Methods cont. Pathogenicity on detached pods Snap bean pods were washed in 0.6% sodium hypochlorite, rinsed twice in distilled water, and allowed to dry. Beans were wounded with a 1 mm needle to the depth of 5 mm. A 5-mm plug from the edge of a 3-day colony of S. sclerotiorum on PDA was placed on the wound. Beans were placed in a moist chamber and stored in the dark at room temperature. After 3 days, the lesions were measured at the longest point along the length of the pod (Fig. 4). Each isolate was used to inoculate 4 pods and the entire experiment was conducted twice.

Pathogenicity on detached leaves Three to four-week old detached bean leaves (var.

were placed in a petri dish with a filter paper moistened with 2 mL sterile distilled water. Leaves were wounded with a 1 mm needle, inoculated as described for the pods, and incubated in the dark at room temperature. At 3 days post inoculation, the percent of the leaf surface covered by lesions was assessed visually (Fig. 5). Data Analysis The effect of isolate was analyzed in both the pod and leaf assays using analysis of variance. Means were separated with

honestly significant differences test (alpha = 0.05) using the agricolae packing in the statistical program, R. The correlation between results from the pod and leaf assays was analyzed by rank correlation test, also using R. In vitro fungicide sensitivity Eight isolates were screened for sensitivity to the fungicide, thiophanate-methyl. Five of the isolates 1103 1102 1104 1099 and 1105 came from a field where thiophanate-methyl was applied and poor control resulted. The remaining isolates were collected at least 20 years earlier and were unlikely to be resistant to thiophanate-methyl. For each isolate, a 5 mm plug from a 3-day old culture was transferred to both unamended PDA, and PDA amended with 10 mg/L thiophanate-methyl. Three replicate plates per isolate were used and incubated at room temperature in the dark for 2 days. Two perpendicular measurements of colony diameter were recorded and averaged. The effect of isolate and fungicide on colony diameter was analyzed by analysis of variance as described above.

Fig 1. Lifecycle of Sclerotinia sclerotiorum, cause of white mold on snap beans (3).

Introduction In crop was worth 55.5 million dollars (1). Snap beans are susceptible to the fungus Sclerotinia sclerotiorum, which causes the disease white mold (Fig. 1, 2). Cultural practices and prophylactic application of fungicides are used for white mold management (3). Thiophanate-methyl is the active ingredient used in a fungicide commonly used to control white mold (Topsin® M). However, suboptimal control has lead to concerns of fungicide resistance. Since 1973, isolates of S. sclerotiorum have been collected from a variety of hosts and locations within New York State. The objectives of this study were to: (i) condition sclerotia for ascospore production to test fungicide efficacy; and (ii) quantify aggressiveness and fungicide sensitivity of a subset of isolates from the collection.

Variation of phenotypic characteristics in populations of Sclerotinia sclerotiorum causing white mold of snap bean in New York

Jenna K. Van Bruggen1 Amara R. Dunn2, Christine D. Smart2, and Sarah J. Pethybridge2

1Calvin College, Grand Rapids, MI 49546; 2Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456

Abstract White mold, caused by Sclerotinia sclerotiorum, is one of the most economically damaging diseases of snap bean (Phaseolus vulgaris) and other vegetables. The goals of this project were to compare aggressiveness on snap beans among different isolates of S. sclerotiorum, and test for resistance to a commonly-used fungicide. Sclerotia were also conditioned to carpogenically germinate for the production of ascospores for use in field trials quantifying fungicide efficacy. Seventeen isolates were used in detached pod and leaf assays which quantified aggressiveness by measuring lesion size. The reproducibility of results between replicates from the pod assay was higher than using leaves. Moreover, in the leaf and pod assays, results were similar for some of the isolates. Significant variation in aggressiveness between isolates was also found. Of the isolates (n = 8), all were sensitive to the fungicide thiophanate-methyl at 10 mg/L based on a mycelial growth assay. Ascospore production and sclerotial conditioning was also conducted, and ascospores field trial.

Methods Thirty-two isolates of Sclerotinia sclerotiorum were cultured on Potato Dextrose Agar (PDA) after 12 to 22 years of storage. Isolates were hyphal tipped using a dissecting microscope to ensure that each isolate was a single genetic individual. These isolates were then used for testing of aggressiveness and fungicide sensitivity. Ascospore Production Ascospores were obtained from 15 isolates (prior to hyphal tipping) following the protocol described by Cobb and Dillard (1; Fig. 3).

