoparasitic nematodes are more often reported to form disease complexes with soilborne fungi than ectoparasitic nematodes. Nematodes such as Meloidogyne spp., Globodera spp., Heterodera spp. and Pratylenchus spp are often reported as forming part of disease complexes with soilborne fungi such as Fusarium spp., Verticillium spp., Phytium spp., Phytophthora spp. and Rhizoctonia spp. (Back et al., 2000). The mechanisms underlying the development of disease complexes are not yet fully known and there are two different theories: the wounding theory and the modification theory. The wounding theory states that nematode feeding and migration within the roots give rise to portals facilitating fungal infection. The modification theory arose from histopathological studies showing that the invading hyphae of fungi did not follow the nematode portals into roots. Instead, the fungus infected the plant at a later stage after the nematodes had induced modifications in the plant physiology (Back et al., 2000). During the spring of 2005, relatively severe infections of R. solani were reported in Sweden. In addition, some previously unidentified symptoms such as thickened root and stem bases, bushiness, decreased tuber setting and fewer stems per plant than normal attracted a lot of attention. The aim of the present study was to investigate possible interactions between R. solani, the cause of stem canker and black scurf, and plant parasitic nematodes on potatoes under field conditions and to determine the consequences of such interactions.

9

Introduction

Rhizoctonia solani on potato, Solanum tuberosum

The young, colourless and later yellow to light-brown mycelium of Rhizoctonia solani consists of several long nuclei cells, branching characteristically at approximately right angles to the main hypha. The cells are further slightly constricted at the junction of the branch. The features of mycelium can be used for identification of the R. solani fungus (CMI, 1974). R. solani is composed of a group of different strains that are more or less related. Different strains are distinguished from each another by anastomosis, the fusion of touching hyphae, which only occurs when the isolates are of the same anastomosis group. The various anastomosis groups are not entirely host-specific. Isolates of AG-3 affect mostly potato, causing stem canker and stolon lesions near the soil line and producing black sclerotia on tubers (Parmeter, 1970; Agrios, 1997).

Life cycle

Figure 1. The life cycle of R. solani (from Agrios, 1997). The fungus is both seedborne and soilborne and is present in most soils (Parmeter, 1970; Agrios, 1997). The pathogen usually overwinters as mycelium or sclerotia in the soil and on infected plant debris, perennial plants or potato tubers (Parmeter, 1970; Olofsson et al., 1996).

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In soil, R. solani exists in either a resting or an actively growing form. The resting form has to germinate before penetration can occur, whereas the actively growing form continues to grow before penetrating the host (Parmeter, 1970). Root exudates diffusing from sprouting potato tubers stimulate the sclerotia in the soil or on infected tubers to germinate and the mycelium may spread over the underground surfaces of the potato plant (Parmeter, 1970; Olofsson et al., 1996). The stimulating effect of the root exudates appears to decrease with increasing physiological age of the host plant. Furthermore, the susceptibility of the host plant towards R. solani decreases with increasing physiological age (Parmeter, 1970).

Mechanism involved in penetration of host tissues

Intact plant tissues constitute a barrier against fungal penetration and R. solani can infect underlying tissues either through mechanical penetration by means of force or through utilising natural openings and wounds (Parmeter, 1970; Back et al., 2002). Mechanical penetration occurs by means of infection cushions. Fine infection pegs develop from the infection cushion at the contact site with the host, enabling the fungus to minimise the force needed for penetration. More than one cell may be infected from one infection cushion, and one cell may be penetrated by more than one peg. After a peg has penetrated, it continues to grow between the cuticle and the epidermal wall. Finally the cuticle and epidermal wall are penetrated, and the infectious fungal organs may extend growth into the cell lumen. R. solani has been recorded to be able to penetrate through intact surfaces of potato tubers (Parmeter, 1970; Demirci & Döken, 1998). Another possible mode of penetration from infection cushions is through the extrusion of hyphal tips from under the cushion. These can grow through the cuticle and between the epidermal cell walls without producing infection pegs (Parmeter, 1970). The fungus may also utilise natural openings such as stomata on stems, cotyledons and leaves or lenticels on potato tubers as entry portals to plant tissues. The growing hypha grows into the opening and fills the underlying cavity before penetrating the new tissues. Wounds can also be used as entry portals, but penetration usually does not occur solely via wounds. The hypha first spread and fill out the wound with densely packed hyphae before penetrating into healthy tissues without the formation of infection structures (Parmeter, 1970; Back et al., 2002). Plant defence mechanisms may stop the fungal infection at the following establishment stages of R. solani:

• Attachment of hypha to plant surface • Formation of infection structures • Penetration of infection pegs • Continued invasion of penetration hyphae can be stopped by hypersensitive

reaction (Parmeter, 1970; Demirci & Döken, 1998).

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Damage to plant tissues

Distinctive browning of cell walls is an early symptom of infection by R. solani (Parmeter, 1970; Anderson, 1982). Hyphal penetration and the collapse of infected host tissue are more or less instant. Intracellular penetration and browning of the protoplasm of host cells is followed by cellular collapse, which leads to extensive growth of the hyphae. The hyphae finally fill the infected cell. Defence mechanisms of the plant may also cause damage to the plant itself due to production of toxic metabolites killing off both the fungal and host tissues. The hypersensitive response of the plant towards fungal infection gives rise to well-defined lesions, which can be seen on infected stem bases, stolons and roots of potato. However the hypersensitive response is not the only defence mechanism; the potato plants may develop phellogen in healthy cells at the margin of the necrotic area. Suberised cells constitute a barrier against the invading hyphae. Recovery from infection is possible to some extent depending on severity of the infection. Plants with infections in the cortical tissues may survive, whereas plants with vascular infection will eventually die (Parmeter, 1970). R. solani can infect the cortex, vascular bundle and pith of potato stems. Sclerotia are formed before the infected cells die in the tissues that sustain the greatest fungal growth. In potato, the cortical cells support the greatest growth (Parmeter, 1970). On emerging sprouts and plant, the outer cortical tissues are subjected to potential fungal pathogens. Tissues infected by R. solani develop elongated, reddish-brown lesions. In severe cases, the sprouts can be burned off. However, new sprouts may develop from healthy tissues beneath the infection site. The emergence becomes delayed and the field shows a patchy emergence pattern. The susceptibility of potato plants to R. solani is greater before the first green leaves appear, since young tissues in general are more susceptible to R. solani (Parmeter, 1970; Olofsson et al., 1996). Phloem and xylem vessels may be partly destroyed during severe infections (Scholte & Jacob, 1989). Yield losses caused by R. solani include both quantitative and qualitative losses (Anderson, 1982; Olofsson et al., 1996), since the flow of assimilates from the above-ground parts are hindered by the lesions on stems and stolons. This causes the plant to set fewer tubers. Tubers are also positioned at a shallower level and in a circle around the stem, causing a greater fraction of green tubers. In some cases the flow of assimilates is hindered by the lesions, which may cause an accumulation of assimilates in the lower leaf axils, giving rise to ‘air tubers’ (Olofsson et al., 1996).

Development of black scurf

Black scurf develops towards the end of the growth season due to changes in nutritional status of the plant, maturity of the periderm and exudate exchange of the tubers (See Figure 2). If infected tubers are stored under moist conditions, sclerotia may continue developing (Olofsson et al., 1996). The inoculum sources of R. solani that are available in the soil are thought to mainly give rise to black scurf on tubers and only under special conditions to damping-off, whereas inoculum on seed potatoes is thought to mainly give rise to damping-off (Bång, 2005).

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Figure 2. Black scurf on potato. © Lars Wiik, 2005.

Influence of the environment

Cold and wet weather favours the fungi and the red-brown lesions can first be seen just below the soil line. The infection may enlarge in all directions and finally includes most of the roots and girdle stem bases of the plant (Parmeter, 1970; Olofsson et al., 1996; Agrios, 1997). During favourable conditions, the perfect stage of R. solani (Thanatephorus cucmeris) can develop on infected mature plants (See figure 3). The perfect stage can be seen as a white mould collar just above the soil line on the stems. The function of the perfect stage is unclear, as the fungus seems to be incapable of producing basidiospores that are able to infect during normal field conditions (Parmeter, 1970; Olofsson et al., 1996; Agrios, 1997).

Figure 3. Thanatephorus cucmeris seen in Swedish potato field during the summer of 2005. © Lars Wiik, 2005

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Mycelium of R. solani can grow within a wide temperature range, 6-32 oC, with an optimum at 25 oC. The optimum temperature for sclerotia development is about 16-18 oC. The optimum temperature for infection and disease development, however, depends on the speed of plant growth and plant defence mechanisms at different temperatures. During field conditions, R. solani is most aggressive at 13-18 oC, with some variations for different soil types. Tubers may exhibit more severe symptoms due to delayed emergence of tubers planted early in cold soil or water stress during warm spring seasons. The pathogen needs moisture for development, and high relative humidity is considered to be optimal for mycelial growth, sclerotia production and infection (Parmeter, 1970; Olofsson et al., 1996; Agrios, 1997). Heavily irrigated young potato plants are subject to an increased risk of becoming infected by damping-off and stem canker (Parmeter, 1970). Wet soils may inhibit sclerotia formation, since mycelial growth is favoured by high relative humidity in combination with good aeration. Soil moisture affects the survival of sclerotia, which under dry conditions can remain dormant for at least six years. Relatively warm and moist conditions cause the sclerotia to germinate and their function as survival organs is thus lost (Parmeter, 1970; Olofsson et al., 1996). Light relatively humus-rich soils are favourable to the fungi, compared to heavier soils. R. solani is tolerant to a wide pH range and grows especially well within the pH levels of most agricultural soils (Parmeter, 1970; Olofsson et al., 1996). However, pH level can affect the pathogen since it affects enzymes and the availability of calcium, magnesium, potassium, nitrogen, iron, phosphorus and micronutrients, which influence the development of the disease. There is an increased potential for infection by R. solani if inorganic potassium, nitrogen or calcium is deficient or if nitrogen and phosphorous levels are excessive (Parmeter, 1970).

Figure 4. The effect of omitting the potato host from the crop rotation on the quantity of R. solani inoculum in the soil (Bång, 2005). The inoculum present in the soil decreases rapidly when other crops are cultivated between the potato crops in the crop rotation (Figure 4).

14

Growing a potato crop every third to fourth year compared to every year reduces the amount of inoculum in soil by more than 25%,. For Sweden, a crop rotation period of five years is recommended (Bång, 2005).

Seed potato treatment

In Sweden, a few fungicides are permitted for use for seed potato treatment, e.g. the active substance pencycuron, which inhibits the growth of the mycelium upon contact. It is important that the whole tuber is completely covered by fungicide in order to disinfect the crop from seedborne inoculum (Bayer CropScience, 2006).

