chapter 6 epidemiological assessments and postharvest disease … · 2010-08-11 · expression of...

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Abstract Postharvest plant disease can be measured by “incidence,” by record-ing the presence or absence of symptoms, and “severity,” the degree to which the symptoms are expressed. Weather and other environmental conditions play a significant role by causing stress in plants and lowering natural defenses, and by creating conditions suitable for pathogens to infect the plants. Specifically for postharvest diseases of fruit, infections can start as early as fruit set and continue until harvest. Although weather conditions influence the epidemiology of a dis-ease in the field, the incidence of postharvest disease depends on the incidence of latent infections that initiate in the field during the season, contamination with fungal propagules during harvest, the effectiveness of postharvest treatments, and storage and marketing conditions. True latent infection, defined as a parasitic relationship that eventually induces macroscopic symptoms (Verhoeff, K., Annu. Rev. Phytopathol. 12:99–107, 1974) plays a major role in both the incidence and severity of postharvest disease. If conditions are favorable, incidence and severity of latent infections will be higher and the risk for postharvest disease development will increase and vice versa. For example, in California kiwifruit there is a posi-tive relationship between the incidence of latent infection of sepals or stem ends, and the incidence of gray mold of fruit in cold storage. We visualize kiwifruit and other kinds of fruit as recording devices that copy the environmental conditions as latent infections. And in some cases, quantification of these latent infections can predict postharvest disease (i.e. BOTMON (Botrytis monitoring in kiwifruit sepals and/or fruit stems and in stems of grape berries) and ONFIT (overnight freezing incubation technique in stone fruit, other fleshy fruit, and in nut crops)). The source of inoculum that can drive an epidemic of a disease in the field can also affect

T.J. Michailides (), D.P. Morgan, and Y. Luo Department of Plant Pathology, Kearney Agricultural Center, University of California-Davis, 9240 South Riverbend Ave, Parlier, CA 93648, USA e-mail: [email protected]

Chapter 6Epidemiological Assessments and Postharvest Disease Incidence

Themis J. Michailides, David P. Morgan, and Yong Luo

D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century Vol. 2, DOI 10.1007/978-1-4020-8930-5_6, © Springer Science + Business Media B.V. 2010

70 T.J. Michailides et al.

the incidence of postharvest decay, by affecting the incidence of latent infection. Reducing the source of inoculum (sanitation) can reduce the incidence of latent infection of fruit, with the ultimate result in reducing postharvest disease (i.e., stone and pome fruit). Environmental conditions during bloom in various crops can have detrimental effects on the incidence of postharvest disease (i.e., grapes, pomegran-ates, prunes, etc.) by affecting the levels of latent infection or affecting the plant host directly. Altering cultural practices that may affect environmental conditions in the field or the physiology and histology of fruit can also affect the incidence of fruit diseases both in the field and postharvest. The development of efficient, accurate, and rapid molecular techniques (including real-time PCR assays) can facilitate the detection and quantification of disease inoculum and latent infec-tion of fruit (i.e., stone fruit) and help predict incidence of postharvest disease. In addition, development of allele-specific RT-PCR methods for rapidly detecting fungicide resistant fungal pathogens will help growers to manage fungicide resis-tance and make correct decisions to reduce postharvest disease. The goal of our laboratory is to develop less expensive molecular techniques that determine latent infections and assess populations of fungi resistant to fungicides and enable us to process large numbers of samples at our laboratory and to provide the protocols to private laboratories.

6.1 Definitions

Before proceeding with this review, we would like to define the various kinds of infection. According to Verhoeff 1974) latent infection of plants by pathogenic fungi is often considered of the highest levels of parasitism, since the host and para-site coexist for a period of time with minimal or no damage to the host. In other words, these infections are successfully established infections some of which will perish while others will survive and resume development as the physiological char-acteristics of the tissues where these infections reside change. A true latent infec-tion involves a parasitic relationship that eventually induces macroscopic symptoms (Verhoeff 1974). Quiescent infection, however, is microscopically visible although mycelial development is arrested after infection and resumes only as the host plant reaches maturity and/or senescence (Sinclair and Cerkauskas 1996). Latent con-tamination, which we prefer the term inoculum load for it, involves fungal spores (or other type of fungal propagules) on the host’s surface which fail to germinate until the host reaches maturity or senescence, or wounded by insects and other means.

Latent infection of plants by parasitic fungi is often considered one of the high-est levels of parasitism, since the host and parasite coexist for a period of time with minimal damage to the host. Latency involves an asymptomatic parasitic phase that eventually gives rise to visible symptoms (Verhoeff 1974) if conditions are favorable for disease development. For instance, if latent infections are at high levels and most develop to active disease symptoms then a disease epidemic can initiate.

716 Epidemiological Assessments and Postharvest Disease Incidence

6.2 Introduction

Postharvest diseases of fresh produce (fruit and vegetables) and mycotoxin contamination of certain susceptible crops contribute to major economic losses to growers, processors, marketers, and consumers. Therefore, management of postharvest disease is needed to avoid losses and increase growers’ revenues.

