Bhabesh Dutta

Transmission of Pantoea ananatis and P. agglomerans, causal agents of center rot of onion (Allium cepa), by onion thrips (Thrips tabaci) through feces.

Frankliniella fusca, the tobacco thrips, has been shown to acquire and transmit Pantoea ananatis, one of the causal agents of the center rot of onion. Although Thrips tabaci, the onion thrips, is a common pest of onions, its role as a vector of P. ananatis has been unknown. The bacterium, P. agglomerans, is also associated with the center rot of onion, but its transmission by thrips has not been previously investigated. In this study, we investigated the relationship of T. tabaci with P. ananatis and P. agglomerans. Surface-sterilized T. tabaci were provided with various acquisition access periods (AAP) on onion leaves inoculated with either P. ananatis or P. agglomerans. A positive exponential relationship was observed between thrips AAP duration and P. ananatis (R² = 0.967; P = 0.023) or P. agglomerans acquisition (R² = 0.958; P = 0.017). Transmission experiments conducted with T. tabaci adults indicated that 70% of the seedlings developed center rot symptoms 15 days after inoculation. Immunofluorescence microscopy with antibodies specific to P. ananatis revealed that the bacterium was localized only in the gut of T. tabaci adults. Mechanical inoculation of onion seedlings with fecal rinsates alone produced center rot but not with salivary secretions. Together these results suggested that T. tabaci could efficiently transmit P. ananatis and P. agglomerans.

Location of Acidovorax citrulli in Infested Watermelon Seeds Is Influenced by the Pathway of Bacterial Invasion

Watermelon seeds can become infested by Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits via penetration of the ovary pericarp or by invasion of the pistil. This study investigated the effect of these invasion pathways on A. citrulli localization in seeds. Seed samples (n = 20 or 50 seeds/lot) from pistil- and pericarp-inoculated lots were dissected into testa, perisperm-endosperm (PE) layer, and embryo tissues and tested for A. citrulli by species-specific polymerase chain reaction (PCR) and by plating on semiselective media. Less than 8% of the testa samples were A. citrulli-positive regardless of the method of seed inoculation. Additionally, the difference in percentages of contaminated testae between the two seed lot types was not significant (P = 0.64). The percentage of A. citrulli-positive PE layer samples as determined by real-time PCR assay was significantly greater for seeds from pistil-inoculated lots (97%) than for seeds from pericarp-inoculated lots (80.3%). The mean percentage of A. citrulli-positive embryo samples was significantly greater for seeds from pistil-inoculated lots (94%) than for seeds from pericarp-inoculated lots (≈8.8%) (P = 0.0001). Removal of PE layers and testae resulted in a significant reduction in BFB seed-to-seedling transmission percentage for seeds from pericarp-inoculated lots (14.8%) relative to those from pistil-inoculated lots (72%). Additionally, using immunofluorescence microscopy, A. citrulli cells were observed in the PE layers and the cotyledons of pistil-inoculated seeds but only in the PE layers of pericarp-inoculated seeds. These results suggest that pericarp invasion results in superficial contamination of the testae and PE layers while pistil invasion results in the deposition of A. citrulli in seed embryos.

Acidovorax citrulli Seed Inoculum Load Affects Seedling Transmission and Spread of Bacterial Fruit Blotch of Watermelon Under Greenhouse Conditions