Plant parasitic nematodes (PPN) in potato

Plant parasitic nematodes are unsegmented, filiform roundworms about 300 to 3000 μm long and 15-35 μm in diameter. A flexible exoskeleton made of cuticle encloses and supports the body fluids and the inner organs. Specialised muscles enable nematodes to control all important body functions. Nematodes have a variety of sensory organs. The most prominent are the amphids, sensory organs situated in the head region and connected to the oesophagus, intestine and reproductive systems by the nervous system. Plant parasitic nematodes feed by piercing and sucking out plant cells through a hollow stylet (Palm et al., 1968; Dropkin, 1980; Agrios, 1997). Bisexual reproduction is common in many nematode species but parthenogenic and hermaphroditic reproduction also occur (Palm et al., 1968; Dropkin, 1980; Agrios, 1997). Although the sexes are often quite similar in appearance, in some species such as in cyst and root-knot nematodes the sexual dimorphism is very marked. The adult females swell up until they become lemon-shaped or round, whereas the males remain eel-shaped. Plant parasitic nematodes can be divided into two groups: soil dwellers and aboveground nematodes. In this paper, only soil dwelling nematodes are considered. The soil dwelling nematodes are further divided with respect to feeding habits into ectoparasitic and endoparasitic nematodes. The ectoparasitic nematodes feed and live outside the roots. Examples of nematodes behaving this way are the pin nematode (Paratylenchus spp.) and the stubby-root nematode (Trichodorus spp.). The endoparasitic nematodes spend most of their lives inside the roots and can be divided into migratory and sedentary species. The migratory endoparasitic nematodes, such as the root lesion nematode (Pratylenchus spp.) invade root tips, move and feed in the cortical parenchyma cells, causing necroses in root tissues. This type of nematode is not known to have any adaptation mechanisms to unfavourable conditions and overwinters in, or close to, roots (Palm et al., 1968; Agrios, 1997). Cyst and root-knot nematodes are sedentary endoparasitic nematodes that migrate into fine roots as second stage juveniles. Inside the root they select a feeding site. Once settled, the females become sedentary for the rest of their lives. The body in Globodera species undergoes some chemical changes and becomes a strong and elastic cyst that protects the eggs from adverse conditions. Favourable conditions cause the juveniles hatch from the eggs inside the cyst and wander off to infect new roots (Palm et al., 1968; Agrios, 1997).

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Life cycle

The generalised life cycle of nematodes is composed of six developmental stages: an egg stage, four larval or juvenile stages (usually referred to as L1, L2, L3 and L4) and an adult stage. The first stage juvenile develops from the embryo inside the egg. The first moult occurs while the juvenile is still within the egg. Second-stage juveniles are the invasive stage, entering host plant roots and starting to feed, while third and fourth stage juveniles continue feeding and increase in size. The sixth stage is the fully developed adult nematode (Palm et al., 1968; Dropkin, 1980; Agrios, 1997).

Environmental factors

The main environmental factors affecting soil dwelling nematodes are temperature, moisture, soil texture, aeration and the chemistry of the soil solution. Temperature affects nematode activities such as hatching, reproduction, movement, development and survival. Most plant parasitic nematodes become inactive at low temperatures (5-15 oC), and at high temperatures (30-40 oC). Optimal temperatures for nematodes are 15-30 oC. Temperature affects the length of the nematode life cycle. Some species develop very fast and under optimal conditions their generation time only takes a few weeks. Nematode populations are also affected by soil moisture content. Moist conditions and irrigation favour nematode reproduction. The active part of nematode life cycle starts with hatching from the egg. The hatched juvenile must then find a suitable host within a few days or starve to death. Plant parasitic nematodes have developed different survival strategies. Cyst juveniles, for example, might stay dormant and enclosed in eggs for many years. In some free living species, L4 can slow down their metabolic activity and become dormant (Palm et al., 1968; Agrios, 1997). Most plant parasitic nematodes inhabit the rhizosphere soil. Root exudates diffusing from roots attract nematodes, stimulate hatching of eggs and help juveniles navigate in the soil when seeking food sources. Very little is known about the chemical nature of nematode attractants. Free water is very important as a medium for distribution of different stimuli needed for hatching of nematode eggs (Palm et al., 1968; Agrios, 1997). The species-specific feeding behaviour of plant parasitic nematodes gives rise to different types of wounds on the roots of host plants. Common to all plant parasitic nematodes is feeding by puncturing host cells with the stylet. During feeding, secretions from the oesophageal glands are injected into the tissues of the host plant, where they cause cytological and biochemical changes. Each nematode species has a specific behaviour and induces different physiological responses in the plant. Ectoparasitic nematodes puncture the outer plant cells with their styles. For example, Trichodorus spp. feed on the outer tissues, whereas Longidorus spp., which have a long, needle-like odontostyle, feed on deeper tissues. The wounds caused by endoparasitic nematodes disturb the roots of the host plants to a larger extent. In roots, the migratory endoparasitic nematodes such as Pratylenchus spp. move intracellularly through the cortex and use the stylet for piercing cell walls.

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Sedentary endoparasites such as Meloidogyne spp. and Globodera spp. have complicated life cycles and feeding strategies. Second stage juveniles enter roots just behind the apex and then move intracellularly (Globodera spp.) or intercellularly (Meloidogyne spp.) to the vascular cylinder, where the juveniles become sedentary and induce feeding sites (Palm et al., 1968; Taylor, 1990; Back et al., 2002). Table 1. Nematode damage threshold levels in potato (Holgado et al., 1998; Dickenson et al., 2000; Guidelines for Nematode Control, 2005) Nematode spp. Damage threshold level

nematodes /250g soil

Meloidogyne spp. 50-250 Globodera spp. 500 Pratylenchus spp. 250 Trichodorus spp. & Paratrichodorus spp.

500

Tylechorynchus spp. 625

Fungi-nematode interactions

In the rhizosphere, the roots are exposed to numerous different soilborne pathogens and microorganisms that occupy the same habitat. It has long been assumed that they influence or interact with each other. Several interactions between nematodes and other soil inhabiting pathogens, including fungi, bacteria, and viruses, have been demonstrated during the last decade (Palm et al., 1968; Taylor, 1990; Back et al., 2002). For example, it has been noted that crops resistant to specific fungal or bacterial pathogens become infected by these pathogens in the presence of nematodes. Interactions between different organisms are a complex matter and the outcome of an interaction can be synergistic, antagonistic or additive. Synergistic interactions are associations between two pathogens resulting in more severe plant damage than the damage caused by either pathogen alone. An interaction is antagonistic when there is less plant damage than could be expected from adding the expected damage from the two pathogens. Additive or neutral interactions are when the plant damage is the same as the sum of the expected pest damage from each pathogen (Back et al., 2002). Several synergistic interactions between endoparasitic nematodes and fungi have been reported. They appear to be more abundant and of greater importance than interactions between ectoparasitic nematodes and fungi. The difference between ecto- and endoparasitic nematodes is probably due to different feeding behaviours and life cycles. Plant parasitic nematodes induce local or systemic modifications in plant cells that transform plant host status to other soilborne pathogens (Back et al., 2002). Endoparasites frequently reported to form synergistic interactions with soilborne fungi are Globodera spp., Meloidogyne spp. (Taylor, 1990; Sugawara et al., 1997; Back et al., 2002), Rotylenchus spp., (Back et al., 2002), and Pratylenchus spp. (Taylor, 1990; Back et al., 2002). The common fungi interacting in disease complexes are Pytium spp., Phytophthora spp., Rhizoctonia spp., Verticilium spp. and Fusarium spp. Most of the disease complexes studied include Meloidogyne spp. and different soil pathogenic fungi (Taylor, 1990; Bertrand et al., 2000; Back et al., 2002).

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There are two generally accepted theories concerning the mechanisms behind fungi-nematode interactions, although the exact mechanisms are still not clear. The wounding hypothesis suggests that nematodes facilitate fungal penetration through feeding and burrowing in roots (Palm et al., 1968; Taylor, 1990; Sugawara et al., 1997; Back et al., 2002). The modification hypothesis suggests that nematodes induce modifications in plants. These modifications change plant defence mechanisms and increase susceptibility to fungal infections (Palm et al., 1968; Taylor, 1990; Sugawara et al., 1997; Back et al., 2002). In trials where plants have been wounded mechanically and then inoculated with fungal pathogen, hyphal penetration has been facilitated by the wounds. However, the mechanisms behind the wounding theory are more complex than just fungi benefiting from wounds. The timing of nematode infection and plant defence mechanisms are also important for the establishment of an interaction. Several examples of nematode-fungi interactions that prove the wounding theory to be valid are described below.

Synergistic interaction

Mechanisms behind the wounding theory

Tobacco roots inoculated with Phytophthora parasitica show increased disease symptoms when they are subjected to artificial wounding. P. parasitica interacts synergistically with Pratylenchus brachyurus and the timing of the inoculation of the two organisms is important for the interaction. When nematodes are inoculated one week before or together with the fungus, the disease symptoms increase. The fungus might utilize the feeding tracts of the nematode to infect the tobacco plants. Feeding tracts made by nematodes inoculated two to three weeks before the fungal infection become necrotic and hyphal penetration is therefore not facilitated (Back et al., 2002). Damping-off in sugarbeet caused by R. solani has been shown to benefit from the invasion tracts of the beet cyst nematode, Heterodera schachtii. A possible explanation for this is that infection structures become unnecessary, since the fungus can easily overcome plant barriers by following the paths of the nematode juveniles (Back et al., 2002). Plant parasitic nematodes make plants more accessible for soilborne fungi through their invasion channels, and by causing other types of mechanical damage. An example of such mechanical damage caused by sedentary nematodes is the rupturing of the cortex by swollen females of cyst or root-knot nematodes. Soilborne pathogens may benefit from such cracks and easily infect the inner tissues. In peanut plants, R. solani has been shown to infect via the cracks made by Meloidogyne hapla females (Back et al., 2002). In potato, interactions involving the wilt fungus Verticillium dahliae and the potato cyst nematode Globodera pallida have been found. This interaction is affected by the resistance of the cultivar to fungal pathogens and the time of nematode attack. The fungus and infective nematode juveniles have to be present in the soil at the same time in order for the interaction to be successful. If the fungus is not present at the time of nematode invasion, the nematode tracts become lignified and sealed (Storey & Evans, 1987; Back et al., 2002).

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The speed of lignification of nematode feeding tracts varies between different potato cultivars. Maris Peer and Maris Anchor are affected by fungi/nematode interaction when both pathogens are present simultaneously, since the invasions tracts became lignified more or less directly after penetration. In the cultivar Pentland Javelin, lignification does not occur and the fungus can benefit from the invasion tracts during a longer period of time (Storey & Evans, 1987; Back et al., 2002).