Postharvest diseases are the result of latent infections that occur in the field dur-ing the growing season and infections from wounding during harvest and handling operations (Eckert and Summer 1967; Michailides and Manganaris 2009). Although postharvest diseases are the continuum and or the result of diseases that initiate in the field, some researchers have emphasized postharvest disease per se without looking into the diseases caused by the same pathogens in the field. A good exam-ple is the brown rot disease of stone fruit caused by Monilinia fructicola or M. laxa. In California brown rot can have four distinct phases which in essence develop as a continuum on stone fruit. At bloom, for instance, if weather is wet and relatively warm, blossoms are infected and blighted: this is the blossom blight phase. Later on, if conditions continue to be favorable for the pathogen, the fungus can invade from the blossoms to the sustaining shoots causing cankers: this is the canker phase (which is more common for M. laxa). If wet conditions prevail, green or maturing fruit get infected and rot on the tree: this is the fruit rot phase. Finally, once fruit is harvested and removed from the orchard still can decay and this is the postharvest fruit rot phase which can occur either in storage, in the market (wholesale and retailed stores), in the household refrigerator, or in customer’s fruit display basket.

Epidemiological knowledge of fungal diseases of fruit trees, nut crops, and vines is essential to help predict disease risk during the growing season and/or at harvest and during postharvest storage. Predicting plant disease is very chal-lenging and requires major research efforts over several years to understand the dynamics of the four main variables involved in disease development: the presence/absence and quantity of the pathogen’s inoculum, stage and suscepti-bility of the crop, environmental conditions, and growers who continuously attempt to change the dynamics of the diseases in their agricultural systems. These major factors are not steady but compoundedly change and make the development of an accurate predictive model difficult, complex, and time con-suming. Although increasingly accurate weather predictions are available through the National Oceanic Atmospheric Agency (NOAA), the other aspects of the disease triangle require a large database of information that includes the pathogens’ inoculum, which can be quantified by either trapping spores or pre-dicted based on disease incidence in previous seasons (historical disease data) or by determining the incidence of latent infections. Susceptibility of the host and the most susceptible stage of the crop can be determined experimentally with periodic inoculations with the pathogen.

In many diseases of fruit trees, nut crops, and vines, latency is an important stage in disease epidemiology and prediction. Although disease prediction depends on the above mentioned factors, it is sometimes accurate, depending on the specific disease and its nature, to predict the disease incidence and severity even without


any recording and consideration of the above mentioned parameters. This is because the success for a latent infection to occur and develop to actual disease depends on all these factors (inoculum level present, susceptibility of the host, and environmental conditions conducive to infection) and these infections then can act as recording devices that “record” and accumulate all these parameters that contrib-ute to disease expression. In this contention, then an infective propagule that has the capability to produce a latent infection and it could be considered as an alarm that will go off as soon as the “right time” has reached based on the accumulated “latency condition of the pathogen” has reached a minimum threshold. A “latency condition” is defined as the status of the pathogen subjected to diminishing adver-sity by the host as it changes physiologically and biochemically and changing conditions of the environment that affect both the host and the pathogen. In this case, the pathogen itself acts as a recording device that records all these changes (of the environment and the host) and develops accordingly, with the final result the expression of disease development. If the latency condition of the pathogen leads to its death, then symptoms of disease will not develop.

Diseases we investigated that have a latency phase include Botrytis gray mold in grapes and kiwifruit caused by Botrytis cinerea (Pers.:Fr.), brown rot in stone fruit caused by Monilinia fructicola (G. Wint.) Honey and/or M. laxa (Aderh.) Honey, panicle and shoot blight of pistachio caused by Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not., and Alternaria late blight of pistachio caused by three species of Alternaria. Latent infections of berries, stone fruit, and nuts remain inactive until these fruit start maturing and environmental con-ditions are favorable for disease development. However, if environmental con-ditions are not favorable, the pathogen may not survive until the stage when the crop becomes conducive to the development of latent infection to disease symp-toms. The incidence of latent infections has been shown to correlate with dis-ease incidence in the field at harvest of fruit and nut crops (Michailides et al. 2000) or with the incidence of decays that develop in storage (Michailides and Morgan 1996a,b).

The degree of success in management of fruit tree diseases depends on the level of disease itself, environmental conditions, effective cultural manipulations, and proper timing and type of fungicides selected against a disease. Fungicide resistance in fungal pathogens of tree fruit and vines also affects the outcome of disease management as well as choices of postharvest treatments. If growers had access to timely information on fungal resistance to fungicides, they could make correct decisions on what fungicide to apply and what rotation schedule would be essential to help avoid a failure of disease control in the field and/or prevent a build-up of resistance in the pathogen’s population in the orchard or vineyard. To base this discussion on personal experience, two examples will be emphasized as follows: the Monilinia fructicola and M. laxa causing brown rot disease of stone fruit; and the Botrytis cinerea causing gray mold of kiwifruit. These examples include good methods for the detection of latent infection during the growing season that predict disease at harvest and postharvest.


