David B. Langston

Investigating intraspecific variation of Acidovorax avenae subsp. citrulli using DNA fingerprinting and whole cell fatty acid analysis.

ABSTRACT To assess the diversity of Acidovorax avenae subsp. citrulli, 121 strains from watermelon, cantaloupe, and pumpkin were compared using pulse field gel electrophoresis of SpeI-digested DNA and gas chromatographic analysis of fatty acid methyl esters. Twenty-nine unique DNA fragments resulted from DNA digestion, and 14 distinct haplotypes were observed. Based on cluster analysis, two subgroups, I and II, were recognized, which accounted for 84.8% (eight haplotypes) and 15.2% (six haplotypes) of the strains, respectively. Results of cellular fatty acid analysis varied quantitatively and qualitatively for the A. avenae subsp. citrulli strains and supported the existence of the two subgroups. Group I includes strains from cantaloupe and pumpkin as well as the ATCC type strain, which was first described in the United States in 1978, whereas group II represents the typical watermelon fruit blotch-causing strains that appeared in the mainland United States in 1989. Knowledge of the two A. avenae subsp. citrulli groups may be useful in screening for watermelon fruit blotch resistance.

Molecular characterization of boscalid‐ and penthiopyrad‐resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

BACKGROUND Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.

Development of an improved isolation approach and simple sequence repeat markers to characterize Phytophthora capsici populations in irrigation ponds in southern Georgia.

ABSTRACT Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.

First Report of a Fruit Rot of Pumpkin Caused by Acidivorax avenae subsp. citrulli in Georgia

In September 1998, a fruit rot was reported affecting pumpkin (Cucurbita pepo) in a commercial field in Terrell Co., Georgia. Symptoms on the surface of fruit occurred as round, necrotic spots or cracks a few millimeters in diameter. With age, the tissue surrounding these lesions became soft and wrinkled. A soft rot expanded into the flesh of the pumpkin, originating from the lesions observed on the surface. In time, infected pumpkins totally collapsed. V-shaped, necrotic lesions occurred at the margin of the leaf and extended inward toward the mid-rib. Samples were collected from the field and bacteria were isolated from fruit and leaf lesions onto Kings medium B (1). The bacterium isolated was rod shaped, gram negative, nonflourescent, oxidase positive, Tween 80 positive, carboxymethyl cellulose positive, β-OH butyrate positive, and malonate negative. The bacterium reacted positively with polyclonal antibodies specific for the watermelon fruit blotch pathogen Acidivorax avenae subsp. citrulli and was identified as A. avenae subsp. citrulli by MIDI (Microbial Identification System, Newark, DE) according to statistical analysis of fatty acid data. Results from polymerase chain reaction (PCR) amplification of the bacterium isolated from pumpkin yielded 360-bp fragments that, when digested with the restriction enzyme HaeIII, had DNA banding patterns identical to those of stock A. avenae subsp. citrulli DNA. Kochs postulates were completed successfully with 2-week-old watermelon seedlings. This is the first report of A. avenae subsp. citrulli causing fruit rot of pumpkin in Georgia. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

First report of Vidalia onion (Allium cepa) naturally infected with tomato spotted wilt virus and Iris yellow spot virus (family Bunyaviridae, genus Tospovirus) in Georgia.

Vidalia onion is an important crop in Georgias agriculture with worldwide recognition as a specialty vegetable. Vidalia onions are shortday, Granex-type sweet onions grown within a specific area of southeastern Georgia. Tomato spotted wilt virus (TSWV) has been endemic to Georgia crops for the past decade, but has gone undetected in Vidalia onions. Tobacco thrips (Frankliniella fusca) and Western flower thrips (Frankliniella occidentalis) are the primary vectors for TSWV in this region, and a number of plant species serve as reproductive reservoirs for the vector or virus. Iris yellow spot virus (IYSV), an emerging tospovirus that is potentially a devastating pathogen of onion, has been reported in many locations in the western United States (2,4). Thrips tabaci is the known vector for IYSV, but it is unknown if noncrop plants play a role in its epidemiology in Georgia. During October 2003, a small (n = 12) sampling of onions with chlorosis and dieback of unknown etiology from the Vidalia region was screened for a variety of viruses, and TSWV and IYSV infections were serologically detected. Since that time, leaf and bulb tissues from 4,424 onion samples were screened for TSWV and IYSV using double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) with commercial kits (Agdia Inc., Elkhart, IN). Samples were collected from 53 locations in the Vidalia region during the growing season between November 2003 and March 2004. Plants exhibiting stress, such as tip dieback, necrotic lesions, chlorosis or environmental damage were selected. Of these, 306 were positive for TSWV and 396 were positive for IYSV using positive threshold absorbance of three times the average plus two standard deviations of healthy negative onion controls. Positive serological findings of the onion tissues were verified by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) for TSWV (3) and RT-PCR for IYSV (1). In both instances, a region of the viral nucleocapsid (N) gene was amplified. The PCR products were analyzed with gel electrophoresis with an ethidium bromide stain in 0.8% agarose. Eighty-six percent (n = 263) of the TSWV ELISA-positive samples exhibited the expected 774-bp product and 55 percent (n = 217) of the IYSV ELISA-positive samples exhibited the expected 962-bp product. The reduced success of the IYSV verification could be attributed to the age and deteriorated condition of the samples at the time of amplification. Thrips tabaci were obtained from onion seedbeds and cull piles within the early sampling (n = 84) and screened for TSWV by the use of an indirect-ELISA to the nonstructural (NSs) protein of TSWV. Of the thrips sampled, 25 were positive in ELISA. While the incidence of IYSV and TSWV in the Vidalia onion crop has been documented, more research is needed to illuminate their potential danger to Vidalia onions. References: (1) I. Cortês et al. Phytopathology 88:1276, 1998. (2) L. J. du Toit et al. Plant Dis. 88:222, 2004. (3) R. K. Jain et al. Plant Dis. 82:900, 1998. (4) J. W. Moyer et al. (Abstr.) Phytopathology 93(suppl.):S115, 2003.

Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.

First demonstration of Koch's postulates for Lasiodiplodia theobromae fruit spot on eggplant (Solanum melongena).

During October 2004, diseased eggplant fruit from a commercial farm in Colquitt County, Georgia, developed circular, tan, water-soaked lesions. Gray, septate mycelia quickly covered the fruit. Diseased fruit became shriveled, spongy, and mummified. Disease incidence in the field was approximately 1%. Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (synonym Botryodiplodia theobromae Pat.) (2) was isolated from the margins of lesions and cultured on acidified potato dextrose agar. The fungus produced grayish colonies with aerial hyphae and black ostiolate pycnidia massed into stroma. Mature elliptical conidia (25.8 × 15.6 μm) were brown, had a single septation, and longitudinal striations. Isolates obtained from peanut and pecan were included in the pathogenicity tests. Mature fruit cv. Nightshade were surface disinfested for 30 s in 70% ethanol, followed by 60 s in 0.5% sodium hypochlorite, rinsed twice in sterile distilled water, and allowed to dry. Inoculations were made by placing an agar plug containing L. theobromae mycelial side down on the surface of the fruit or wounding with a sterile toothpick containing mycelium of the fungus. Fruit similarly inoculated with agar plugs or sterile toothpicks served as controls. There were a total of three replicates. Fruit were placed in plastic containers lined with moistened paper towels. Containers were placed in a dew chamber and incubated (28°C, relative humidity >95%) for 3 days, and then evaluated. Symptoms identical to those observed on naturally infected fruit developed on inoculated fruit. Controls remained disease free. L. theobromae was reisolated from all symptomatic tissue, satisfying Kochs postulates. Disease damage on wounded fruit was twice that of nonwounded fruit. However, seven of nine inoculations with agar plugs containing L. theobromae resulted in infection. Lesion lengths from wound inoculations were 9.8, 7.3, and 5.2 cm for isolates from peanut, pecan, and eggplant, respectively. Generally, L. theobromae is considered a facultative wound pathogen or a secondary invader (3). However, this study suggests that direct infection can occur. Although fruit spot has been reported previously on eggplant (1), to our knowledge, this is the first report verifying L. theobromae as the causal agent. References: (1) S. A. Alfieri et al. Index of Plant Diseases in Florida. Fla. Dep. Agric. Consum. Serv. Bull. 11, 1984. (2) H. L. Barnett and B. B. Hunter. Illustrated Guide of Imperfect Fungi. 4th ed. The American Phytopathological Society St. Paul, MN, 1998. (3) P. M. Phipps and D. M. Porter. Plant Dis. 82:1205, 1998.

Interactions of Seedborne Bacterial Pathogens with Host and Non-Host Plants in Relation to Seed Infestation and Seedling Transmission

The ability of seed-borne bacterial pathogens (Acidovorax citrulli, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria, and Pseudomonas syringae pv. glycinea) to infest seeds of host and non-host plants (watermelon, tomato, pepper, and soybean) and subsequent pathogen transmission to seedlings was investigated. A non-pathogenic, pigmented strain of Serratia marcescens was also included to assess a null-interacting situation with the same plant species. Flowers of host and non-host plants were inoculated with 1×106 colony forming units (CFUs)/flower for each bacterial species and allowed to develop into fruits or umbels (in case of onion). Seeds harvested from each host/non-host bacterial species combination were assayed for respective bacteria by plating on semi-selective media. Additionally, seedlots for each host/non-host bacterial species combination were also assayed for pathogen transmission by seedling grow-out (SGO) assays under greenhouse conditions. The mean percentage of seedlots infested with compatible and incompatible pathogens was 31.7 and 30.9% (by plating), respectively and they were not significantly different (P = 0.67). The percentage of seedlots infested with null-interacting bacterial species was 16.8% (by plating) and it was significantly lower than the infested lots generated with compatible and incompatible bacterial pathogens (P = 0.03). None of the seedlots with incompatible/null-interacting bacteria developed symptoms on seedlings; however, when seedlings were assayed for epiphytic bacterial presence, 19.5 and 9.4% of the lots were positive, respectively. These results indicate that the seeds of non-host plants can become infested with incompatible and null-interacting bacterial species through flower colonization and they can be transmitted via epiphytic colonization of seedlings. In addition, it was also observed that flowers and seeds of non-host plants can be colonized by compatible/incompatible/null-interacting bacteria to higher populations; however, the level of colonization differed significantly depending on the type of bacterial species used.