Infested seed are typically the primary source of inoculum for bacterial fruit blotch (BFB) of cucurbits. An inoculum threshold of 1 infested seed per 10,000 seeds is widely used in seed health testing for Acidovorax citrulli. However, the influence of seed inoculum load on BFB seedling transmission has not been elucidated. In this study, watermelon seedlots (128 seeds/lot) containing one seed inoculated with A. citrulli at levels ranging from 1 × 101 to 1 × 107 CFU were used to investigate the effect of seed inoculum load on seedling transmission and spatiotemporal spread of BFB under greenhouse conditions. The relationship between A. citrulli seed inoculum load and frequency of BFB seedling transmission followed a sigmoidal pattern (R2 = 0.986, P = 0.0047). In all, 100 and 96.6% of seedlots containing one seed with 1 × 107 and 1 × 105 CFU of A. citrulli, respectively, transmitted the pathogen to seedlings; in contrast, the proportion of seedlots that yielded BFB-infected seedlings was lower for lots with one seed infested with 1 × 103 (46.6%) and 1 × 101 (16.7%) CFU of A. citrulli. The relationship between A. citrulli seed inoculum load and frequency of pathogen detection in seedlots using immunomagnetic separation combined with a real-time polymerase chain reaction assay also followed a sigmoidal pattern (R2 = 0.997, P = 0.0034). Whereas 100% of samples from seedlots (10,000 seeds/lot) with one seed containing ≥1 × 105 CFU tested positive for A. citrulli, 75% of samples from lots with one seed containing 1 × 103 CFU tested positive for the pathogen, and only 16.7% of samples with one seed containing 10 CFU tested positive. Because disease transmission was observed for lots with just one seed containing 10 A. citrulli CFU, zero tolerance for seedborne A. citrulli is recommended for effective BFB management. The seedling transmission experiments also revealed that temporal spread of BFB in 128-cell seedling trays increased linearly with A. citrulli inoculum load (r2 = 0.976, P = 0.0037). Additionally, the frequency of spatial spread of BFB from an inoculated seedling in the center of a planting tray to adjacent healthy seedlings over one-, two-, or three-cell distances was greater for lots with one seed infested with at least 1 × 105 CFU than for lots with one seed infested at lower inoculum loads (1 × 101 and 1 × 103 CFU/seed).

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.

Role of Blossom Colonization in Pepper Seed Infestation by Xanthomonas euvesicatoria

Colonization of Xanthomonas euvesicatoria was investigated in pepper blossoms and the relationship between inoculum concentrations and seed infestation was determined. Inoculation of blossoms resulted in asymptomatic pepper fruit. However, real-time polymerase chain reaction detected X. euvesicatoria in 39% of the seed lots assayed and viable colonies were recovered from 35% of them. Successful transmission occurred in 16% of the seed lots tested. In a separate experiment, X. euvesicatoria reached populations of up to 1 × 10(5) CFU/blossom on stigmas 96 h after inoculation. Bacteria colonized stylar and ovary tissues with populations ranging from 1 × 10(5) to 1 × 10(6) CFU/blossom 96 h after inoculation. A positive correlation existed between inoculum concentration and percentage of infested seedlots. Blossoms inoculated with Acidovorax citrulli also resulted in infested pepper seedlots. Furthermore, A. citrulli colonized pepper blossoms significantly better than X. euvesicatoria by 96 h postinoculation. It was concluded that pepper blossoms can be a potential site of ingress for X. euvesicatoria into seed, and blossom colonization may be involved in pepper seed infestation. Data also indicated that seed infestation via blossoms may be nonspecific because nonhost plants can be colonized by incompatible pathogens. Thus, host-pathogen interactions may not be important for bacterial ingress through blossoms.

Interactions Between Frankliniella fusca and Pantoea ananatis in the Center Rot Epidemic of Onion (Allium cepa)

An Enterobacteriaceae bacterium, Pantoea ananatis (Serrano) Mergaert, is the causal agent of an economically important disease of onion, center rot. P. ananatis is transmitted by an onion-infesting thrips, Frankliniella fusca (Hinds). However, interactions between F. fusca and P. ananatis as well as transmission mechanisms largely remain uncharacterized. This study investigated P. ananatis acquisition by thrips and transstadial persistence. Furthermore, the effects of bacterial acquisition on thrips fitness were also evaluated. When thrips larvae and adults were provided with acquisition access periods (AAP) on peanut leaflets contaminated with the bacterium, an exponentially positive relationship was observed between AAP and P. ananatis acquisition (R(2) ≥ 0.77, P = 0.01). P. ananatis persisted in thrips through several life stages (larvae, pupae, and adult). Despite the bacterial persistence, no significant effects on thrips fitness parameters such as fecundity and development were observed. Immunofluorescence microscopy of adult thrips with P. ananatis-specific antibody after 48 h AAP on contaminated food revealed that the bacterium was localized only in the gut. These results suggested that the pathogen is not circulative and could be transmitted through feces. Mechanical inoculation of onion seedlings with fecal rinsates produced center rot symptoms, whereas inoculation with rinsates potentially containing salivary secretions did not. These results provide evidence for stercorarian transmission (transmission through feces) of P. ananatis by F. fusca.