Mechanisms behind the modification theory

It has been shown by anatomical studies that fungal infections are not always facilitated by wounding caused by nematodes. In cotton, Fusarium oxysporum is not able to infect via entry tracts of Meloidogyne incognita. If the nematode invades the plant three to four weeks prior to the fungus, the symptoms become severe. Wounding theory cannot explain the interactions in this case, since it is more likely that the nematodes induce some kind of physiological changes in plants. This and other results gave rise to the modification theory. The mechanisms behind the modification theory suggest a complex interaction, since plant induced modification is often both nematode- and plant species-specific and the fungus must be present during a specific event of the nematode life cycle in order for an interaction to take place (Taylor, 1990;Back et al., 2002). The specialised feeding sites close to the vascular bundle induced by sedentary nematodes develop into giant cells or syncytia. Giant cells are specialised cells with high metabolic activity from which the female nematodes feed. The total protein, amino acid, lipid and DNA levels are much higher in such cells compared to uninfected cells. Soilborne pathogenic fungi have been found to colonise giant cells. The nutrient-rich cell contents make a perfect substrate for fungi infection. From colonised giant cells, the hyphae can spread and infect surrounding cortical vascular tissues. In several crops Rhizoctonia solani has been found to develop sclerotia on encrusted galls of M. incognita. The giant cells become colonised after the hyphae have grown out of the sclerotia, penetrated the cortex and extended intercellularly to the giant cells (Taylor, 1990; Back et al., 2002). Pratylenchus spp. have been shown to induce changes in nutritional and hormonal balance in host plants. The modifications induced by Pratylenchus spp. in potato influence the host physiology, making the host either resistant or susceptible to Verticillium (Riedel et al., 1985). It is still not fully understood whether nematode induced physiological modifications are systemic or local. Some evidence points towards a systemic change, whereas other findings indicate a local change in plant physiology (Parmeter, 1970; Back et al., 2002).

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The role of root exudates

Plant parasitic nematodes and soilborne fungi are both attracted by root exudates (Taylor, 1990; Bowers et al., 1996; Back et al., 2002). The chemical composition and the quantity of root exudate may be influenced by nematode infestation. A change in the chemical composition may affect the behaviour and development of fungal pathogens in the rhizosphere. The role of root exudate changes in the synergistic interaction is still not clear, but it might be one of the initial mechanisms underlying synergistic interactions (Taylor, 1990). R. solani has been proven to aggregate around root-knot galls on many plants. This may be explained by an increased leakage of metabolites from the galls of Meloidogyne, which attracts the hyphae of the fungus. R. solani is not the only soilborne pathogen attracted to galls of Meloidogyne spp. Fusarium oxysporum f.sp. lycopersici has been shown to be attracted to the galls of M. javanica (Back et al., 2002).

Complexity of interaction

Although a disease complex often involves only two pathogenic organisms, several further species may be involved. In New Zealand, researchers have found a disease complex involving a number of different fungi and nematodes that cause severe damage in white clover pastures (Back et al., 2002; Zahid et al., 2002). Another example of a disease complex consisting of more than two organisms has been found in potatoes (Scholte & Jacob, 1989). It contains Verticillium dahliae, Rhizoctonia solani and either Meloidogyne spp. or Pratylenchus neglectus. The reasons for an interaction of this kind are difficult to explain, but it has been suggested that the combination of R. solani and V. dahliae increases the aggressiveness of the fungal pathogens. The role of the nematodes and the mechanisms behind this kind of interaction are still unclear (Scholte & Jacob, 1989).

Negative effects on nematode reproduction

Fungi-nematode interactions may not be favourable to nematodes, because plants infected by a disease complex are more prone to early senescence and death. This has an unfavourable impact on the reproduction of nematodes. Fungi involved in a disease complex may affect nematode reproduction negatively by colonising giant cells and thereby disturbing their function (Back et al., 2002; Zahid et al., 2002). Another factor that can affect nematode reproduction is that pathogenic fungi may produce metabolites that have a inhibitory effect on the hatching of nematode eggs (Zahid et al., 2002). The decline in nematode populations involved in a disease complex with fungi may also be explained by competition for nutrients and root space between the two organisms (Back et al., 2002).

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Species specificity

Nematode-fungi interactions have in some cases been shown to be species-specific. It is thought that fungal genotype and nematode species can affect the establishment and aggressiveness of a disease complex. Species specificity may be explained by the local prerequisites for survival at the location of the interaction (Riedel et al., 1985; Bowers et al., 1996; Back et al., 2002).

Effectiveness of resistance against pathogens in host plants

Reaction of a certain pathogen to a defined plant resistance may alter when that pathogen forms a part of a disease complex. In some cases the nematode invasion may give rise to plant damage, facilitate pathogen penetration and make the plant susceptible to fungal infection (Back et al., 2002). Resistance effectiveness may also be a matter of physiological alterations that make the plants more susceptible to the pathogen, as has been demonstrated in split-root experiments (Back et al., 2002). The genetic background is of great importance when it comes to the effectiveness of resistance. Polygenic resistance in plants is easier to overcome (Chang et al., 1997; Sugawara et al., 1997; Back et al., 2002).

Effect of physical and chemical soil factors

Abiotic factors, such as soil moisture and soil temperature, have been proven to influence the development and severity of fungus-nematode interactions (Taylor, 1990; Sugawara et al., 1997; Scherm et al., 1998). Abiotic factors and host-pathogen interactions play a major role in the infection and development of disease complexes (Back et al., 2002). Temperature has been shown to influence nematode-fungi interactions, since it may affect one or all the organisms involved in the disease-complex (Sugawara et al., 1997; Scherm et al., 1998). Soil factors such as mineral particles, pH, cation exchange capacity, humus content, water, air, etc. are of great importance for infection and the development of interactions (Back et al., 2002; Agu, 2002).

Disease complexes involving PPN and R. solani in potato

In England, Back et al. (2002) have found that disease complexes involving R. solani and plant parasitic nematodes increase the severity of damping-off, stem canker and black scurf in potato. In Denmark it is believed that Trichodorus spp. might form a disease complex with R. solani (Nielsen et al., 2004). A synergistic interaction between common scab on potato and Pratylenchus spp. has been proven in Norway (Holgado et al., 1998). This has given rise to the suspicion that effects of interaction between R. solani and plant parasitic nematodes can lie behind some of the problems that producers have encountered during the last decade.

21

Materials and Methods

Soil samples from 41 potato fields in south-west Sweden were collected with an auger during the summer of 2005 in the growing potato crop. Roots for a nematode count and determination of mycelia coverage were collected by digging out four randomly selected plants in each field.

Quantification of Rhizoctonia solani in the field

In the field studies, the potato plants were scored for Rhizoctonia solani. This was done to get a measure of the amount of infection in the field. The quantification of R. solani was performed according to a method developed and commonly used in Sweden (Lars Wiik, pers. comm. 2005).

Figure 5. Healthy plant vs. plant severely infected by R. solani. © Lars Wiik The method involves visual grading of symptoms typical for the fungus, such as root and stolon rot appearing as reddish brown lesions. The following scoring scale from 0.0 to 5.0 was used: 0.0= no symptoms; 0.1= small symptoms of rot on the roots; 1.0= few, shallow lesions on occasional stems and stolons; 2.0= a well-defined shallow necrosis that does not girdle on most of the stems and stolons, fine roots destroyed; 3.0= one quarter to one half of the stems and stolons are girdled by necroses are girdled by lesions,;

22

deep wounds on some of the stems; 5.0= the plant is dead or all root parts attacked and destroyed, (See Figure 5). Four random locations were chosen in each field and at each location the visual symptoms of R. solani were quantified on 10 plants.

Quantification of Rhizoctonia solani on tubers from closed containers

To determine the presence of R. solani in the soil samples collected, the closed container method was used. A portion of 250 g of soil from each sample was placed in a plastic container and 37 ml of water was added. A micropropagated potato tuber was then placed in each container. The containers were closed and left in a dark chamber at 18-20 oC for 7-10 days. Then the root development was measured. The containers were monitored regularly to avoid problems with excess or lack of water. The stems, roots and micropropagated tubers were washed after 3.5 months and the R. solani symptoms were quantified using the same method and scoring scale as in the field. The roots were stained, the number of endoparasitic nematodes in the roots was calculated and the amount of hyphae on the roots was quantified.

Nematode extractions

Free living nematodes

From each soil sample, a representative 250 g subsample was taken and processed using a semiautomatic elutriator for extraction of free living nematodes according to Seinhorst (1962). The extracted nematodes were then identified to genera and counted (Appendix I).

Cyst nematodes

The collected soil samples were manually disaggregated and mixed carefully before 500 g subsamples were taken and extracted using a semiautomatic elutriator for cyst nematodes as recommended by Seinhorst (1964). Cysts were furthermore separated from organic debris using a method developed by Andersson (1970). Eggs and juveniles were freed from cysts using a tissue grinder and placed in counting dishes. Only vigorous juveniles and eggs of potato cysts nematodes were counted (Appendix I).

Staining of nematodes in roots

About 2 g of fine roots were chopped off randomly from the roots of four collected plants from each field and washed under tapwater in a coarse sieve. The washed roots were dried using absorbent paper. Infective nematodes were made visible by staining roots with acid fuchsine according to Byrd et al., (1983) and then counted. The presence of R. solani hyphae on the stained roots was also recorded and quantified.

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Isolation of free living nematodes from plant material

From a field with especially interesting symptoms, such as coiled and thickened roots, 10 potato plants were collected. Five plants had a normal appearance above ground, based on the plant length, number of stems per plant, etc., and the other five did not. In order to determine the cause of symptoms, the presence of plant parasitic nematodes in the roots was examined. The roots from each plant were washed, chopped into smaller pieces and processed according to the mistifier extraction technique (Seinhorst, 1950) (Appendix I).

Questionnaire

Commercial producers were asked to provide background information about the field in a questionnaire. Questions concerned origin and status of seed potatoes, seed treatment, previous R. solani problems in the field, amount of black scurf on the tubers, crop rotation, irrigation and application of animal manure (Appendix III).

Nutritional status and soil type

Soil samples were sent AnalyCen in Kristianstad, in order to classify the soil type and nutritional status.

Statistical methods

The statistical calculations were carried out using the computer programme minitab (MINITAB, 2006)

Correlations

The correlation coefficient was calculated for all factors tested.

Analysis of variance

One-way ANOVA and Turkey’s honestly significant difference test were used to compare means of different factors. The different factors were divided into 2-4 groups depending on the variation of the values. The groups were pair-wise compared to each other.

