Search

Alternaria brassicicola is part of a complex of Alternaria species that causes leaf blight and head rot in brassica crops such as broccoli, kale, cabbage, cauliflower, and collards. Seed can serve as a potential source of inoculum for the transmission of A. brassicicola in broccoli as demonstrated earlier; however, seed-to-seedling transmission of pathogen was never characterized empirically. So, the objectives of this study were to (i) re-evaluate the effect of artificial seed infestation on seed germination and seed-to-seedling transmission of A . brassicicola in broccoli; (ii) determine the effect of A. brassicicola- seed inoculum levels on seed-to-seedling transmission; (iii) evaluate if variations in A. brassicicola aggressiveness affect A . brassicicola seed-to-seedling transmission; and (iv) evaluate seed treatments that can reduce seed-to-seedling transmission of A. brassicicola in broccoli. Artificially infested seedlots were generated by inoculating broccoli seeds with a spore suspension of 1 × 10 5 conidia/ml of A. brassicicola using the vacuum infiltration method. Inoculated ( n = 10 seedlots; 300 seeds/seedlot) or control seedlots in three replicates were planted on two layers of sterile blotter paper saturated with sterile water in transparent plastic boxes and incubated at 20°C and >90% relative humidity (RH) under continuous fluorescent light. Percent seed germination and percent seed-to-seedling transmission were recorded every other day for 21 days. Percent seed germination was significantly affected with artificial pathogen inoculation. One hundred percent of the seedlots transmitted the pathogen to broccoli seedlings, and seed-to-seedling percentages of the seedlots varied considerably. A strong linear and significant relationship between A . brassicicola inoculum level and seed-to-seedling transmission (%) within each seedlot was observed. Interestingly, variations in aggressiveness of A. brassicicola isolates did not affect seed-to-seedling transmission, as 100% of the seedlots were able to transmit the pathogen. Seed treatment with Miravis (a.i. pydiflumetofen 18.3%) significantly increased seed germination and reduced seed-to-seedling transmission percentages in A. brassicicola- inoculated seedlots. These results indicate that artificial seed inoculation with A. brassicicola can result in consistent seed-to-seedling transmission with significant impact on seed germination. Seed inoculum density of ≥10 4 conidia/ml is necessary for reliable transmission of A . brassicicola . Further seed-to-seedling transmission is not dependent on aggressiveness of A. brassicicola isolates and seed treatment with Miravis can significantly reduce pathogen transmission in broccoli seedings. Overall, this study provides detailed characterization of seed-to-seedling transmission of A. brassicicola in broccoli that can be further used to determine inoculum threshold, which has potential applications in seed-health testing and sample size determination. Furthermore, we also provide options for effective seed treatments that can significantly reduce A. brassicicola seed-to-seedling transmission and may potentially aid in managing seedborne fungal infection., Competing Interests: The author(s) declare no conflict of interest.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures ( n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent ( n = 133) of the isolates formed a cluster with a known A . brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates ( n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates ( n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC 50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC 50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC 50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola . We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields., Competing Interests: The author(s) declare no conflict of interest.

Sida golden mosaic virus (SiGMV), an obligate pathogen that infects snap beans ( Phaseolus vulgaris ), is known to infect prickly sida ( Sida spinosa L.), which is a common weed in agricultural farms in Georgia. Prickly sida has also been reported as a suitable host of sweetpotato whitefly ( Bemisia tabaci ), the vector of SiGMV. Despite being a host for both SiGMV and its vector, the role of prickly sida as a reservoir and inoculum source for SiGMV in snap bean farms has not been evaluated. This study was conducted to document the occurrence of SiGMV-infected prickly sida plants and to assess its potential role as a source of SiGMV inoculum in snap bean farms. A survey of 17 commercial snap bean farms conducted in spring 2021 confirmed the presence of SiGMV-infected prickly sida in southern Georgia. In fall 2021 and 2022, on-farm field trials were conducted in four commercial farms where SiGMV-infected prickly sida plants were documented earlier as a part of survey in spring 2021. The spatial distribution and temporal patterns of adult whiteflies and SiGMV on snap bean were compared between macroplots (13.7 × 30.5 m) "with prickly sida" or "without prickly sida" that were at least 232 m apart from each other. We did not observe any consistent differences in counts of adult whiteflies between macroplots with or without prickly sida in the four commercial farms. SiGMV infection was detected earlier and with higher incidences in snap bean macroplots "with prickly sida" compared with macroplots "without prickly sida." An apparent disease gradient was observed in two of the four farms assessed. Higher SiGMV incidences were observed on the edges of macroplots "with prickly sida." These findings indicate prickly sida as a potential natural reservoir and a source for SiGMV spread in snap bean farms in southern Georgia., Competing Interests: The author(s) declare no conflict of interest.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut ( Arachis hypogaea ) has been a focus of molecular breeding for the U.S. industry-funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping population has been genotyped with the peanut 58K single-nucleotide polymorphism (SNP) array and phenotyped for LLS severity in the field for 3 years. Joint linkage-based quantitative trait locus (QTL) mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained up to 47.7%. A genome-wide association study identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 to 1,520 kb (184 kb) on chromosome B02 and from 1,026.9 to 1,793.2 kb (767 kb) on chromosome B03, designated as peanut LLS resistance loci, PLLSR -1 and PLLSR -2, respectively. PLLSR -1 contains 10 nucleotide-binding site leucine-rich repeat disease resistance genes. A nucleotide-binding site leucine-rich repeat disease resistance gene, Arahy.VKVT6A , was also identified on homoeologous chromosome A02. PLLSR -2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat, acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as nested association mapping for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding., Competing Interests: The author(s) declare no conflict of interest.

