First Report of Pythium deliense Associated with Peanut Pod Rot in Georgia.

In fall 2012 and 2013, peanut (Arachis hypogaea L.) grown in commercial fields in Tift County, GA, showed pod rot symptoms. The disease was primarily damaging pods and kernels and symptoms included brown to black, water-soaked lesions on pods and blackened pegs with white fluffy mycelia. Ten random symptomatic pods collected from the field were plated on potato dextrose agar after surface sterilization with 0.5% NaOCl. The plates were incubated at 25°C for 5 days in the dark. Whitish fungus-like cultures with non-septate mycelium grew from all the pods. Single hyphal tip cultures were obtained on pimaricin-ampicillin-rifampicin-pentachloronitrobenzene (PARP) medium. Isolates on PARP agar plates had three different growth patterns: two groups of isolates produced sporangia and the third group produced oogonia. The isolates were identified as Pythium spp. based on growth pattern and sporangial and oogonial structures (2). DNA of one representative isolate from each group was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA were amplified and sequenced with primers ITS1 and ITS4 (1). ITS sequences of the isolates shared 99 to 100% similarity with Pythium ultimum, P. vexans, and P. deliense isolates in GenBank (Accession Nos. KC689906, GU133594, and HQ643521, respectively). The isolates were identified as P. ultimum var. ultimum, P. vexans, and P. deliense based on molecular analysis and morphological characteristics. P. ultimum produced plenty of spherical sporangia, but no oogonia in the culture, P. deliense produced characteristic terminal oogonia and aplerotic oospores with oogonial stalks curved towards the antheridia, and P. vexans produced spherical sporangia and aplerotic oospores. ITS sequences of three isolates representing each of the three species were deposited in GenBank (KF500573, KF500574, and KF500572). Pathogenicity of one representative isolate from each group was tested on peanut under greenhouse conditions (30°C day and 20°C night). Nine 10-week-old peanut seedlings (cv. GA07W) grown in 20-cm pots (2:1 ratio of potting mix/sterilized field soil) were inoculated with the isolates separately by applying 5 ml of respective Pythium-infested beet seeds. Nine untreated plants were used as a control. Pods were washed off 1 month after inoculation for disease assessment. All plants inoculated with the isolates showed pod rot similar to those observed in the field. The three Pythium species were re-isolated from respective symptomatic pods and the identity was confirmed by morphological characteristics and molecular analysis. The untreated plants did not show typical pod rot symptoms and the Pythium species were not isolated from these plants. P. ultimum and P. vexans have been reported to be associated with peanut pod rot in the United States (3). To our knowledge, this is the first report of P. deliense causing peanut pod rot. Georgia is the top peanut producer in the United States and the occurrence of pod rot caused by the Pythium spp. needs to be taken into account in developing disease management programs in peanut production. References: (1) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (2) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:51, 1981. (3) T. A. Wheeler et al. Peanut Sci. 32:9, 2005.

Induction and Maintenance of Systemic Acquired Resistance by Acibenzolar-S-Methyl in Three Cultivated Tobacco Types.

Induction and maintenance of systemic acquired resistance (SAR) in 'N' gene containing burley, flue-cured, and oriental tobacco cultivars were assessed by monitoring decreases in the number of local lesions caused by Tobacco mosaic virus (TMV) following treatment with acibenzolar-S-methyl (ASM). Leaf samples were collected from lower, middle, and top positions on seedlings at 3-day intervals over 21 days following ASM treatment and subsequent inoculation with TMV under laboratory conditions. Local lesion number for each leaf was recorded 7 days postinoculation. Reductions in TMV local lesion numbers on ASM-treated versus nontreated tobacco varied over time, and differed for each tobacco type. Based on reduced local lesion numbers, SAR was induced in burley and flue-cured tobacco by 3 and 6 days postinoculation, respectively, while oriental tobacco responded by 9 days. SAR was maintained in burley tobacco from 3 to 9 days after ASM application, and from 9 to 15 days after application in oriental tobacco. ASM treatment reduced local lesion numbers in flue-cured tobacco significantly at 6, 12, and 21 days postapplication, but not at 15 and 18 days after treatment. The SAR response was similar among lower, middle, and top leaves with no effect of ASM on response by leaf position, although TMV local lesion numbers were greater on lower leaves than on middle and top leaves 6 days after treatment, but significantly less on lower leaves 18 days after treatment compared to middle and top leaves.

First Report of Alternaria Leaf Spot of Banana Caused by Alternaria alternata in the United States.