The asterisk system

The significance is given by the P-value expressed in accordance with the asterisk system. The asterisk system from Olsson & Engstrand, 2002: If p > 0.05 the result is given as “not significant”, ns If 0.01 <p≤ 0.05 the result is “significant at the 5% level”, * If 0.001 <p≤ 0.01 the result is “significant at the 1% level”, ** If p≤ 0.001 the result is “significant at the 0.1% level”, ***

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Results

All results for the different factors can be seen in the raw data table in Appendix IV. A table explaining the meaning of the different factors can be found in Appendix II.

Correlations

The correlation data in Tables 2 to 13 below show how different factors correlate with each other in either a positive or negative manner. The number of fields, n, for the different factors varied from 39 to 41. There is a relationship between two variables if large values of one of the variables is likely to be corresponded by large values of the other variable (positive correlation) or if small values of one of the variables is likely to be corresponded by small values of the other variable (negative correlation). Some of the variables with a significant correlation in table 2 can be used as examples for how to interpret the correlation of between different factors. For example the significant negative correlation of -0.391(P< 0.01) between previous stem canker and black scurf problems and the yield of the field was due to that fields with no or few previous stem canker and black scurf problems had usually a high yield, which is why the correlation value is closer to zero than to one. Fields with few previous stem canker and black scurf problems had a low yield, which explains why there is a negative correlation between the two variables. The variables stem canker symptoms (0.326, P< 0.05), black scurf on tubers (0.779, P< 0.001), and ecto PPN (0.315, P< 0.05) in the field had a significant positive correlation with the variable previous stem canker and black scurf problems. Fields with previous stem canker and black scurf problems developed more stem canker symptoms, black scurf on tubers, and had a greater population of ecto PPN in the field.

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Table 2. Correlation for previous R. solani problems vs. various factors Previous stem canker and/ or black scurf vs.

Pearson correlation coefficients and significance

Stem canker symptoms, field 0.326* R. solani mycelia on roots, field 0.092 Black scurf on tubers, field 0.779*** PCN population, field -0.073 Pratylenchus spp., field 0.074 Ecto PPN, field 0.315* Endo PPN in plant roots, field -0.123 Yield, field -0.391** Stem canker symptoms, container 0.108 R. solani mycelia on roots, container 0.079 Black scurf on micropropagated tubers, container -0.244 Black scurf on roots, container -0.194 Endo PPN in the plant roots, container -0.103 Crop rotation, field -0.087 Table 3. Correlation for stem canker symptoms in the field vs. various factors Stem canker symptoms, field vs. Pearson correlation coefficients

and significance R. solani mycelia on roots, field 0.226 Black scurf on tubers, field 0.434** PCN population, field 0.095 Pratylenchus spp., field 0.020 Ecto PPN, field 0.073 Endo PPN in plant roots, field 0.165 Yield, field -0.294 Stem canker symptoms, container 0.384* R. solani mycelia on roots, container 0.538*** Black scurf on micropropagated tubers, container 0.304 Black scurf on roots, container 0.185 Endo PPN in the plant roots, container -0.055 Crop rotation, field -0.112

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Table 4. Correlation for R. solani mycelia on stained roots from the field vs. various factors R. solani mycelia on roots, field vs. Pearson correlation coefficients

and significance Black scurf on tubers, field 0.059 PCN population, field 0.030 Pratylenchus spp., field -0.016 Ecto PPN, field -0.280 Endo PPN in plant roots, field -0.204 Yield, field 0.091 Stem canker symptoms, container 0.014 R. solani mycelia on roots, container -0.115 Black scurf on micropropagated tubers, container -0.004 Black scurf on roots, container 0.009 Endo PPN in the plant roots, container 0.357* Table 5. Correlation for black scurf on harvested tubers vs. various factors Black scurf on tubers, field vs. Pearson correlation coefficients

and significance

PCN population, field -0.195

Pratylenchus spp., field -0.008

Ecto PPN, field 0.235

Endo PPN in plant roots, field -0.057

Yield, field -0.399**

Stem canker symptoms, container 0.181

R. solani mycelia on roots, container 0.169

Black scurf on micropropagated tubers, container -0.154

Black scurf on roots, container -0.181

Endo PPN in the plant roots, container -0.068

Crop rotation, field -0.019

27

Table 6. Correlation for the PCN population in the field vs. various factors PCN population, field Pearson correlation coefficients

and significance Pratylenchus spp., field -0.099 Ecto PPN, field -0.099 Yield, field -0.164 Stem canker symptoms, container -0.094 R. solani mycelia on roots, container -0.022 Black scurf on micropropagated tubers, container -0.014 Black scurf on roots, container 0.414** Endo PPN in the plant roots, container 0.028 Table 7. Correlation for the Pratylenchus spp. population in the field vs. various factors Pratylenchus spp. Pearson correlation coefficients

and significance Ecto PPN, field 0.084 Yield, field 0.135 Stem canker symptoms, container -0.027 R. solani mycelia on roots, container 0.097 Black scurf on micropropagated tubers, container -0.132 Black scurf on roots, container -0.104 Table 8. Correlation for the population of ectoparasitic nematodes in the field vs. various factors Ecto PPN Pearson correlation coefficients

and significance Endo PPN in plant roots, field -0.016 Yield, field -0.165 Stem canker symptoms, container -0.185 R. solani mycelia on roots, container -0.056 Black scurf on micropropagated tubers, container -0.058 Black scurf on roots, container -0.162 Endo PPN in the plant roots, container -0.037

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Table 9. Correlation for endoparasitic nematodes in roots from the field vs. various factors Endo PPN in plant roots, field Pearson correlation coefficients

and significance Yield, field -0.193 Stem canker symptoms, container -0.159 R. solani mycelia on roots, container 0.318* Black scurf on micropropagated tubers, container 0.304 Black scurf on roots, container 0.206 Endo PPN in the plant roots, container -0.068 Table 10. Correlation for stem canker symptoms in the container vs. various factors Stem canker symptoms, container Pearson correlation coefficients

and significance R. solani mycelia on roots, container 0.168 Black scurf on micropropagated tubers, container 0.109 Black scurf on roots, container 0.075 Endo PPN in the plant roots, container -0.109 Table 11 Correlation for R. solani mycelia on roots from closed container vs. various factors R. solani mycelia on roots, container vs. Pearson correlation coefficients

and significance Black scurf on micropropagated tubers, container 0.352* Black scurf on roots, container -0.118 Endo PPN in the plant roots, container -0.429** Table 12. Correlation for black scurf on micropropagated tubers from closed containers vs. various factors Black scurf on micropropagated tubers, container vs.

Pearson correlation coefficients and significance

Black scurf on roots, container 0.398** Endo PPN in the plant roots, container -0.127 Table 13. Correlation for sclerotia on roots in the closed containers vs. endoparasitic nematodes in the closed container. Black scurf on roots, container vs. Pearson correlation coefficients

and significance Endo PPN in the plant roots, container 0.076

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Linkage between different correlation coefficients

The dendrogram in Figure 6 shows the linkage and similarity between the correlation coefficients of the different factors. The dendrogram can be divided into 12 steps at which one or more factors are joined into clusters. The dendrogram is thus a schematic representation of the correlations.

Variables

Sim

ilari

ty

Pratylenchus/25

0g so

il

Endo PP

N conta

iner

Mycelia fie

ld

Ecto PPN

Endo PP

N

Sclerotia r

oots conta

ine

Scler

otia tubers c

ontainer

PCN pop.

field

Stem

canker co

ntaine

r

Mycelia co

ntaine

r

R. solan

i field

Black

scurf

on ha

rvest

Earlie

r R. prob

lems

55.30

70.20

85.10

100.00

Dendrogram with Single Linkage and Correlation Coefficient Distance

Figure 6. Dendrogram with Single Linkage and Correlation Coefficient Distance.

Analysis of variance

Fungi

The use of seed treatment significantly reduced the R. solani symptoms during the growth season. Plants from untreated seed potatoes had a higher degree of R. solani symptoms (1.12±0.7) in the field compared to plants from treated seed potatoes (0.63±0.59) (P< 0.05). There was no significant difference in the degree of black scurf on new tubers from untreated seed potatoes compared with new tubers from treated.

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The R. solani disease history of a field was important when it came to the development of black scurf on the tubers (Figure 7). More black scurf was found in fields with previous problems with stem canker and/or black scurf. The quantity of black scurf on the tubers generally increased with increasing previous R. solani problems (P< 0.001).

Black scurf on tubers vs stem canker in the field

00.5

11.5

22.5

33.5

0 0.5 1 1.5 2 2.5 3 3.5

Black scurf on tubers, 0-4

Stem

can

ker i

n th

e fie

ld,

0.0-

5.0

Figure 7. The figure shows the relation of the mean values of the two variables. The trend line is a polynomial line of the second order. The development of black scurf was not only affected by the history of R. solani disease in the field, but also by the severity of the R. solani symptoms in the field during the growth season. Fields with a low degree of R. solani symptoms in the field developed no or a very limited amount of black scurf on the tubers, while fields with the highest degree (2.0-3.0) of R. solani symptoms developed considerably more black scurf on the tubers (P< 0.05). No differences in development of black scurf on the tubers could be seen when fields with low and intermediate levels of stem canker symptoms and fields with intermediate and high levels of stem canker symptoms were compared. The fungus survived in the sampled soils, infected the roots and could be seen as mycelia on the stained roots in the closed containers. The quantity of mycelia on the roots increased with increasing degree of stem canker symptoms in the field. For soils developing a low amount of mycelia on the roots in the containers, no differences in the degree of stem canker symptoms could be seen in the field (P< 0.001). Stem canker symptoms developed on the coleoptiles and roots of the micropropagated tubers in the closed container. The extent of the stem canker symptoms could be related to the severity of R. solani symptoms in the field. The stem canker symptoms did not differ in severity for fields with low degrees of stem canker symptoms in the field, but fields with high degrees of stem canker symptoms had significantly more stem canker in comparison to the others (P< 0.01).