Relationship Between Fungicide Sensitivity and Control of Gummy Stem Blight of Watermelon Under Field Conditions

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is the most destructive disease of watermelon and is managed primarily with fungicides. D. bryoniae has developed resistance to many fungicides that were once very effective, including azoxystrobin, boscalid, and thiophanate-methyl. Field experiments were conducted in Tifton (TN) and Reidsville (RV), GA in 2009 and 2010 to establish a relationship between frequency of resistance to a fungicide based on in vitro assays and its efficacy in the management of GSB. Frequency of resistance to boscalid, thiophanate-methyl, and azoxystrobin was >0.80 in isolates collected from nontreated plots in both locations and years. All isolates collected after six applications of boscalid, thiophanate-methyl, or azoxystrobin were resistant to the respective fungicide. All isolates collected from treated and nontreated plots were sensitive to tebuconazole and difenoconazole. GSB severity was assessed on a weekly basis from 63 days after planting. GSB severity in plots treated with boscalid, thiophanate-methyl, or azoxystrobin was not significantly different from that in the nontreated plots (39%, TN-2009; 45%, TN-2010; and 16%, RV-2010). GSB severity in tebuconazole-treated plots (27%, TN-2009; 14%, TN-2010; and 4%, RV-2010) was significantly lower than all other treatments and the nontreated control. There was a consistent negative association between frequency of fungicide resistance and disease control in the field. Thus, knowledge of the frequency of fungicide resistance in the pathogen population will be helpful in selecting the most effective fungicides for the management of GSB in watermelon fields.

First Report of a Leaf Spot and Stem Canker Caused by Myrothecium roridum on Watermelon in the United States

Myrothecium roridum Tode:Fr, pathogenic to a number of cucurbit species, causes fruit rots, cankers on crowns and stems, and leaf spots. Hosts include cantaloupe and honeydew (Cucurbita melo) and cucumber (Cucumis sativus) (1,3). In June 2004, following a period of heavy rainfall, numerous round-to-oblong, brown lesions with concentric rings were observed on leaves of watermelon (Citrullus lanatus) cv. Desert King at the Blackshank Farm in Tifton, GA. Disease was localized in the field and severity was low (<5% of leaf area affected). No symptoms were observed on fruit. Sections of tissue were removed from the margin between healthy and diseased tissue and plated on acidified, 25% potato dextrose agar (aPDA). A small plug of agar and mycelium were removed from colonies that emerged from lesions and were transferred to aPDA. Isolated colonies were characterized by a white, floccose mycelium with concentric, dark green-to-black rings of sporodochia bearing viscid masses of conidia. Conidia were cylindrical with rounded ends and measured 6 to 8 × 1.5 to 2.5 μm. The features of the fungus were consistent with the description of Myrothecium roridum (1,2). Pathogenicity tests were conducted in a temperature-controlled greenhouse. Twenty-five watermelon plants (cv. Desert King) were inoculated with a conidial suspension of M. roridum (5 × 105 conidia per ml) plus 0.1% vol/vol Tween 20. Inoculum was applied on leaves and stems until runoff with a hand-held mister, and plants were placed in a dew chamber for 72 h. Ten plants were sprayed with sterile, distilled water to serve as controls. Inoculated and noninoculated control plants were removed from the dew chamber and maintained at 25 to 28°C. Symptoms appeared 8 days after inoculation and were characterized by round, dark lesions with concentric rings; noninoculated plants were symptomless. Sections of symptomatic tissue were plated, and M. roridum was reisolated. Although M. roridum is a common pathogen of melons and cucumber, to our knowledge, this is the first field report of a leaf spot caused by M. roridum on watermelon in the United States. No further occurrences of the disease on watermelon have been observed in Georgia since the initial discovery of M. roridum in 2004; however, losses could be potentially severe if widespread infection of fruit were to occur. References: (1) B. D. Bruton. Crater Rot. Pages 49-50 in: Compendium of Cucurbit Diseases. T. A. Zitter et al., eds. The American Phytopathological Society, St. Paul, MN, 1996. (2) M. B. Ellis. Page 552 in: Dematiaceous Hyphomycetes. CAB International, Wallingford, UK, 1971. (3) D. F. Farr et al. Page 809 in: Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.