6.2.1 Brown Rot-Monilinia fructicola and M. laxa

Brown rot of stone fruit in California is caused by Monilinia fructicola and M. laxa. The latter species was the predominant in the early nineteenth century while in late century, M. fructicola became the dominant species, at least in prune orchards (Michailides et al. 1987). The disease expresses itself in two major phases, infec-tion of blossoms leading to blossom blight and infection of fruit leading to fruit rot (both immature, green fruit and mature fruit infection). A third phase is the latent infection that can occur from bloom to harvest. The occurrence of latent infection by Monilinia spp. is known since the early 1960s various reports provided good examples of latent infection on apricot (Wade 1956; Tate and Corbin 1978; and Wade and Cruickshank 1992). On cherry (Curtis 1928; Förster and Adaskaveg 2000), and on peach (Tate and Corbin 1978; Michailides et al. 2000). The best examples of latent infection by Monilinia spp. are on plum (Rosenberger 1983; Northover and Cerkauskas 1994) and on dried plum (Luo and Michailides 2001, 2003; Luo et al. 2001). Specifically, on dried plum, infections by the brown rot fungus can be divided in (1) latent infections in blossoms; (2) latent infections and quiescent infections in green fruit, and (3) quiescent infections on leaves. Quiescent infections on leaves are small brown spots that upon isolations on acidified PDA the majority of them will produce colonies of Monilinia spp. Quiescent infections on immature dried plum fruit (prunes) are raised pin-head black specks (Fig. 6.1). The majority of the latent infections do not develop to disease; however, some can overcome the inhibiting factors encountered in green fruit and form a brown decay lesion in immature, green fruit. Latent infections will also affect directly the inci-dence of postharvest decay (see below). Because of the direct relationships between latent infection and brown rot disease, detecting latent infections could be a useful assay to determine risk of brown rot at harvest and postharvest.

Fig. 6.1 Quiescent infections of Monilinia spp. on ‘Howard Sun’ plum


6.2.1.1 Conventional Methods Used to Detect Latent and Quiescent Infections of Monilinia spp.

There are two types of methods for the detection of quiescent and latent infections. These include conventional (direct isolation – incubation) and molecular (PCR and real time PCR) techniques. These techniques have been used in our studies in the last several years with great success.

Direct Agar Plating Technique (DAPT) This technique is commonly used to isolate plant pathogens from plant tissues. The detection and quantification of the Monilinia sp. pathogen in quiescent infections can be determined using this tech-nique. The agar medium commonly used for the DAPT technique in our laboratory is potato dextrose agar acidified with lactic acid (2.5 mL of a 25% vol./vol. lactic acid per liter of medium), resulting in a pH of 3.5 that is inhibitory to the majority of bacteria, but it allows the Monilinia spp. to grow when incubated at 20°–25°C (68°F to 77°F) for several days. The traditional way is to cut the plant tissue in 3 × 3 × 3 mm cubic pieces, surface disinfect them in a 5–10% chlorine solution (prepared from household bleach, which is a 5.25% solution of NaOCl) for 1 to few minutes, rinse them with sterile water once or twice, blot them on clean paper towels, and place 5–10 pieces in a 55- or 90-mm in diameter Petri plate. The plates are usually incubated at 25°C (77°F) for 5 to 7 days and recorded for the presence/absence of Monilinia spp. from the isolations. The DAPT has been used for isolating latent infections and quiescent infections from the skin of various stone fruits, fruit-to-fruit contact areas, stylar and basal ends of the fruit, flower petals, and leaf blades. For instance, when the weather is unusually wet, quiescent infections show as small rusty spots on the leaf blade and 3 × 3 mm square pieces of the blade can be surface sterilized and plated as described above.

Flower Incubation Technique (FIT) A second conventional technique for the detection of latent infections of brown rot in flowers is by collecting 100–150 ran-dom flowers per field, surface sterilized them in 1% chlorine solution (prepared from household bleach containing 5.25% NaOHCl), and lay them either on wet sterile paper towels or on sterile plastic screens in clean containers. Usually, the containers used in our laboratory are made of hard plastic and measure 24.5 × 18.0 × 8.0 cm. The containers with the flowers are incubated at room temperature or at 25°C for 5 days when latent infections develop on the hypatheum showing charac-teristic sporulation. The incidence of flowers with latent infection by M. fructicola is then determined within 5–7 days incubation. This technique can also be per-formed by using short (15–20 cm) twigs bearing flowers and placing flat four to five twigs on top of the plastic screens in the plastic containers. All the other steps for this procedure, incubation and determination of latent infections are the same as those used above for individual flowers (Luo et al. 2001).

Overnight Freezing – Incubation Technique (ONFIT) (Table 6.1) This is another conventional technique which developed and frequently used in our labo-ratory and can detect and quantify latent infections of Monilinia fructicola and M. laxa (the fungi that cause brown rot in stone fruit), Botrytis cinerea in grapes