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The presence and extent of mycelia on the roots was affected by the degree of black scurf on the micropropagated tubers in the containers. Soils supporting high sclerotia development on the tubers had comparably more mycelia on the roots than soils with lower degrees of sclerotia on the tubers (P< 0.01). A correlation between the development of sclerotia on micropropagated tubers and on roots could be seen in the containers. Containers in which there was no development of sclerotia on the roots had a lower degree of sclerotia on the micropropagated tubers than containers with sclerotia development on the roots (P< 0.05). The infection level of R. solani in the field, quantified by the degree of symptoms, had an effect on the development of sclerotia on the roots in the containers. The degree of sclerotia on the roots did not differ for varying degrees of R. solani symptoms in most of the cases. It could however be noticed that symptoms of a higher degree caused a greater sclerotia growth on the roots than in soils with a lower degree of symptoms (P< 0.05). The degree of previous R. solani problems did not significantly affect the shifting levels of R. solani symptoms in the field, mycelia on stained roots, stem canker on coleoptiles in containers, mycelia on roots in container, sclerotia on tubers in containers or sclerotia on roots in containers found in the different soils. Furthermore, no significant differences in severity could be observed for: mycelia on stained roots, stem canker on coleoptiles in containers, mycelia on roots in containers, sclerotia on tubers in containers or sclerotia roots in containers for soils with different degrees of R. solani symptoms in the field. The degree of mycelia coverage on roots from the field did not significantly vary with different degrees of black scurf on harvested tubers, stem canker on coleoptiles in containers, mycelia on roots in containers, sclerotia on tubers in containers or sclerotia on roots in containers. The quantity of black scurf on harvested tubers did not cause significant corresponding variations in severity of stem canker on coleoptiles in containers, mycelia on roots in containers, sclerotia on tubers in containers and sclerotia on stems and roots in containers, when comparing black scurf with black scurf plus the other factors. There were no significant correlations between the severity of stem canker on coleoptiles in containers, mycelia on roots in containers, sclerotia on tubers in containers and sclerotia on stems and roots in containers.

Fungi and nematode

There were indications of Pratylenchus spp. and R. solani interaction (Figure 8). Intermediate populations of Pratylenchus spp. appeared to favour development of stem canker symptoms in the field. When different population sizes of Pratylenchus spp. were compared, it became evident that populations of 50-99 Pratylenchus spp./250 g soil favoured stem canker symptoms to the highest degree, while other population sizes seemed to influence the fungus development equally (P< 0.01). The amounts of mycelia on the roots in the closed containers were also affected by the population of Pratylenchus spp.

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Mycelial growth was most favoured by intermediate populations of Pratylenchus spp. (51-100/250 g soil), whereas other population densities appeared to affect mycelial growth equally (P< 0.05).

Pratylenchus spp.vs Stem canker symptoms

-0.50

0.51

1.52

2.53

3.5

0 50 100 150 200 250 300

Pratylenchus spp./250g soil

Stem

can

ker

sym

ptom

s in

th

e fie

ld, 0

.0-5

.0

Figure 8. The figure shows the relation of the mean values of the two variables. The trend line is a polynomial line of the second order. In the containers, the presence of endoparasitic nematodes had a negative impact on the development of mycelia on the roots. Roots without any endoparasitic nematodes inside were more covered with mycelia than roots containing endoparasitic nematodes (P< 0.01). Furthermore, the population density of endoparasitic nematodes in the roots was affected by the degree of sclerotia on the tubers in the same container. It could be seen that containers with a low level of sclerotia on the tubers had a greater number of endo-parasitic nematodes in the roots compared to containers that had developed a higher level of sclerotia on the tubers (P< 0.05). The population sizes of PCN in the field, ectoparasitic nematodes, Pratylenchus spp. and other endoparasitic species and endoparasitic nematodes in the roots of the container did not show significant correlations with different levels of previous R. solani problems or mycelia on roots from the field. Fields with varying levels of stem canker symptoms did not differ significantly in population sizes of PCN, ectoparasitic and endoparasitic nematodes or endoparasitic nematodes in the roots of the containers. Different levels of sclerotia on tubers and sclerotia on roots in the containers were found to be uninfluenced by the PCN populations in the field, Pratylenchus spp., hatched PCN, ectoparasitic and endoparasitic nematodes in the field, and endoparasitic nematodes in the roots from the containers. These results were however not significant. The mycelia coverage on the roots was also not significantly influenced by PPN, except for Pratylenchus spp., which, as mentioned earlier, had an influence on the coverage of mycelia.

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Tuber yield

Stem canker symptoms vs Yield

01020304050607080

0 0.5 1 1.5 2 2.5 3 3.5

Stem canker symptoms in the field, 0.0-5.0

Yiel

d, to

n/ha

Figure 9. The figure shows the relation of the mean values of the two variables. The trend line is a polynomial line of the second order. The size of the potato yield reflected the R. solani infection level in the field (Figure 9). Soils with a low yield had more R. solani symptoms in the field than fields with normal to high yields, but there was no difference in the degree of R. solani symptoms between fields with a normal yield and high yielding fields (P< 0.01). The presence and extent of black scurf on the tubers may also have affected the size of the tuber yield. Fields with a higher degree of black scurf on the tubers had lower yields than fields without black scurf on the tubers (P< 0.05). No significant differences in yield size could be observed for varying levels of previous R. solani problems or mycelial coverage on the roots from the field.

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Pratylenchus spp. vs Yield

01020304050607080

0 50 100 150 200 250 300

Pratylenchus spp. No/250g soil

Yiel

d, to

n/ha

Figure 10. The figure shows the relation of the mean values of the two variables. The trend line is a polynomial line of the second order. When the population of Pratylenchus spp. exceeded certain levels, reductions in tuber yield could be seen (Figure 10). Fields with a population of 150-250 Pratylenchus spp./250 g soil had a significantly lower yield than fields with a population of 50-99 Pratylenchus spp./250 g soil. For Pratylenchus spp. populations varying from 50 and 149/250 g soil, no correlation between population size and yield reduction could be seen (P< 0.05). There were no significant differences in tuber yield from fields with different population densities of PCN in the field, endoparasitic nematodes in stained roots, ectoparasitic nematodes and endoparasitic nematodes in roots from the containers. With crop rotations of four years, higher yields were achieved than with crop rotations of one to three years (P< 0.01). Fields irrigated with more than 50 mm of water had higher yields than fields irrigated with 0-49 mm (P< 0.001).

Soil factors

Soil factors such as nutritional status of the soil, soil type and management practices had some impact on the disease development of R. solani in the field. The nutritional status of the soil appeared to be of less importance for the development of R. solani, since no significant difference in degree of R. solani symptoms in the field could be seen for varying levels of pH, available soil phosphorous, available potassium or potassium class, available magnesium, potassium/magnesium ratio or available calcium. However, both available and bound phosphorus were important for the development of R. solani symptoms in the field. Soils of P-class 3 had more symptoms compared to those with P-class 4 or 5 (P< 0.01).

35

The soil content of sand and silt did not appear to affect the development of R. solani symptoms in the field. However, the severity of R. solani symptoms found in the field did appear to be affected by the clay content of the soil (P< 0.05). Differences in the degree of stem canker symptoms could only be seen between fields with 10-14% and 15-24% clay. Soils with 10-14% clay had fewer symptoms than soils with 15-24% clay. Management practices such as the length of the crop rotation and the use of a catch crop in the crop rotation system did not significantly affect the level of previous R. solani problems in the field, R. solani symptoms in the field, mycelia stained roots, black scurf, stem canker in containers, mycelia in containers, sclerotia on tubers in containers and sclerotia on roots in containers. Irrigation applied in the fields did not significantly affect the development of R. solani symptoms. The size of the population of PCN in the field, Pratylenchus spp., endoparasitic nematodes in stained roots, ectoparasitic nematodes and endoparasitic nematodes in roots from the containers were not significantly affected by differences in the crop rotation lengths or whether irrigation was applied in the field.

36

Discussion

Field studies

External growth factors can affect the establishment of a disease complex greatly. Since this problem arises in the field, it was considered important to study the action of the disease complex and mechanisms behind it in vivo.

Choice of fields

Forty-one potato fields were included in the study and many different parameters were studied. Had more locations been included, data would have been more reliable and exceptional values would not have had the same great impact as they did here. However, time was a limiting factor and a total of 41 fields were considered the maximum possible in practice. Many of the results obtained were contradictory, mainly due to a few exceptional values and the lack of sufficient data for some of the categories. This may have led to somewhat misleading results, so it is difficult to draw any reliable conclusions. However despite the lack of data, the study gives an overview of the situation and indications of interactions between PPN and R. solani. A more thorough study examining the situation in a greater number of locations is needed in order to prove whether interactions between PPN and R. solani are common in Swedish potato fields. Most of the fields were chosen due to their known infection status with respect to PCN. Fields with and without PCN populations were chosen in order to allow possible synergistic interactions between PCN and R. solani in potato to be investigated. Unfortunately few fields had a PPN population exceeding the damage threshold. This was also the case for the free living nematodes (Pratylenchus spp. and ectoparasitic nematodes). The fact that the PPN populations were so low might be one of the reasons why it was so difficult to prove PPN-R. solani interactions in this study. Bowers et al. (1996) found that low populations of Pratylenchus penetrans and Verticillium dahliae did not cause any damage individually, but acted together and caused severe disease symptoms and great yield losses. It is possible that low nematode populations can cause great problems in Sweden and therefore they might be important if they act together with another pathogen. Some of the fields were part of other studies in which e.g. different pesticides, applied as liquid tuber treatment, were tested for their effects against R. solani. Other fields were included due to emergence problems and severe R. solani symptoms. The study area extended from south-east Scania to south-west and northern Scania, southern Halland and southern Dalsland. Costal areas on the Swedish west coast, belonging to Swedish climate zones 1-3, were mainly covered. The summer temperature conditions of growth zone 1-3 are within the optimal range for R. solani (Parmeter, 1970; Olofsson et al., 1996; Agrios 1997) and plant parasitic nematodes (Palm et al., 1968; Agrios, 1997) (Appendix V).

37

Of the 41 locations studied, 19 were treated with fungicides active against R. solani. This might have interfered with the results, since the compound used reduces the inoculum of R. solani on the tubers significantly. This might have had an effect on the possible interaction with nematodes, due to the lower infection potential of the fungus.

Influence of cultivar

A number of different potato cultivars were grown on the fields included in this study. These were Asterix, Bintje, Ceraster, Ditta, Eloge, Faxe, Frieslander, Fulva, GMO-seedtubers, King Edward, Kardal, Kuras, Producent, Rocket, Satina, Saturna, Secura, Ukama and Victoria. This mixture of different cultivars might have influenced the results of this study, since:

• There are different criteria for the quantity of top yields for the different varieties. • Some of the cultivars are produced and harvested as early potatoes. • The cultivars differ to some extent in resistance towards R. solani (Hallqvist,

1987) and nematodes.

Materials and Methods

The methods used for extraction of PCN and free living nematodes are well-known and well-proven and can therefore be regarded as reliable. The use of the closed container method, which is normally used for studies of reproduction of PCN, is new. Ulla Bång (pers. comm. 2006) uses another method to quantify the amount of R. solani infection in the soil in which micropropagated potato tubers are cultivated in pots with sample soil. In the greenhouse, the climatic conditions can sometimes be difficult to control, and with the closed container the environment is probably kept more stable. The micropropagated tubers used are not sterilised since they are guaranteed free from disease. Normal tubers could also be used, but there is always a risk that they will carry some disease even if they have been surface sterilised using diluted hypochlorite. There is also a risk of the meristematic tissues on the tubers being damaged by the sterilisation.