Fig. 3. Chronological description of ascospore production for Sclerotinia sclerotiorum. (A) agar plugs from 2-3 day old colonies were placed on cornmeal-based media; (B) sclerotia were harvested with a sieve, and allowed to dry; (C) sclerotia were placed in cheese (D) sclerotia were placed on wet, sterile sand in petri dishes where the apothecia emerge; (E) apothecia forcibly ejected ascospores into the air; (F) ascospores were collected with a

funnel and vacuum filtration; and (G) ascospores were filtered onto 2 filter paper, and stored at -15ºC.

A B C D E F G

Results Ascospores were collected from 15 different isolates and were used for field inoculations on 24 July to quantify fungicide efficacy for disease management. The aggressiveness of isolates on pods varied significantly (P <0.0001). The aggressiveness of isolate

the noninoculated control, and three isolates and longer lesions than the other isolates. Results from the leaf assay were less

reproducible than the pod assay. For example, only 5 of the 13 isolates differed significantly from the noninoculated control (Table 1). There was a significant correlation in isolate aggressiveness between the bean pod and leaf assays ( = 0.67, P = 0.025; Fig. 6). There was little growth on thiophanate-methyl-amended PDA from any of the isolates. Moreover, colony

other isolates (Fig. 7).

c

a

ab

d

abc

bc

abc

ab

e

e

e

e

e

e

e

e

0.00 2.00 4.00 6.00 8.00

1105

48 x

1099 A

307 x

25 A

1104 A

1102 A

1103 B

Colony Diameter (cm)

Isol

ate

Nam

e

PDA + 10 mg/L of Thiophanate-methylPDA

Fig. 7. Effect of isolate and fungicide on in vitro growth of Sclerotinia sclerotiorum. Means followed by the same letter are not significantly

different; alpha = 0.05.

Conclusions Isolates of S. sclerotiorum differ in aggressiveness on snap bean pods and leaves. - and were consistently more aggressive in both assays.

Results from the pod assays were more reproducible than the leaf assay. Isolates tested were sensitive to thiophanate-methyl.

Fig. 4. Detached pod assay used for quantifying aggressiveness of

Sclerotinia sclerotiorum.

Fig. 5. Detached leaf assay used for quantifying aggressiveness of

Sclerotinia sclerotiorum.

Fig. 6. Correlation between leaf and bean pod

correlation test.

Table 1. Aggressiveness of Sclerotinia sclerotiorum isolates based on bean pod and leaf assays. Means

followed by the same letter are not significantly different; alpha = 0.05.

Isolate Mean lesion length

on pod (cm) Mean % lesion

on leaf 531 B 7.5 a 1107 A 6.8 ab 53.8 ab 1124 B 6.0 abc 74.8 a

48 x 4.8 bcd 38.4 abc 29 4.6 bcd 28.4 bcd

1102 A 4.6 bcd 1104 A 4.4 cd 55.0 ab 1099 A 4.2 cd 73.8 a 529 B 4.0 cd 1108 3.8 cd

1103 B 3.4 de 19.4 bcd 1105 A 3.3 de 9.3 cd 1110 A 3.2 de 307 x 3.1 de 10.5 cd 532 B 3.1 de 528 A 3.0 de 25 A 1.2 ef 24.8 bcd

Noninoc. 0.0 f 0.0 d 0

1

2

3

4

5

6

7

8

0 20 40 60 80

Leng

th o

f les

ion

on b

ean

pod

(cm

)

% of leaf covered with lesion

Fig. 2. Sclerotinia sclerotiorum infecting snap beans in the field.

Photo courteously of the Dillard lab.

= 0.67 P = 0.025

Literature Cited 1. USDA-NASS. 2014. Vegetables: 2013 summary. USDA, National Agricultural Statistics Service, Washington, DC. 2. Cobb and Dillard, 2004. The Plant Health Instructor. DOI: 10.1094/PHI-T-2004-0604-01. 3. Heffer Link, and Johnson. 2007. The Plant Health Instructor. DOI: 10.1094/PHI-I-2007-0809-0

Acknowledgments I would like to thank all of those who helped make this project possible, including Professor Helene Dillard for maintaining the S. sclerotiorum collection, and the members of the C. Smart and S. Pethybridge labs. This project was supported by the New York Vegetable Research Association/Council and by the Specialty Crops Research Initiative competitive grant No. 2012-51181-20001 from the USDA National Institute of Food and Agriculture.

Apothecia emerge from sclerotia located near soil surface.

Ascospores are released and blown by wind

Ascospores infect aerial plant parts,

aided by food energy

obtained from flower petals

Lesion development and expansion

Maturation of sclerotia on and in diseased tissue

Hyphae produced by sclerotia infect crowns and basal stems of nearby plants

Sclerotia accumulate in soil as plant material decays

Sclerotium overwintering in soil