causing bunch rot, and B. dothidea and Alternaria species causing blight diseases in pistachio. The rationale of the technique is based on the fact that killing the fruit tissues at a stage when the tissues do not favor disease development triggers the development of latent infections to active disease symptoms or accelerates the growth of hidden colonists. The herbicide paraquat (1,1´ diemthyl-4.4´ bipyridin-ium dichloride) has been used in the past to kill plant tissues and trigger latent fungal infections in soybean (Cerkauskas and Sinclair 1980) and modified later for the detection of latent infections of plums by M. fructicola (Northover and Cerkauskas 1994). We also used paraquat in our laboratory initially, but because it is a toxic herbicide, we looked for an innocuous alternative. In contrast, the ONFIT is a safe technique because it does not require the use of any noxious pesticides, only surface disinfectants such as dilutions of household bleach and disinfectants, such as ethyl alcohol, which are generally regarded as safe. But, it is still necessary to thaw and then incubate the frozen fruit for a number of days under laboratory conditions (75°–77°F) until latent infections develop into symptoms and signs of the pathogen can easily be recorded (Fig. 6.2a) and results become available within 5–7 days. Latent infections of brown rot can also develop to disease symptoms, but fruit needs to be incubated under high humidity for at least 2–3 weeks. Using the ONFIT, a grower would need to wait only 5–7 days until he would be able to make a decision on disease risk in the field (Luo and Michailides 2003). For instance, the incidence of Monilinia spp. determined with the ONFIT performed with immature

Table 6.1 Protocol of overnight freezing incubation technique (ONFIT)a to reveal latent infections by Monilinia fructicola or M. laxa in stone fruit

1. Collect 100 immature fruit from the orchard and bring to the laboratory in an ice chest. 2. Disinfect cleaned plastic screen racks and plastic containers (2 per site) in 1:20 bleach

(5.0% sodium hypochlorite: water) solution for 5 min. 3. Place 50 fruit in the plastic mesh-stretch bags, label as desired, and secured mesh bags with

plastic clips. 4. Prepare bleach solution in large (20 L) plastic containers. Solution is prepared 1:10 with 0.5

mL of Tween 20 per liter of tap water. Typical preparation is 5 L of solution with 0.125 mL of Tween 20. Place the plastic container in the sink to minimize unwanted splashing of the bleach solution.

5. Prepare 1 L of 70% ethanol in a 2 L beaker. 6. Place the samples in the ethanol solution for 10 s, shake off quickly and place in the bleach/

Tween-20 solution for 4 min. Swirl the bags in the solution for 5–10 s during every minute the bags are in solution.

7. Remove the bags, shake off the excess liquid in the sink and quickly place the bags in the appropriately labeled containers; replace the lid.

8. Arrange fruit in the containers under a hood, pour 200 mL of tap water, and cover the containers.

9. Place all plastic containers at 3°–4°F (−16°C) freezer for about 15 h (17:00 h to 8:00 h) overnight.

10. Remove containers from the freezer and place on a laboratory counter at about 77°F (25°C).11. Record and count fruit showing brown rot symptoms and sporulation of the pathogen after

5–7 days.aONFIT can be used for any stone fruit such as apricot, cherry, nectarine, peach, plum, and prune


prune fruit collected in May/June correlated linearly with the incidence of the brown rot developed in the field at harvest (Fig. 6.3). In other words, the incidence of Monilinia determined with the ONFIT can predict the risk for fruit brown rot at harvest. Waiting one week is still a long waiting time, therefore, more efficient and quicker techniques than the conventional ones are urgently needed for the detection of latent infections in tree fruits, nut crops, and vines in California. The timing for performing ONFIT depends on various factors. One important factor is the inoculum potential of Monilinia spp. in the orchard. For example, when the inoculum potential is high in an orchard the timing of ONFIT can range from mid May to early July,

Fig. 6.2 (a) ONFIT on French prunes to detect latent infections after 7 days incubation, (b) incu-bation of surface sterilized French prunes without freezing for 4 weeks at room temperature


when the inoculum potential is low, the best timing will be in mid June, and when the inoculum potential is moderate, the best timing can be at any time in the month of June. But in general, the critical period to determine latent infection for prunes is in June (Luo and Michailides 2003).

Incubation of fruit after surface sterilization following the steps that are used for the ONFIT procedure without freezing requires a long time until latent infections in green fruit are triggered to develop. For instance, when immature prune fruit were collected from two different sides of a large prune orchard, surface sterilized, and incubated as above (without freezing the fruit), latent infections started devel-oping after 8–10 days incubation, reached to 5–7% levels in 15 days and to a maxi-mum after 30 days (Fig. 6.2b). Even this procedure if it is done in May provides sufficient time for determining the risk for disease in an orchard and making deci-sions for pre-harvest sprays. There is linear correlation of the incidence of latent infection and postharvest decay for at least some of the stone fruit (i.e., prunes).

6.2.2 Botrytis Monitoring (BOTMON) - Botrytis cinerea (Table 6.2)

The California kiwifruit industry and other kiwifruit industries suffer tremendous losses due to postharvest gray mold caused by Botrytis cinerea. Although this disease does not show any symptoms and or signs in the field in California, postharvest gray mold is considered as the number one disease of kiwifruit in cold storage. Since no disease symptoms develop in the field, it is most likely that infections

PBFR = −2.6 + 0.3355 ILI

Incidence of latent infection (%)

Per

cent

bra

nche

s w

ith fr

uit r

ot (

PB

FR

)