38

Figure 11, 12. Potato plants infected by Erwinia spp. and Verticillium spp. © Lars Wiik Visual quantification of R. solani in the field can in some cases be somewhat unreliable since confusion with other soilborne fungi symptoms is possible. Figure 11 and 12 are examples of potato diseases, occurring in the field that can cause symptoms somewhat similar to those caused by R. solani. However, this is not very likely, since R. solani causes very typical brown lesions and black scurf symptoms. The method of quantifying R. solani symptoms in the field is commonly used in Sweden and the readings were always carried out together with a person with experience of quantifying R. solani symptoms in the field. In this preliminary study a questionnaire answered by growers gave some background information about the production history, but information about previous nematode populations and the degree of previous stem canker symptoms and black scurf were lacking. Data on previous PPN and previous R. solani infections would have made it possible to find out more about how the organisms interrelate.

R. solani

The amount of soilborne inoculum of R. solani appeared to be important for the development and severity of R. solani disease. The theory that soilborne inoculum gives rise to black scurf on tubers (Bång, 2005) seems to be true according to the results of the present study. There was a positive correlation between previous R. solani problems and black scurf (Table 2), where the degree of black scurf varied with the degree of previous R. solani problems. The fact that there was no difference in black scurf between untreated and treated seed potatoes also supports the theory that the inoculum is soilborne. Fields where there had been problems

39

with R. solani in previous years developed black scurf on the tuber. The severity was determined by the degree of earlier problems. The development of black scurf on the tubers in the field was also affected by the symptoms of R. solani seen in the field earlier in the season. A plant that is heavily infected with R. solani develops black scurf later during the season when the metabolic activity of the plant changes due to maturation (Parmeter 1970; Olofsson et al., 1996). The inconsistent results regarding the relationship between R. solani and the development of black scurf in the field may, except for exceptional values, have been due to the fact that early potatoes were produced on some of the fields and that treated seed was used in almost half the fields studied. The development of sclerotia in the containers was also mainly on the tubers. The development of black scurf in the closed containers was negatively affected by the degree of previous R. solani problems in the field, which was probably due to the soil having been collected before new black scurf had developed. The inoculum source in the containers was consequently both resting hyphae and actively growing hyphae. The untreated tubers had a higher degree of R. solani symptoms in the field compared to treated tubers. This fits well with the theory that inoculum of R. solani on the tubers gives rise to damping-off (Bång, 2005). The influence of R. solani symptoms on the different R. solani parameters studied in the containers can be explained by the survival of the hyphae in the sampled soil. The fungus might lose some of its viability during the storage, which might be the reason for some of the irregular results. Previous R. solani problems did not appear to affect disease development in the containers, probably due to the changed amount of R. solani inoculum in the soil as a result of the influence of various factors during the growth period prior to the collection of soil. The relationship between the different R. solani factors in the field and in the containers can be explained by the life cycle of the fungus and the disease development in potatoes (Parmeter, 1970; Olofsson et al., 1996). The degree of mycelia found on the stained roots from the field reflected the degree of R. solani symptoms found in the field at the same time. The R. solani infection giving rise to the symptoms survived in the sampled soil and infected the coleoptiles, covered the roots and coleoptiles, and developed into sclerotia on tubers, stems and roots in the closed containers, which is why there was a positive correlation between these factors. The degree of R. solani symptoms in the field affected the amount of mycelia found on the roots in the closed containers, since the hyphae causing the symptoms survived in the soil during storage and then infected the roots in the closed containers. The mycelia formed infection structures and infected the roots and coleoptiles in the closed containers and therefore the R. solani symptoms found in the closed containers were also affected by the degree of symptoms in the field. The amount of mycelia found on the potato plants in the closed containers affected the degree of sclerotia on the tubers in the containers, since the mycelia develop into sclerotia on the tubers when the energy of the tubers changes, which cause the fungus to initiate the production of survival structures (Parmeter, 1970; Olofsson et al., 1996). This also caused the development of sclerotia on the roots and coleoptiles in the containers, but there was only development of sclerotia on the coleoptiles and roots when there were a lot of sclerotia on the tubers.

40

The inoculum of R. solani in the closed containers derived from the degree of R. solani infection in the field, and the degree of sclerotia on the tubers of the containers was therefore affected by the degree of R. solani symptoms in the field when the soil was sampled. A healthy crop rotation appears to be important for the quantity of soilborne inoculum, as was indicated by the negative correlation between previous R. solani problems, R. solani symptoms in the field and black scurf on the tubers.

Nematodes

The population of PCN affected the populations of Pratylenchus spp. and ectoparasitic nematodes negatively, probably because they compete for root space and nutrition. The population of Pratylenchus appeared to be able to coexist with ectoparasitic nematodes without negative effects on the population size. The negative correlation between endoparasitic nematodes in the roots from the field and roots from the closed containers might be explained by the fact that the most endo-parasitic nematodes in fields were already inside the potato roots during the time of soil collection.

Interactions

Interactions between PCN and R. solani were difficult to prove in this study, since some of the varieties included in the study were resistant to PCN. In soybean, the severity of brown stem rot increases in the presence of Heterodera glycines, but this enhancing effect of the nematode is not observed when the H. glycines resistant soybean cultivar Peking is cultivated (Sugawara et al., 1997). The same effect of PCN resistance in disease complexes might also occur in potatoes. It has also been shown that nematodes can assist the fungus by making the fungi resistance of the potato plant less effective. For example, the natural defence of the potato varieties Maris Anchor, Maris Peer towards V. dahliae becomes less effective in the presence of PCN juveniles, where the fungus has been seen to follow in the digestion tracts of the nematodes and infect the nearby tissues (Storey & Evans, 1987). However, it appears that there is a synergistic interaction between Pratylenchus spp. and the degree of R. solani symptoms in the field. A synergistic interaction seems to occur in soils with a population density of 50-99 per 250 ml soil. Large populations of nematodes might interfere with the growth of mycelia on the root surfaces (Back et al., 2002; Zahid et al., 2002). The mechanisms behind the interaction between R. solani and Pratylenchus sp in potato might be due either due to wounding by the nematode or to modifications caused by Pratylenchus influencing the potato plant physiology. Several interactions between Pratylenchus and various fungi on different crops have been recorded and explained by either the wounding theory or the modification theory (Riedel et al., 1985; Back et al., 2002). Bowers et al. (1996) studied the mechanisms behind the disease complex of P. penetrans and V. dahliae in potato. They speculated whether the nematode causing root damage that resulted in increased root growth and thus increased exudation might stimulate higher amounts of roots to come in contact with inoculum. The exact mechanism between an

41

interaction between R. solani and Pratylenchus sp. on potatoes needs to be studied in greater detail. The populations of endoparasitic nematodes appeared to have a negative effect on the development of fungal sclerotia on tubers when the species occurred together in the containers. This might be due to the competition for nutrients and root space between the two organisms (Back et al., 2002). According to the literature, fungi-nematode interactions sometimes have a negative impact on nematode reproduction. The negative impact on the reproduction might be explained by shorter lifetime for diseased plants, disturbed function of giant cells infected by the fungi, possible production of metabolites with a suppressing effect on nematode egg hatching by the fungus, or competition for root space and nutrients between the organisms (Back et al., 2002; Zahid et al., 2002). The competition for root space and nutrients might explain why the frequency of mycelia on the roots in the closed containers seems to be negatively affected by the presence of endoparasitic nematodes. According to the results from the correlation matrix, there are synergistic interactions between R. solani and PPN, but the results from the analysis of variance are ambiguous. The diverging results are probably due to the small statistical material and the large variation in some parameters. It seemed, however, that there were synergistic interactions between R. solani and the endoparasitic and ectoparasitic nematodes included in the study, especially endoparasitic nematodes, a finding which fits well with the literature (Back et al., 2002). In further studies on the interaction between R. solani and nematodes in potatoes, it would be interesting to study whether interactions might be species-specific or more general, whether the initial mass of fungal inoculum is important and whether there might be interactions between the fungus and more than one nematode type.

Tuber yield

The flow of assimilates from the top of the plant is hindered by the lesions caused by R. solani, and the potato plant sets fewer and more shallow tubers. R. solani infection also causes misshapen tubers since the development of the peel is disturbed by infections of the fungus in the cortex, causing it to break (Parmeter 1970; Olofsson et al., 1996). These effects were also apparent in the present study, where the yield decreased with increasing degree of R. solani infection. Plants with air tubers as well as misshapen and green tubers were observed. The relationship between low yield and high degree of black scurf can be explained by the negative effect of the mycelia before the development of black scurf. It is important to mention that early potatoes were produced on some fields, which probably influenced the results since early potatoes are harvested while the tubers are still small and immature. The population of PCN affected the yield negatively even before the damage threshold limit of 2 eggs/g soil commonly accepted for Swedish conditions is exceeded. Ecto-parasitic nematodes and endoparasitic nematodes also affect yield negatively, but the effect does not reduce the yield to any greater extent before the damage threshold levels are exceeded (Holgado et al., 1998; Dickenson et al., 2000; Guidelines for Nematode Control, 2005). In this study, the populations of

42

Pratylenchus found in the fields were very low and generally did not exceed the damage threshold level of 250 Pratylenchus/250g soil. From the analysis of variance it was found that fields with 50-99 Pratylenchus spp./250g soil had a higher yield than all other fields. This can be explained by the exceptionally high yields in these fields due to a combination of favourable external growth factors. It is a well-known fact that crop rotation plays an important role in pest management and consequently determines the yield size. In this study, fields with a crop rotation of more than 5 years between the potato crops had higher yields. The field soils included in the study probably differed in water holding capacity and precipitation rates, which may explain some of the varying effects of irrigation on the yield. Unlimited irrigation, or even any irrigation, may not always be possible due to the natural conditions of the place where the field is situated, and irrigation equipment may also be lacking. Heavily irrigated fields had a higher yield and no enhancing effects of irrigation on the quantity of stem canker symptoms in the field could be observed. Therefore irrigation probably had a positive effect on plant vigour and made them less susceptible to infection by the fungus.