Research efforts were initiated in 2003 to identify and introduce banana (Musa spp.) cultivars suitable for production in Georgia (1). Selected cultivars have been evaluated since 2009 in Tifton Banana Garden, Tifton, GA, comprising of cold hardy, short cycle, and ornamental types. In spring and summer of 2012, 7 out of 13 cultivars (African Red, Blue Torres Island, Cacambou, Chinese Cavendish, Novaria, Raja Puri, and Veinte Cohol) showed tiny, oval (0.5 to 1.0 mm long and 0.3 to 0.9 mm wide), light to dark brown spots on the adaxial surface of the leaves. Spots were more concentrated along the midrib than the rest of the leaf and occurred on all except the newly emerged leaves. Leaf spots did not expand much in size, but the numbers approximately doubled during the season. Disease incidences on the seven cultivars ranged from 10 to 63% (10% on Blue Torres Island and 63% on Novaria), with an average of 35% when a total of 52 plants were evaluated. Six cultivars including Belle, Ice Cream, Dwarf Namwah, Kandarian, Praying Hands, and Saba did not show any spots. Tissue from infected leaves of the seven cultivars were surface sterilized with 0.5% NaOCl, plated onto potato dextrose agar (PDA) media and incubated at 25°C in the dark for 5 days. The plates were then incubated at room temperature (23 ± 2°C) under a 12-hour photoperiod for 3 days. Grayish black colonies developed from all the samples, which were further identified as Alternaria spp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 23 to 73 µm long and 15 to 35 µm wide, with a beak length of 5 to 10 µm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures of four isolates from four different cultivars were obtained and genomic DNA was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA (562 bp) were amplified and sequenced with primers ITS1 and ITS4. MegaBLAST analysis of the four sequences showed that they were 100% identical to two Alternaria alternata isolates (GQ916545 and GQ169766). ITS sequence of a representative isolate VCT1FT1 from cv. Veinte Cohol was submitted to GenBank (JX985742). Pathogenicity assay was conducted using 1-month-old banana plants (cv. Veinte Cohol) grown in pots under greenhouse conditions (25 to 27°C). Three plants were spray inoculated with the isolate VCT1FT1 (100 ml suspension per plant containing 105 spores per ml) and incubated under 100% humidity for 2 days and then kept in the greenhouse. Three plants sprayed with water were used as a control. Leaf spots identical to those observed in the field were developed in a week on the inoculated plants but not on the non-inoculated control. The fungus was reisolated from the inoculated plants and the identity was confirmed by morphological characteristics and ITS sequencing. To our knowledge, this is the first report of Alternaria leaf spot caused by A. alternata on banana in the United States. Occurrence of the disease on some banana cultivars in Georgia provides useful information to potential producers, and the cultivars that were observed to be resistant to the disease may be more suitable for production. References: (1) E. G. Fonsah et al. J. Food Distrib. Res. 37:2, 2006. (2) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

First Report of Leaf Spot on Onion Caused by Fusarium acuminatum in Georgia.

An unknown disease was observed on shallot onions (Allium cepa L.) in Tattnall County, Georgia, in September of 2012. Disease symptoms included leaf tip die back and scattered light brown lesions on leaves, oval to round in shape, with a size ranging from 0.5 to 2.0 cm. The disease occurred in approximately 1.2 ha of commercial onion fields, and disease incidence ranged from 50 to 70%. Infected leaf tissues from 10 plants were surface sterilized with 0.5% NaOCl and plated on quarter-strength potato dextrose agar (QPDA). The fungus grew on all plates, producing bright orange to rose pigmentation in the medium after incubation at 25°C for 5 days. The fungus produced sickle shaped macroconidia with 3 to 6 septa, 4 to 5 µm wide, and 30 to 54 µm in length. Chlamydospores were formed in chains and averaged 20.4 × 16.8 µm. The fungus was identified as Fusarium sp. based on morphological characteristics (3). Genomic DNA was extracted from single conidial cultures of three representative isolates and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS2) were amplified and sequenced with primers ITS1 and ITS4 (2). MegaBLAST analysis of the sequences showed that they were 100% identical to a Fusarium acuminatum isolate (Accession No. JN624894). ITS sequence of an isolate FAON-1 was deposited in GenBank (KC477845). Pathogenicity tests were performed with the three isolates grown on QPDA at 25°C for 7 days. Eight-week-old shallot onion seedlings were inoculated by foliar spray with conidial suspensions at 2 × 106 spores/ml (5 ml per plant). Ten plants were inoculated with each isolate and 10 plants were sprayed with water as a control. The plants were incubated in a humidified chamber at (25 ± 3°C) with >95% relative humidity and 12-h photoperiod for 48 h after inoculation, and then kept in a greenhouse at 22 ± 2°C. Inoculated plants started to show symptoms identical to those observed in the field 7 days after inoculation, and disease incidence reached 100% within 14 days. No disease occurred on the control plants. The fungus was reisolated from the diseased plants and confirmed to be F. acuminatum based on morphological characteristics and molecular identification. Onion was previously reported to be a host of F. acuminatum in Montana (4). To our knowledge, this study is the first report confirming F. acuminatum causing disease on onion in Georgia. Onion is a major vegetable crop in Georgia with an annual production of approximately 5,000 ha (1). Occurrence of leaf spot caused by F. acuminatum and the impact of the disease needs to be considered in developing and implementing disease management programs in onion production. References: (1) S. R. Boatright and J. C. Mckissick, Univ. of Georgia CAES Publication AR-10-02, 2010. (2) M. A. Innes et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 1990. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) C. G. Shaw. Washington State Univ. Agric. Exp. Sta. Bull. 765:12, 1973.