0 10 20 30 40 500

2

4

6

8

10

12

14

16

r2 = 0.82, P = 0.002

Fig. 6.3 Linear correlation between incidence of latent infections (ILI) and percentage of branches with fruit rot (PBFR) caused by Monilinia fructicola on prune, detected by the ONFIT technique. Each data point represents an average value of multiple locations and inoculations


by B. cinerea of kiwifruit are primarily latent. Michailides and Morgan (1996a) found that these infections occur on the fruit sepals and receptacles during the growing season starting 1 month after bloom and continuing until harvest. Furthermore, there are major differences in the epidemiology of Botrytis gray mold of kiwifruit in New Zealand and California. For instance, because of the dry climatic conditions in California, latent infections is of primary importance for the postharvest gray mold while in New Zealand, it is the infection of stem wound that affect the levels of the postharvest decay and not the latent infections of sepals by B. cinerea. Therefore, in California to develop more efficient methods for controlling gray mold in kiwifruit, it was necessary to understand the relationship between B. cinerea latent infections initiated in the field and the incidence of gray mold in cold storage. The BOTMON technique was developed and used to detect B. cinerea, causing latent infections of kiwifruit (Actinidia deliciosa) sepals in the field. The rationale of the technique is based on the fact that the incidence of latent infection is a good predictor of gray mold in cold storage (Michailides and Morgan 1996a,b; Michailides and Elmer 2000).

BOTMON involves the collection of fruit samples with stems attached from the kiwifruit vineyard, removal of sepals or stem ends (receptacles; Fig. 6.4), and plating the fruit samples in plates with APDA (Fig. 6.5). It was determined that 1 month before harvest is the best time for sampling immature fruit to perform BOTMON and gray mold prediction, since the correlation coefficients of B. cinerea colo-nization of sepals and stem ends and gray mold in cold storage are the highest (r = 0.92–0.98). Interpretation of the technique’s results was given in previous

Table 6.2 Protocol of Botrytis monitoring (BOTMON) to reveal latent infections by Botrytis cinerea by plating symptomless sepals and stem ends of kiwifruit

1. Harvest 60 kiwifruit from each 1-ha field (avoid any wounding) 4 months after fruit set (about 1 month before harvest).

2. Remove sepals (by hand) and stem end (with a cork borer) from each fruit. 3. Surface disinfect sepals and stem ends in 0.5% chlorine household bleach plus 2 drops of

Triton-X-100 surfactant (per liter water). 4. Rinse the above, surface-sterilized fruit sepals and stem ends in sterile water and dry them in

a positive-flow hood for 10–15 min. 5. Plate the above in Petri plates containing acidified potato-dextrose agar (pH = 3.2–3.5). 6. Incubate the petri Plates at 7°C for 6 days and record B. cinerea colonies growing from the

sepals and stem ends in each plate (first Botrytis recording). 7. Move and incubate the petri Plates at 23°C for 3 more days and record additional B. cinerea

colonies in each plate (second Botrytis recording). 8. Combine data from the two recordings (= total B. cinerea colonies) and determine incidence

(%) of colonization of sepals or stem ends. 9. Use prepared tables or regression lines for sepal or stem end colonization to predict Botrytis

gray mold after expected to develop after 3 or 5 months storage of fruit in controlled atmosphere.

10. Make decisions: (a) yes or no preharvest fungicide spray(s); (b) which fruit to store longer; (c) yes or no resorting and re-packing; etc.


publications (Michailides and Morgan 1996a,b). The results of the colonization of sepals and/or stem ends by B. cinerea grown become available to the grower in 9 days. After obtaining the results, growers can use Table 6.3 to predict the levels of gray mold in cold storage for a specific lot of fruit. In California more and more kiwifruit growers have been using the BOTMON technique to make decisions on the need for pre-harvest vinclozolin spray(s) since this technique uses fruit collected one month before harvest, allowing for sufficient time to apply a fungicide. Additionally, packinghouse

Fig. 6.4 Sepals and stem-ends of kiwifruit cut from the fruit to be used in BOTMON to monitor the incidence of colonization of Botrytis cinerea

Fig. 6.5 BOTMON technique: plates containing acidified potato-dextrose agar where sepals (left) and stem ends (right) were plated to reveal colonization by Botrytis cinerea


operators and shippers can use the results to decide on the need for fruit sorting and re-packing to minimize secondary spread of the disease in storage and plan on the timing for marketing these fruit. When growers spray only when it is needed and only those vineyards which have a high incidence of latent infections, they reduce costs and contamination of the environment with pesticides. The only dis-advantage of the technique is that it is still time consuming (6 days pre-incubation of the plated sepals and stem ends on APDA at 45°F and 3 more days at 77° F (see protocol in Table 6.2). Therefore, there is still a need for a quick technique that will provide results within one day or even within a few hours. BOTMON has been used also to detect B. cinerea in latent infections of apples, figs, grapes, pears, pistachios, pomegranates, and various stone fruit (cherry, nectarine, peach, plum, and prune).

Prusky et al. (1981) developed a pre-harvest assessment of latent infections by Alternaria alternata in mango fruit and found that there was a positive correlation between the relative surface of fruit infected by latent Alternaria at harvest and the incidence of black spots that developed on the fruit during postharvest storage.