Distribution and Survival of Pseudomonas sp. on Italian Ryegrass and Curly Dock in Georgia

Yellow bud, caused by Pseudomonas sp., is an emerging bacterial disease of onion. A polymerase chain reaction assay based on the coronafacate ligase (cfl) and HrpZ genes was used to detect initial suspected bacteria on weeds. Growth on an agar medium, ability to cause a hypersensitive response in tobacco, pathogenicity on onion, and sequence analysis of 16S ribosomal RNA and cfl genes were used to confirm the identity of Pseudomonas sp. recovered from 10 asymptomatic weed species in the Vidalia onion-growing zone (VOZ) of Georgia. Among the weeds identified as epiphytic hosts for Pseudomonas sp., Italian ryegrass (Lolium multiflorum) and curly dock (Rumex crispus) were prominent because ≥73% of the samples from five sample sites were positive for the bacterium. These weeds are commonly found throughout Georgia and, thus, were selected to assess their role in yellow bud epidemiology. Samples of the two weed species were collected from sites along the perimeter of and within the VOZ (n = 5 sites) during late June, August, and September 2012 and 2013, which represented the time interval between onion growing seasons. Samples (n = 10/weed species/site) were collected and processed for bacterial detection as described above. In June (2012 and 2013), Pseudomonas sp. was detected from Italian ryegrass and curly dock in 100 and 40% of the sample sites, respectively. During the months of August and September (2012), the bacterium was recovered from Italian ryegrass in 60 and 10% of the sample sites, respectively; whereas, in August (2013), Pseudomonas sp. was recovered from 40% of the sample sites. However, the bacterium was not recovered from any of the sites in September (2013). In contrast, during August and September (2012), Pseudomonas sp. was recovered from curly dock in 20 and 80% of the sample sites, respectively. Similarly, in August and September (2013), the bacterium was detected from 40 and 100% of the sample sites, respectively. These data demonstrated that the Pseudomonas sp. responsible for yellow bud can survive as an epiphyte on Italian ryegrass and curly dock between onion crops. Furthermore, using artificially infested onion seed, we demonstrated that Pseudomonas sp. can be transmitted through contaminated seed.

Embryo Localization Enhances the Survival of Acidovorax citrulli in Watermelon Seeds

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits has been observed to survive for >34 years in stored melon and watermelon seeds. To better understand this remarkable longevity, we investigated the bacteriums tolerance to desiccation and the effect of bacterial localization in different watermelon seed tissues on its survival. We compared the ability of A. citrulli to tolerate desiccation on filter paper discs and on host (watermelon) and nonhost (cabbage, corn and tomato) seeds to two seedborne (Xanthomonas campestris pv. campestris and Pantoea stewartii subsp. stewartii) and one soilborne (Ralstonia solanacearum) plant-pathogenic bacteria. A. citrulli survival on dry filter paper (>12 weeks) was similar to that of X. campestris pv. campestris but longer than P. stewartii subsp. stewartii. Ralstonia solanacearum survived longer than all other bacteria tested. On all seeds tested, A. citrulli and X. campestris pv. campestris populations declined by 5 orders of magnitude after 12 weeks of incubation at 4°C and 50% relative humidity, while R. solanacearum populations declined by 3 orders. P. stewartii subsp. stewartii was not recovered after 12 weeks of incubation. To determine the effect of tissue localization on bacterial survival, watermelon seeds infested with A. citrulli by flower stigma inoculation (resulting in bacterial localization in the embryo/endosperm) or by ovary pericarp inoculations (resulting in bacterial localization under the testa) were treated with peroxyacetic acid or chlorine (Cl2) gas. Following these treatments, a significantly higher reduction in BFB seed-to-seedling transmission was observed for seeds generated by ovary pericarp inoculation (≥89.5%) than for those generated by stigma inoculation (≤76.5%) (P<0.05). Additionally, higher populations of A. citrulli survived when the bacteria were localized to the embryo/endosperm versus the seed coat, suggesting that tissue localization is important for bacterial survival in seed. This observation was confirmed when P. stewartii subsp. stewartii survived significantly longer in stigma-inoculated (embryo/endosperm-localized) watermelon seeds than in vacuum-infiltrated (testa-localized) seeds. Based on these results we conclude that A. citrulli cells are not intrinsically tolerant to desiccation and that localization of the bacterium to testa tissues does not enhance A. citrulli survival. In contrast, it is likely that embryo/endosperm localization enhances the survival of A. citrulli and other bacteria in seeds.