First Report of Target Spot Caused by Corynespora cassiicola on Cotton in Georgia.

In 2005, crop consultants in southwestern Georgia reported an unusual occurrence of leaf spot in cotton (Gossypium hirsutum L.). Initial symptoms first developed as brick red dots that led to the formation of irregular to circular lesions with tan-to-light brown centers. Lesions further enlarged and often demonstrated a targetlike appearance formed from concentric rings within the spot. Observations included estimates of premature defoliation up to 70%, abundant characteristic spots on the leaves and bracts, and losses of several hundred kg of lint/ha. When symptomatic leaves were submitted to the University of Georgia Tifton Plant Disease Clinic in Tifton, GA, for identification in 2008, the causal agent was tentatively diagnosed as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei on the basis of similar symptoms and signs previously reported on cotton (3). In September 2011, symptomatic leaves were obtained from diseased cotton within a field (var. DP 1048B2RF) near Attapulgus, GA. Symptomatic tissue from diseased leaves was surface disinfested in 0.5% sodium hypochlorite for 1 min and plated on potato dextrose agar (PDA). Ten isolates were incubated at 21.1°C for 2 weeks with a 12/12 h light/dark cycle using fluorescent light located approximately 70 cm above the cultures. After 1 week, two isolates were transferred to quarter strength PDA for enhanced sporulation and were grown under the same conditions. Conidiophores from the isolated fungus were simple, erect, intermittently branching and septate, and gave rise to single, subhyaline conidia. Conidia had 4 to 17 pseudosepta and were 50 to 197 µm long and 7 to 16 µm wide, straight to curved, and obclavate to cylindrical. Pathogenicity tests were conducted by spraying 10 cotton seedlings (DP 555BR and DP 1048B2RF, two to four true leaf stage) until runoff with a blended suspension from a 2-week-old pure culture of the fungus diluted with 100 mL of sterile water. Five plants were sprayed with sterile water as noninoculated controls. Cotton seedlings were then incubated in a moist chamber at 21.1°C for 48 h. Within 1 week, all inoculated plants showed symptoms similar to those of diseased field plants. Symptoms were not observed on noninoculated control plants. The fungus was reisolated five times from symptomatic leaves and grown in pure culture. Conidia and conidiophores were identical to the morphology of the original isolates, and were similar to descriptions of C. cassiicola (2). To confirm the identity of the pathogen, DNA was extracted from a week-old culture and amplified with specific primers for loci "ga4" and "rDNA ITS" (1). DNA sequences obtained with the Applied Biosystems 3730xl 96-capillary DNA Analyzer showed 99% identity to C. cassiicola from BLAST analysis in GenBank. The resulting sequence was deposited into GenBank (Accession No. JQ717069). To our knowledge, this is the first report of this pathogen in Georgia. Given the increasing prevalence of this disease in southwestern Georgia, its confirmation is a significant step toward management recommendations for growers. Because foliar diseases caused by C. cassiicola are commonly referred to as "target spot" in other crops (e.g., soybeans), it is proposed that Corynespora leaf spot of cotton be known as "target spot of cotton." References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) M. B. Ellis and P. Holliday. CMI Description of Pathogenic Fungi and Bacteria, 303, 1971. (3) J. P. Jones. Phytopathology 51:305, 1961.

Population Structure, Mating Type, and Mefenoxam Sensitivity of Phytophthora nicotianae in Virginia Tobacco Fields.

Black shank is an important disease of tobacco (Nicotiana tabacum) caused by the fungus-like organism, Phytophthora nicotianae. Three physiological races (0, 1, and 3) have been documented in the United States. Shifts in the pathogen population structure have become a concern due to the widespread use of cultivars possessing resistance to race 0 arising from a single gene (Php or Phl). A comprehensive statewide survey conducted throughout major tobacco-growing areas during summer 2006 and supplemented by additional isolates in 2007 and 2008 yielded 217 isolates from flue-cured, burley, and dark fire-cured tobacco fields. After determining species identity using a single-strand conformational polymorphism fingerprinting technique, the race identity of isolates was assessed via greenhouse tests using three differential cultivars (Hicks, L8, and NC1071). Approximately 76% of the isolates belonged to race 1, 21% to race 0, and the remaining 3% were race 3. This race structure was comparable with those in the other tobacco-producing states in the United States. Approximately 94% of isolates belonged to A2 mating type and merely 6% were A1. These data suggest that it is unlikely that sexual recombination serves as a major mechanism enhancing the genetic diversity of the pathogen in Virginia. All isolates were also evaluated against mefenoxam at 5 µg/ml. None were insensitive; 98% of isolates were either highly sensitive or sensitive and the remaining 2% were intermediately sensitive. These results indicate that mefenoxam remains effective for control of black shank in Virginia. The results of this study can assist breeders to develop cultivars possessing the most appropriate set of disease resistance traits, as well as extension specialists, county agents, and tobacco growers in their decision-making process to manage tobacco black shank in Virginia.