6.2.2.1 Molecular Techniques

The polymerase chain reaction (PCR) and the development of thermocyclers have revolutionized the molecular biology since they were first described in 1985. PCR-based techniques have been used in various biological studies. Specifically in plant pathology, PCR has been used in identifying pathogens and determining pathogen population structures, taxonomy, and classification. Additionally, in the last several

Table 6.3 Relationship of levels of sepal and stem-end colonization by Botrytis cinerea in kiwifruit samples collected 4 months after fruit set and number of vineyards with low (<1%), moderate (1–3%), and high (>3%) levels of Botrytis gray mold decay after 3 months in controlled atmosphere storage (31°F (−0.5°C) and eight parts per billion ethylene) in 2 years

Number of vineyards with fruit showing different levels of postharvest gray molda

Sampled Colonization Low (£1%) Moderate (1–3%) High (³3%)

Plant part Level Colonization Yr 1 Yr 2 Yr 1 Yr 2 Yr 1 Yr 2

%Sepals Low 0–15 6 3 0 1 0 0

Medium 16–50 0 0 2 3 1 0High >50 0 0 0 0 0 1

Stem ends Low 0–15 4 2 0 0 0 0Medium 16–50 2 1 2 3 0 0High >50 0 0 0 1 1 1

a In year 1 a total of nine vineyards were sampled and in year 2 a total of eight vineyards (fruit from one vineyard in Butte County was not sampled because of prescheduled grower’s harvest). Percentage of colonization was determined by plating 300 sepals and 60 stem ends per sampling in each orchard.


years, techniques that quantify the DNA of pathogens have been developed, and these can be very useful when the relationship of quantities of pathogens’ DNA, latent infections, and disease levels are established. Such techniques could aid in estimating spore inoculum that determines disease potential and levels of latent infections that relate to disease risk at harvest or after postharvest storage. Following, we present a few examples of molecular techniques we developed in the plant pathology laboratory at the Kearney Agric. Center for the early detection of pathogens and fungicide resistant pathogen genotypes and providing answers to critical epidemiological questions that help predict disease risk in the field and pathogen resistance to fungicides. We also discuss how these techniques could be used in agribusiness as decision making tools.

PCR-based assays that detect M. fructicola and M. laxa in stone fruit Brown rot, caused by M. fructicola and or M. laxa, is a destructive disease of stone fruit (Prunus spp.) in California causing initially blossom blight and later fruit rot. When the microclimatic conditions in the orchards are unfavorable for further disease development, infected blossoms develop into young fruit with latent infections. These fruits may drop naturally or be thinned, and when humidity is high, these dropped fruit produce numerous conidia which can cause fruit infections in mid-season. Later, when favorable conditions and maturation of the fruit occur, a num-ber of the latent infections may develop into fruit rot. Inoculum potential in the orchards is an important factor affecting both blossom blight and fruit infections (Luo and Michailides 2001, 2003). Thus, determination of inoculum potential (amount of pathogen’s spores in the orchard) in early- and mid-season is critical for predicting and managing brown rot.

Inoculum potential is the most difficult parameter to determine in a stone fruit orchard. Spore traps have historically been used to determine the spore density for air-borne disease agents including M. fructicola. Because samples from traps require microscopic examination, it is both very time consuming and requires spe-cial training to recognize and count spores in the spore samples. Additionally, spore counts may be an unreliable indicator of inoculum potential because of the abun-dance of dust and other fungal species (i.e., Botrytis cinerea) having spores similar to those of M. fructicola. Furthermore, culturing airborne spores collected on spore-trap tapes or slides is also tedious and subject to frequent contamination problems. Thus, such classical methods are impractical for recording large number of spore trap samples required for a large-scale disease management.

PCR-based assays have the potential to monitor airborne inoculum levels of plant pathogens because they are highly specific and sensitive. Recently, we devel-oped a nested PCR method for the detection of M. fructicola on spore-trap tapes (Ma et al. 2003b). Nested-PCR primer pairs (an external primer pair EMfF + EMfR and the internal primer pair IMfF + IMfR) were designed based on the sequence of a microsatellite region generated by a microsatellite primer M13 (5¢-GAG GGT GGC GGT TCT-3¢). In specificity tests, we observed that the primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571- and a 468-bp DNA fragment, respec-tively, from all tested M. fructicola isolates collected from different stone fruit hosts


at different locations in different years. No fragments were amplified from any other fungus associated with stone fruit. Sensitivity tests showed that the nested PCR assay could detect the specific fragment in as little as 1 fg of M. fructicola DNA (Fig. 6.6a) or in DNA from only two spores of M. fructicola (Fig. 6.6b). This nested PCR method can detect 200 spores in a spore-trap tape sample (equivalent to two spores/PCR reaction) collected from a commercial prune orchard. Using these species-specific primers, we can also detect latent infections in fruits caused by M. fructicola within hours, while direct plating on agar media, or overnight freezing-incubation technique would require at least one week (see details below:

Fig. 6.6 PCR using specific primers to detect (a) M. fructicola DNA and (b) DNA from spores


Table 6.4). Since the nested-PCR assay cannot quantitatively detect the number of M. fructicola spores on a spore-trap tape, we are now working on a real-time PCR technique that can quantify spores of M. fructicola. The efficient, accurate, and feasible real-time PCR method could help growers in making timely decisions for fungicide application and reduction of unnecessary sprays.