Unlike most characterized bacterial plant pathogens, the broad-host-range plant pathogen Pantoea ananatis lacks both the virulence-associated type III and type II secretion systems. In the absence of these typical pathogenicity factors, P. ananatis induces necrotic symptoms and extensive cell death in onion tissue dependent on the HiVir proposed secondary metabolite synthesis gene cluster. Onion (Allium. cepa L), garlic (A. sativum L.), and other members of the Allium genus produce volatile antimicrobial thiosulfinates upon cellular damage. However, the roles of endogenous thiosulfinate production in host-bacterial pathogen interactions have not been described. We found a strong correlation between the genetic requirements for P. ananatis to colonize necrotized onion tissue and its capacity for tolerance to the thiosulfinate "allicin" based on the presence of an eleven-gene, plasmid-borne, virulence cluster of sulfur redox genes. We have designated them "alt" genes for allicin tolerance. We show that allicin and onion thiosulfinates restrict bacterial growth with similar kinetics. The alt gene cluster is sufficient to confer allicin tolerance and protects the glutathione pool during allicin treatment. Independent alt genes make partial phenotypic contributions indicating that they function as a collective cohort to manage thiol stress. Our work implicates endogenous onion thiosulfinates produced during cellular damage as major mediators of interactions with bacteria. The P. ananatis-onion pathosystem can be modeled as a chemical arms race of pathogen attack, host chemical counterattack, and pathogen defense., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 2632 T and P. stewartii subsp. stewartii LMG 2715 T 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 2632 T 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.

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.

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 bacterium's 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.

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 × 10(6) 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.

Center rot, caused by Pantoea ananatis, has been one of the most important bacterial diseases of onion leading to considerable economic losses. Symptoms can be expressed in the onion foliage and bulb, with the pathogen moving from the infected leaves to bulb scales. However, little is known regarding which growth stage the plant is most susceptible to bulb infection and if there are differences in susceptibility to bulb infection among sweet onion cultivars. In this study, five cultivars of sweet onion (Pirate, Sweet Harvest, 1518, Granex YPRR, and 1407) were inoculated by clipping the tips of onion foliage and depositing 1 ml of 1 x 108 CFU/ml of P. ananatis suspension into the central leaf cavity. The inoculations were done at three growth stages (first leaf senescence, bulb initiation, and bulb swelling). Center rot incidence was assessed for precured and cured onion bulbs. In addition, total bulb incidence of center rot for each cultivar inoculated at three growth stages were also calculated. Total bulb center rot incidence was significantly higher for Granex YPRR (84%) compared with other cultivars. Also, cultivars 1518 (49%) and 1407 (33%) had significantly lower incidence of bulb infection compared with other tested cultivars. Onions were significantly more susceptible to bulb infection when inoculated during first leaf senescence (62%) as compared with bulb initiation (37%) and bulb swelling (31%) stages in precured bulbs (P = 0.041 ). Significantly higher incidence of center rot was observed for bulbs whose foliage were inoculated during first leaf senescence stage (64%) compared with bulb initiation (55%) and bulb swelling (52%) stages (P = 0.048). Interactions between onion cultivai' and inoculation stage on center rot bulb incidence were not significant (P ≥ 0.218), when evaluated at different assessment periods. However, different cultivars displayed significant variability in susceptibility to bulb infection. The outcomes of this study may have implications in devising management strategies aimed at protecting most susceptible onion growth stages against P. ananatis. [ABSTRACT FROM AUTHOR]