External growth factors

In this study only a few external growth factors appeared to have some influence on the establishment and development of a disease complex, but not to any great extent. The study was carried out in fields where potatoes are commercially grown and where water and nutrient supply are ample, which is probably why so few effects of external growth factors were seen. The soil phosphorous class appears to be important for development of R. solani disease according to the results of the field study, where fields with P-class 3 had a higher degree of R. solani symptoms in the field (P-class 3 = 4-8 mg available P/100 g air-dried soil). According to the literature, excessive levels of phosphorous increase the potential for disease development of R. solani (Parmeter, 1970). All the fields taking part in the study had sandy soils, and could therefore be considered potential risk soils for R. solani infection, as R. solani thrives in light, relatively humus-rich soils (Parmeter, 1970; Olofsson et al., 1996). It was therefore no surprise that the fields with the greatest clay content had the lowest degree of R. solani symptoms. Crop rotation and the use of a catch crop in the crop rotation appeared to be of minor importance when it came to disease development of R. solani, nematodes and the interaction between the organisms in the study. However, these results cannot be given any greater attention, since the study was performed during one year only and the history of the fields was not fully known. It is a well-known and often demonstrated fact that a sound crop rotation and the use of catch crops is of major importance when it comes to pest management. The misleading results in the present study are probably due to the small statistical sample, some exceptional data and the production history of the fields. The weed management at the production sites may also have interfered with the results, as the pests are able to survive and reproduce on weeds (Olofsson et al., 1996).

43

The pH and nutrient status of the soils included in the study were within the general recommendations for potato production and no difference in R. solani symptoms could be seen for varying nutrient levels in the fields. According to the literature, R. solani has a wide pH range (Parmeter, 1970; Olofsson et al., 1996) and the soils of the study had a pH of 5.5-8. Neither the fungus nor the nematode population appeared to be affected by different quantities of applied irrigation. This can perhaps be explained by the local moisture conditions at the various fields, situated within different geographical precipitation zones. Both the fungus (Parmeter, 1970; Olofsson et al., 1996; Agrios 1997) and the different nematode types (Palm et al., 1968; Agrios 1997) are adapted to survive water stress and adverse climate conditions and the summer of 2005 was not an exceptionally dry summer. It is not easy to give any advice on how to manage interactions of R. solani and PPN, since the general advice commonly accepted today deals with the management of each pathogen individually. When different pathogens interplay in disease complexes, considerably lower population densities of pathogens cause more serious damage than when pathogens act separately (Bowers et al., 1996). New guidelines for management of R. solani and PPN are needed. Further studies in which the potential risks for disease complexes are investigated and new thresholds for each pathogen are determined would be of great use in modern potato production. Healthy plants are less susceptible and a well-planned crop rotation decreases quantitative and qualitative yield losses.

44

Conclusions

Seed treatment and use of certified seed potatoes are valuable strategies to control R. solani. The origin of the R. solani inoculum (soilborne or seedborne) is significant for disease development and the symptoms in the field. The potential for development of black scurf on the tubers is greater in fields with soilborne inoculum and severe R. solani symptoms. R. solani infection in the field gives rise to great quantitative and qualitative harvest losses. Synergistic interactions between R. solani and plant parasitic nematodes might be present in Swedish potato fields, but more field studies need to be done confirm this finding. Nematode populations below the threshold limit appear to play a part in interactions with the fungus. New guidelines and damage threshold limits are needed for pathogens in a disease complex in order to determine when to apply pest management.

45

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Acknowledgements

I would like to thank: Sanja Manduric and Lars Wiik for all their enthusiasm, support and help.

Lennart Pålsson for helping me with the readings in field.

Personnel at the Nematology Department for helping me with extracting nematodes, determining nematode types and growing potatoes in closed containers.

Svensk Potatisforskning Alnarp for financing my Master’s thesis.

Farmers for their interest and for allowing me to take samples in their fields.

Appendix I

Descriptions of the PPN extraction methods used

Extraction of cyst nematodes

A sample emptied into the elutriator is met by an upwards water stream which causes heavy particles to sink to the bottom and light particles such as cysts and plant debris to float. The floating particles are taped off into a sieve and dried. A filter paper is placed in a funnel with a tap. The funnel is filled with ethanol to about 1.5-2 cm from the filter paper margin. The dried and warm light particles are slowly emptied into the ethanol and carefully stirred so that the floating particles settle off at the edges of the filter paper. The tap is opened and the ethanol is allowed to slowly run off into a beaker without moving the particles that have settled at the edges of the funnel. The filter paper is lifted out and placed on a glass plate. After the ethanol is vaporised, the sample is examined under a dissecting microscope. The cysts present in the sample can be found at the margin of the settled particles as they easily float when dry and warm. The cysts found in the sample are picked out and placed on a wet filter paper in a Petri dish.

Extraction of free living nematodes

The sample is first wet sieved through a coarse sieve and into a glass flask in order to remove stones and other larger particles. The flask with the water suspended sample is placed on top of the water column of the elutriator. An upwards water stream allows the heavy particles in the suspension to sink to the bottom and the swimming nematodes to stay in the water column. The water in the column is collected and sieved through a set of sieves. The nematodes are rinsed from the sieves and poured through a funnel onto a 2 layer thick wool filter where they are captured. The fabric sieve is placed in a Petri dish with 20 ml of water and left overnight to allow the nematodes to swim through the filter into the water.

Staining of nematodes in roots

The washed roots are chopped into finer pieces and put into boiling fuchsin acid for five minutes. The content of the fuchsin acid is 2 parts of glycerol, 1 part of lactic acid and 1 part of distilled water. The stained roots are then rinsed with water, dried with absorbing paper and put in a Petri dish with a solution composed of equal parts of glycerol and water. Free living nematodes inside the stained roots are counted in one quarter of the stained roots under a dissecting microscope.

Isolation of free living nematodes from plant material

The roots from each plant are washed, chopped into smaller pieces, and processed according to the mistifier extraction technique. They are then placed on a filter paper in a sieve. The sieve is placed on 3 rubber corks in a plastic container with an opening 2 cm from the bottom. The plastic container with the sieve is placed under a mistifier for 48 h.

The nematodes move, facilitated by the moisture, out from the roots through the filter paper and into the sieve following the water. In the container, the nematodes sink to the bottom. Excess water is allowed to run off through the opening without any nematodes escaping from the container. After the 48 h the sieve with the roots is removed and the water in the container poured through a fine mesh sieve in which the nematodes are captured. The nematodes are transferred into a beaker by rinsing with tap water. The extracted nematodes are then poured into a large Petri dish with a painted grid, identified and counted.

Appendix II

Explanation of factors

Factor Explanation Unit Certified, Uncertified Whether certified or farm

saved seed was used. Yes or No

Treated, Untreated Whether seed treatment was used on the seed potatoes or not.

Yes or No

Previous R. solani Estimated degree of R. solani problems during the former production periods.

0-4

Stem canker symptoms, field

Quantification of the degree of visible symptoms of R. solani performed in the field in summer, 2005.

0.0-5.0

Mycelia, field Quantification of the amount of hyphae on the stained roots from the plants collected in the field in summer, 2005.

0-3

Sclerotia, field The quantified amount of black scurf on tubers harvested in autumn 2005.

0-4

PCN Density of PCN population in the field in the sampled soil.

Eggs and juveniles/g soil

Pratylenchus Number of Pratylenchus spp. found in the sampled soil.

Number of specimens/ 250 g soil

Ecto PPN Number of ectoparasitic nematodes found in the sampled soil. Types found: Longidorus spp., Tylenchorynchus spp., Trichodorus spp. Heliocotylenchus spp., Paratylenchus spp.

Total number of specimens of ectoparasitic nematodes/ 250g soil.

Endo PPN Number of endoparasitic nematodes in the stained roots from the plants collected in the field in summer, 2005.

Total number of specimens/ 2g roots

Yield Size of potato yield Tonnes/ ha R. solani, container, c Quantification of the degree of

visible symptoms of R. solani on the potato coleoptiles and roots in the closed containers.

0.0-5.0

Mycelia, c Quantification of the amount of hyphae on the potato coleoptiles and roots in the closed containers.

0.0-5.0

Sclerotia tuber, c The quantified amount of black scurf on the mini-tubers in the

1-4

closed containers. Sclerotia root, c The quantified amount of black

scurf on the potato roots and coleoptiles in the closed containers.

1-4

Endo PPN, c Number of free-living Pratylenchus spp. and cyst juveniles found in the stained roots from the closed containers.

0-2

Crop rotation The number of years since potatoes were produced in that particular field.

No. of years

Catch crop The use of a catch crop after one or more of the crops in the crop rotation.

Yes or No.

Irrigation The amount of irrigation applied to the field.

mm

Manure 2004 Type and amount of manure applied in the field before the growth season of 2004.

Tonnes/ha

Manure 2005 Type and amount of manure applied in the field before the growth season of 2005.

Tonnes/ha

pH The pH of the soil from the field.

P-AL The amount of available phosphorus in the soil of the field.

mg/100g air-dried soil

P-class The phosphorous class of the soil based on the amount of available phosphorus and bound phosphorus in the soil.

Class

K-AL The amount of available potassium in the soil of the field.

mg/100g air dried soil

K-class The potassium class of the soil based on the amount of available potassium and bound potassium in the soil.

Class

Mg-AL The amount of available magnesium in the soil of the field.

mg/100g air dried soil

K/Mg quota The ratio of potassium/magnesium.

Ratio

Ca-AL The amount of available calcium in the soil of the field.

mg/100g air dried soil

Organic matter content The fraction of organic matter in the soil.

%

Clay content The fraction of clay in the soil. % Silt content The fraction of silt particles sin

the soil. %

Appendix III

Questionnaire sent to producers

Bakgrund, fältinventering Namn: Gårdsnamn: Fält: Sort: Utsädeskälla: Certifierat utsäde Ja Nej (ange certifierings klass) Eget utsäde Ja Nej Antal år uppförökning av eget utsäde Betat utsäde: Betningsmedel: Obetat: Växtföljd de senaste 4 åren: År 2004 År 2003 År 2002 År 2001 När odlades potatis senast? Mellangröda/or (ange vilken): Har du haft problem med groddbränna tidigare? 0=inga problem; 1= små problem; 2= problem; 3= stora problem; 4= mkt. stora problem Stallgödsel: Typ? Mängd? År 2004 År 2005 Ungefärlig storlek på årets skörd i ton per ha? Lackskorv på årets skörd: 0=inga problem; 1= små problem; 2= problem; 3= stora problem; 4= mkt. stora problem Bevattning, antal gånger och genomsnittlig mängd/tillfälle:

Appendix IV

Data Field Variety Variety Certified Own seed Year propg. Early 1¸Late 2 Y/N Y/N years 1 K. Edward 1 1 0 0 2 K. Edward, Ukama 1 0 1 1 3 Ukama, Ditta, Satina 1 0 1 1 4 Satina, Ditta 1 0 1 1 5 K.Edward, Eloge 1 0 1 1 6 Victoria 1 0 1 1 7 Ceraster 2 1 0 0 8 Bintje 1 1 0 0 9 K. Edward 1 1 0 0 10 K. Edward 1 0 1 1 11 Ukama 1 1 0 0 12 Bintje 1 1 0 0 13 Rocket 1 0 1 1 14 Rocket 1 0 1 1 15 Bintje 1 0 1 1 16 Bintje 1 0 1 1 17 Kuras 2 0 1 2 18 Kuras 2 0 1 1 19 Producent 2 1 0 0 20 Producent 2 1 0 0 21 Kuras 2 1 0 0 22 Kuras 2 1 0 0 23 Fulva 2 0 1 1 24 Asterix, K. Edward 1 0 1 1 25 Faxe 1 1 0 0 26 Faxe 1 1 0 0 27 Secura 1 0 1 2 28 K. Edward 1 1 0 0 29 Bintje 1 1 0 0 30 Bintje 1 1 0 0 31 Bintje, victoria, asterix 1 1 0 0 32 Ukama, Rocket 1 1 0 0 33 Kardal 2 1 0 0 34 K.Edward 1 1 0 0 35 Kardal 2 1 0 0 36 Saturna 1 1 0 0 37 Frieslander 1 0 1 1 38 GMO utsäde 2 1 0 0 39 GMO utsäde 2 1 0 0 40 Bintje 1 1 0 0 41 Kardal 2 1 0 0

Field Seed treatment

Previous R. solani

Stem canker, field

Mycelia, field

Black scurf, field

Y/N 0/4 0/5 0/3 0/4 1 1 1 0.145 0 1 2 1 0 1.1225 0 0 3 1 1 0.3025 2 0 4 1 1 0.6925 0 0 5 0 1 0.4275 1 0 6 0 1 0.15 0 1 7 1 0 0.055 0 0 8 1 0 0.48 0 0 9 0 1 1.0425 0 2 10 0 4 1.3475 0 3 11 0 1 1.0875 1 0 12 0 0 0.78 1 0 13 0 1 1 1 14 0 1 2 1 15 0 0 1.7025 1 0 16 0 0 1.38 2 0 17 0 1 2.225 1 1 18 0 1 0.641667 1 1 19 0 1 0.241667 0 0 20 1 2 0.308333 1 0 21 0 1 0.7 2 0 22 1 1 1.116667 0 1 23 1 1 0.555 1 1 24 1 0 0.095 1 0 25 1 0 0.3725 0 0 26 1 2 0.54 1 1 27 0 3 2 3 28 1 3 0.7 1 1 29 1 0 0.24 1 0 30 1 0 0.535 1 0 31 1 1 0.8575 1 1 32 0 1 1.2425 2 1 33 0 0 0.77 2 0 34 0 0 0.840625 2 0 35 0 0 0.55 1 0 36 1 0 0 0 0 37 0 3 2.225 3 2 38 1 2 2.515 0 1 39 1 2 1.265 0 1 40 0 1 2.147493 1 41 0 1 0.71875 1 1

Field PCN pop., field

Pratylenchus spp.

Ecto PPN, field

Endo PPN, field

Crop rotation

juv/g soil no/250g soil no/250g/soil no/2g years 1 0.00356 65 366 120 42 2 1.62624 100 191 170 4 3 0.9348 7 44 150 4 4 0.0108 19 5 90 4 5 3.8981 26 23 80 4 6 0 22 80 12 4 7 0.70928 15 16 24 4 8 0 8 8 12 19 9 0 44 144 56 6 10 0 74 213 172 6 11 36.4455 9 69 76 3 12 0.018 7 136 52 5 13 0 0 0 20 1 14 0 0 0 12 2 15 13.7632 25 135 356 4 16 0.78854 41 127 4 4 17 0 16 154 36 4 18 0.0084 100 84 12 4 19 0 2 401 36 3 20 0 250 43 12 4 21 0 9 69 12 4 22 0 34 209 8 4 23 0.06675 1 18 53 5 24 0 10 2 12 4 25 0 15 35 28 12 26 0.13392 22 102 4 5 27 0.06072 25 294 4 4 28 0 6 498 0 4 29 2.0976 150 216 48 4 30 0 150 186 16 4 31 2.46428 150 61 40 4 32 0 11 19 0 1 33 0.0779 13 49 0 4 34 0 100 156 0 12 35 0 41 88 0 12 36 0 21 227 92 5 37 0.00776 63 36 28 1 38 0 55 362 32 18 39 0 68 584 16 4 40 0 68 80 5 41 0 49 605 4 5

Field Manure 2004

Manure 2005 Yield

Irriga -tion pH P-AL

ton/ha ton/ha ton/ha mm mg/100g 1 0 0 40 150 5.7 13 2 0 0 45 150 6.2 20 3 0 0 50 150 5.9 33 4 0 0 50 175 6.3 22 5 0 0 50 150 7.4 17 6 0 0 30 150 6.5 12 7 27 27 45 156 6 26 8 0 27 72 224 5.5 15 9 0 0 35 40 5.5 12 10 0 0 15 80 5.5 20 11 5 5 30 75 6.8 16 12 0 0 45 150 7.4 23 13 0 0 25 40 6.3 21 14 0 0 30 40 6.3 25 15 0 0 35 75 6.5 21 16 0 0 35 75 6.1 17 17 23 35 47 160 7.5 28 18 60 40 55 125 6.1 31 19 0 0 40 66 7.5 15 20 21 33 45 100 8 16 21 15 25 65 108 7.1 19 22 0 40 55 120 7.1 14 23 0 0 50 100 6.4 23 24 0 0 40 100 5.6 12 25 0 0 30 0 7 17 26 0 0 50 100 6.1 16 27 25 0 43 140 7.9 19 28 25 0 44 160 77 24 29 0 0 52 200 7.4 17 30 0 0 64 240 7.2 11 31 0 0 50 125 6.9 19 32 0 0 28 150 6 15 33 25 0 60 60 7.5 33 34 0 0 45 120 8 17 35 0 0 45 120 7.7 18 36 20 20 35 50 5.9 11 37 0 0 25 18 6.3 31 38 0 0 18 0 39 0 0 29 0 40 0 11 51 100 6.7 7.2 41 0 0 46 90 5.7 11

Field P-class K-AL K-class Mg-AL K/Mg

kvot Ca-AL OM

content class mg/100g class mg/100g qvota mg/100g % 1 4 7 2 3.2 2.2 80 2.5 2 5 20 4 7.1 2.8 150 3 3 5 51 5 20 2.6 280 5.4 4 5 20 4 4.9 4.1 85 1.9 5 5 13 3 19 0.7 480 3.7 6 4 14 3 11 1.3 220 2.3 7 5 19 4 9.5 2 110 2.3 8 4 23 4 11 2.1 140 2.3 9 4 17 4 9.5 1.8 90 3.3 10 5 17 4 7.5 2.3 61 3.1 11 4 19 4 11 1.7 300 2 12 5 15 3 14 1.1 550 1.9 13 5 11 3 17 0.6 87 2.8 14 5 13 3 18 0.7 110 2.6 15 5 11 3 5.8 1.9 150 2.2 16 5 12 3 6.3 1.9 160 2.1 17 5 7.3 2 15 0.5 1300 3.7 18 5 21 4 6.4 3.3 140 2.7 19 4 6.9 2 11 0.6 470 3.5 20 4 8.3 3 27 0.3 2000 2.4 21 5 8.4 3 9.6 0.9 350 3.7 22 4 12 3 11 1.1 200 3 23 5 14 3 6.7 2.1 120 1.9 24 4 23 4 10 2.3 120 3 25 5 15 3 9.7 1.5 210 2.5 26 4 14 3 5.8 2.4 100 1.6 27 5 17 4 60 0.3 2000 2.8 28 5 16 3 50 0.3 2000 2.8 29 5 8.7 3 11 0.8 360 2.5 30 4 9.2 3 11 0.8 330 2.5 31 5 12 3 6 0.2 200 1.8 32 4 13 3 4.2 3.1 99 1.6 33 5 12 3 14 0.9 790 4.1 34 5 8.5 3 43 0.2 2000 3.1 35 5 5.9 2 23 0.3 2000 3 36 4 11 3 16 0.7 180 4.7 37 5 17 4 15 1.1 110 2.4 38 39 40 3 15 3 6.9 2.2 95 2.4 41 4 11 3 13 0.8 130 5.7

Field Clay content Silt content Catch crop

Stem canker, container

Mycelia, container

% % Y/N 0.0-5.0 0.0-5.0 1 6 80 0 0.5 0.1 2 9 70 0 1 0.2 3 24 38 0 1.5 0 4 6 83 0 1.5 0.1 5 16 48 0 1 0.1 6 12 53 0 1.4 0 7 4 83 0 0.5 0.1 8 10 66 0 2 0.1 9 7 73 1 1.7 0.1 10 3 88 1 0.7 0.2 11 8 66 1 1 0.1 12 10 66 1 0.5 0.1 13 5 78 1 1.4 0.1 14 6 84 0 2 0.1 15 8 74 0 0.5 0.2 16 8 76 0 1.7 0.1 17 5 83 0 1.4 0.1 18 4 86 0 1.5 0.1 19 14 66 0 0.5 0 20 14 60 0 1.2 0.1 21 6 78 1 0.6 0.1 22 7 66 1 1 0.1 23 6 79 0 1.2 0.1 24 12 64 0 1.5 0.1 25 8 73 0 1.5 0.1 26 6 80 0 0.6 0.1 27 8 80 0 1.5 0.1 28 8 80 0 0.4 0.1 29 11 62 0 1.5 0.1 30 16 53 0 0.6 0 31 9 63 0 0.8 0.1 32 8 72 0 0.6 0.1 33 5 82 0 1.3 0.2 34 8 73 1 0.3 0 35 13 63 1 0.5 0 36 9 65 0 0.5 0 37 7 71 1 1.7 0.1 38 0 3.5 0.2 39 0 0.8 0.2 40 8 62 0 1.4 1 41 13 73 0 0.8 0.1

Field Sclerotia tuber container Sclerotia root container Endo PPn, container 0-4 0-4 0-2 1 2 0 1 2 2 1 1 3 3 1 1 4 2 0 1 5 1 0 0 6 1 1 1 7 1 1 0 8 2 0 0 9 1 1 1 10 1 0 0 11 1 2 1 12 1 0 1 13 2 0 1 14 1 1 1 15 4 2 1 16 3 1 0 17 2 1 2 18 2 0 1 19 1 0 1 20 1 1 1 21 1 1 1 22 4 3 0 23 2 0 1 24 2 0 1 25 3 1 1 26 2 0 1 27 1 0 1 28 2 0 1 29 1 0 1 30 2 0 1 31 1 0 1 32 2 1 2 33 2 1 1 34 2 1 2 35 1 0 1 36 1 0 1 37 1 0 1 38 2 1 1 39 1 0 0 40 3 0 0 41 2 0 1

Appendix V

Geographical spread of sampling sites