6.3 Use of Species-Specific PCR to Detect M. Fructicola in Fruit and Flowers (Comparison of Conventional with PCR Techniques)

In 2001, in a plum orchard cv. Howard Sun, the presence of numerous visible qui-escent infections (Fig. 6.1) suggested the presence of even more latent (invisible) infections. In order to compare the direct plating technique of visible quiescent infections, with the ONFIT of invisible latent infections and a species-specific PCR technique, in mid May fruit were observed in the field and their fruit-to-fruit con-tact surface was marked with a permanent pen. All these fruit were collected and brought to our laboratory at Kearney Agricultural Center, surface disinfected in 10% bleach solution for 3 min, rinsed with sterile water twice, and placed on clean paper towels. The fruit samples were split in three subsamples; one subsample was used for the DAPT, the second for the ONFIT, and the third subsample for the species-specific PCR technique.

(a) For the direct plating technique, latent infections (small pieces of green tissue of the fruit surface) and visible quiescent infections (small black specks on the fruit skin) were excised with a sterile razor, and plated on APDA plates as described in DAPT. (b) For the ONFIT, fruit without any visible infections were processed fol-lowing the protocol in Table 6.1, and recorded for brown rot development on the fruit after 7–9 days incubation. And (c) for the species-specific PCR method, invisible latent and visible quiescent infections were excised as in (a) above, pre-incubated at 77°F for 1 day, and then DNA was extracted and diagnosed using M. fructicola specific PCR following published protocols (Boehm et al. 2001). Results from the PCR method after isolating fungal DNA can be completed in 6 h, i.e., in 30 h since the initiation of the procedure (Table 6.4).

Table 6.4 Techniques to detect latent and quiescent infections by Monilinia fructicola in ‘Howard Sun’ plums

Technique Latent infections (%) Quiescent infections (%)Time required for results (days)

PCR 7.9 60.5 1.25a

ONFIT 6.7 – 7–9DAPT – 54.3 5–7a Time includes 1-day preincubation of sample.


Using the PCR technique, 7.9% of the samples with invisible latent infections were positive for DNA of M. fructicola and 6.7% of the fruit processed with ONFIT developed brown rot. Similarly, as expected when visible quiescent infections were used, 60.5% were positive for M. fructicola with the PCR technique and 54.3% of those plated on APDA developed colonies of M. fructicola. Interestingly, the tradi-tional techniques required 5–9 days to completion while the PCR technique made results known within only 1.25 days (Table 6.4).

In another experiment, plum flowers (cv. Royal Diamond) were collected in March-April from a commercial orchard in Reedley, CA. Flowers were divided into three groups based on visual symptoms: (+) flowers were heavily infected with M. fructicola and showed obvious signs of fungal sporulation on the stem and calyx surface; (+/−) flowers displayed brown patches on the petals but showed no external signs of fungal sporulation; and (−) flowers were asymptomatic, without any evidence of brown discoloration or fungal infection. DNA extractions from indi-vidual plum flowers were obtained using the Fast-Prep System FP-120 biohomog-enizer instrument, following the manufacturer’s instructions for plant DNA extraction (Q-BIOgene, Inc.). In planta polymerase chain reaction (PCR) detection from flowers used the species-specific primer pair 210F1 + 210R1 at the high annealing temperature for retention of species specificity. The results indicated that all (100%) of the (+) series of flowers had much stronger amplification signals than either the (+/−) (80%) or the (−) flowers (only 10% of the flowers carrying M. fructicola (Fig. 6.7). Thus in approximately 8 h, one can determine the percentage of asymptomatic flowers carrying M. fructicola. Knowing the percentage of flowers latently infected by M. fructicola should prove a useful estimate of the inoculum potential in stone fruit orchards necessary for determining disease risk, assessment and blossom blight incidence, and in developing pre-harvest and postharvest chemical control strategies against brown rot. The PCR technique can replace the flower incubation technique (FIT) that can also provide an estimate of inoculum potential in stone fruit orchards.

6.4 Techniques to Monitor Resistance of Fungal Pathogens to Fungicides

Fungicides are commonly used to manage plant diseases. However, the frequent use of fungicides with single mode of action incurs a high risk of selecting resistant genotypes of plant pathogens. To determine levels of resistance to fungicides in fungal populations, the most common conventional technique used is the direct plating of single-spore isolates in media amended with the fungicide of interest. Single-spore isolates of the pathogen are grown on PDA or acidified PDA or other specialized media under conditions that favor their sporulation. Media such as PDA or water agar (WA) amended with increasing concentrations of the test fungicide are used to determine either the EC

50 of inhibition of hyphal growth (after placing

a 5-mm in diameter mycelial plug in the center of the Petri plate) and/or EC50

of


spore germination (after spreading a 0.5 mL of a dense spore suspension of the fungus on the surface of the plate) in comparison with the respective medium that is not amended with the test fungicide. EC

50 is the concentration of the fungicide

that provides 50% inhibition of mycelial growth or spore germination of the patho-gen to be tested. This technique was used in our laboratory for detecting fungicide resistance in plant pathogens such as M. fructicola and M. laxa, Botryosphaeria dothidea, Fusarium moniliforme, B. cinerea, Alternaria alternata, A. tenuissima, and A. arborescens. This technique is time consuming, and obtaining the results of the tested isolates is usually very critical, especially since growers would rely on such results to decide what fungicide programs to use, depending on the presence and levels of resistance in a field.