Isolation and characterization of novel Pantoea stewartii subsp. indologenes strains exhibiting center rot in onion

Center rot of onion is an economically important disease caused by three Pantoea spp.: Pantoea ananatis, P. agglomerans, and P. allii. Symptoms caused by these three species are similar and include white streaking and necrosis of foliage; and, in some cases, the bacterium may enter the bulb, causing liquefaction and rot of bulb scales. Two bacterial strains were isolated from onion expressing symptoms indicative of center rot from two different outbreaks in Toombs County, GA in 2003 (PNA 03-3) and 2014 (PNA 14-12). These strains were initially identified as P. ananatis based on physiological and specific polymerase chain reaction (PCR) assays; however, further 16S ribosomal RNA (rRNA) and multilocus sequence analysis showed that the strains were more closely related to P. stewartii subsp. stewartii and P. stewartii subsp. indologenes. Further characterization using phylogenetic analysis, a P. stewartii subsp. indologenes-specific PCR assay, indole test, and pathogenicity on onion and pearl millet were conducted. Phylogenetic analyses (16S rRNA and atpD, gyB, infB, and rpoB genes) revealed that these strains formed a distinct cluster with the type strains of P. stewartii subsp. indologenes LMG 2632T and P. stewartii subsp. stewartii LMG 2715T separate from P. ananatis, P. agglomerans, and P. allii. Furthermore, onion strains were amplified with the P. stewartii subsp. indologenes-specific PCR assay. The pathogenicity assays with onion strains showed that they were pathogenic on onion and pearl millet, a known host of P. stewartii subsp. indologenes. However, the type strain of P. stewartii subsp. indologenes LMG 2632T was pathogenic only on pearl millet but not on onion. These results suggest that the onion strains PNA 03-3 and PNA 14-12 can potentially be novel P. stewartii subsp. indologenes strains capable of producing symptoms on onion. Hence, we recommend the inclusion of P. stewartii subsp. indologenes as the fourth member in the center rot complex of onion, along with P. ananatis, P. agglomerans, and P. allii.

First Report of Myrothecium Leaf Spot Caused by Myrothecium roridum on Pepper in the United States

In September, 2015, an outbreak of leaf spot on pepper (Capsicum annuum) on variety cv. 9325 under conventional production (Pernezny et al. 2003) was reported from the Colquitt County, Georgia (approximately 5% of a 0.5 ha field was symptomatic). Infected foliage displayed round to oblong, dark brown lesions with distinct concentric rings (8-10 mm in diameter). Discoloration of the stem was also observed. Ten symptomatic leaves were collected and a section of each symptomatic leaf tissue was placed onto potato dextrose agar (PDA) media amended with 35 mg/L streptomycin. After incubation at 25o C for four days, floccose buff colonies with black-colored sporodochia in concentric rings were observed, similar to the genus Myrothecium (Seebold et al. 2005) were observed. Smooth, hyaline, and narrowly ellipsoid conidia (dimension: 7.5-8.8µm×2.1-2.7µm) with rounded ends were observed through light/compound microscope. To confirm the iIdentity of isolated pathogen was confirmed after DNA extraction and amplificat...