Depending on the pathogen species, in order to obtain mycelial plugs or adequate amounts of spores, the entire test can take 7–10 days, if not longer, if one takes into account the time required to isolate the pathogens from infected plant tissues. In 2004, a new technique was reported using a Spiral Plate® gradient dilu-tion method to determine EC

50 values that still requires 1–5 days for mycelial

growth assays and 14–20 h for the spore inhibition studies to reveal results plus 2–5 days for sporulation in culture (Förster et al. 2004). Although results with this tech-nique are available sooner than the classical technique, both the high cost of the Spiral Plate® equipment and the long time period to obtain results when one wants to check multiple isolates are prohibitive. Therefore, more efficient and rapid tech-niques are needed for checking resistance of pathogens to fungicides.

An example will be the resistance in Monilinia spp. Since benzimidazole resis-tance in M. fructicola and M. laxa has been shown to be associated with point mutations in the b-tubulin gene (Ma et al. 2003a), we developed an allele-specific real-time PCR method for rapidly detecting benzimidazole-resistant M. fructicola and M. laxa in stone fruit orchards. A similar procedure was used for the detection

Fig. 6.7 PCR using specific primers to detect DNA of Monilinia fructicola in latent and quiescent infections of plum flowers


of Alternaria resistance to azoxystrobin. This technique was used with blossoms collected from peaches in spring (March) and successfully detected the level of resistance to benzimidazoles in M. fructicola. Obviously, such rapid and quantita-tive detections of fungicide resistance in the fungal pathogen populations will be valuable for growers to manage fungicide resistance in fruit tree orchards and vineyards.

6.5 Conclusions and Future Prospects

The presented examples represent a part of the methodology used at the Kearney Agricultural Center to access latent infections to help predict diseases at harvest and in postharvest storage and guide growers to disease management decisions and packinghouse operators to proper marketing of fruit. Although the conventional techniques can be accurate and may be less expensive because of specialized reagents (enzymes and equipment are not needed), only minimal information con-cerning a few isolates becomes available and only after 1–2 weeks. This “waiting-for-results” time often can be a very critical time for growers who need to make a decision on disease management, fungicide timing and frequency, type of fungi-cide, and resistance management program selection in their fields. Because of these serious drawbacks in the last 5 years we have focused our efforts on research to develop molecular techniques, which although are more costly, they can replace the conventional ones and have the potential to finally provide very accurate and timely information for disease management decisions.

As shown from the examples above, molecular technology has proven very valu-able in our plant pathology research program at the Kearney Agricultural Center. One disadvantage of the molecular methodology is that it does require specialized reagents, enzymes, and costly equipment (Polymerase Chain Reaction and Real Time PCR machines) and may be more expensive than the conventional methodology. However, when affordable, portable real-time PCR instruments and simple protocols are developed, routine and efficient diagnosis of many crop diseases or fungicide resistance in pathogen populations can be made on site and within one day, thus reducing the total costs of such tests. In general, molecular technology also helps us in understanding the biology and population structures of plant patho-gens and provides quick and accurate answers to epidemiological questions on plant diseases. Subsequently, these techniques help us in developing effective strategies for disease control.

With many diseases, latent infections were correlated with disease levels in the field and or postharvest. Although significant progress has been made in the dis-covery of conventional methods that help detect latent infections, latent infection detection is based mainly upon subjecting the infected tissues to surface sterilants, and tissue damaging agents (paraquat) or conditions (freezing) followed by incuba-tion. Additionally, there are many variations in the type, number, duration and sequence of these processes. There is a need for faster and more efficient methods


of latent infection detection and the use of real time polymerase chain reaction (RT-PCR) can provide the basis for efficient, accurate, and more rapid detection of pathogens.

There are still gaps in our understanding on the trigger that activates the patho-gen’s growth in latent infections. It is hoped though molecular techniques will eventually replace conventional ones in other patho-systems, help elucidate gaps in epidemiological research, and improve our understanding of plant disease. Future goals of our research are to develop more efficient, accurate, and rapid molecular procedures using RT-PCR and replace the conventional ones. Furthermore, our goal is to reduce costs for processing samples by using such techniques in large numbers of samples, a process that will provide accurate answers to epidemiological ques-tions and allow the expansion of molecular epidemiology in plant disease.

Acknowledgments We thank M. Doster, D. Felts, L. Boeckler, H. Reyes, R. Puckett, and J. Windh for technical assistance. We also thank Drs. Z. Ma, H. Avenot, and E. Boehm, Postdoctoral Associates, and visiting Professor M. Yoshimura, for their research contributions. Funding for this research was provided by the California Kiwifruit Commission, California Pistachio Commission, California Dried Plum Board, California Tree Fruit Agreement, and the California Fig Institute.

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