Ron Gitaitis

Transmission of Iris Yellow Spot Virus by Frankliniella fusca and Thrips tabaci (Thysanoptera: Thripidae)

ABSTRACT Thrips-transmitted Iris yellow spot virus (IYSV) (Family Bunyaviridae, Genus Tospovirus) affects onion production in the United States and worldwide. The presence of IYSV in Georgia was confirmed in 2003. Two important thrips species that transmit tospoviruses, the onion thrips (Thrips tabaci (Lindeman)) and the tobacco thrips (Frankliniella fusca (Hinds)) are known to infest onion in Georgia. However, T. tabaci is the only confirmed vector of IYSV. Experiments were conducted to test the vector status of F. fusca in comparison with T. tabaci. F. fusca and T. tabaci larvae and adults reared on IYSV-infected hosts were tested with antiserum specific to the nonstructural protein of IYSV through an antigen coated plate ELISA. The detection rates for F. fusca larvae and adults were 4.5 and 5.1%, respectively, and for T. tabaci larvae and adults they were 20.0 and 24.0%, respectively, indicating that both F. fusca and T. tabaci can transmit IYSV. Further, transmission efficiencies of F. fusca and T. tabaci were evaluated by using an indicator host, lisianthus (Eustoma russellianum (Salisbury)). Both F. fusca and T. tabaci transmitted IYSV at 18.3 and 76.6%, respectively. Results confirmed that F. fusca also can transmit IYSV but at a lower efficiency than T. tabaci. To attest if low vector competency of our laboratory-reared F. fusca population affected its IYSV transmission capability, a Tomato spotted wilt virus (Family Bunyaviridae, Genus Tospovirus) transmission experiment was conducted. F. fusca transmitted Tomato spotted wilt virus at a competent rate (90%) suggesting that the transmission efficiency of a competent thrips vector can widely vary between two closely related viruses.

EXPERIENCES WITH A FOOD PRODUCT X-RAY INSPECTION SYSTEM FOR CLASSIFYING ONIONS

Maintaining product quality is critical for success in fresh fruit and vegetable marketing. Some onion packinghouses are considering the addition of x-ray inspection systems to their existing optical inspection systems. X-ray systems enable detection of voids that are likely to be associated with the presence of various bacterial or fungal rots in onions. A series of tests were conducted at the University of Georgia Vegetable and Vidalia Onion research and education center in Toombs County, Georgia, with a commercially available x-ray inspection machine. In 2001, two 100-onion batches of medium-sized onions and 100 jumbo onions were machine-inspected, and then halved for a visual internal evaluation. In each series of tests, the accuracy rate was greater than 93% and the false positives were less than 6%. In 2002, two 100-onion batches were run on a similar machine as in 2001. Additionally, in 2002 and 2004, multiple onions with slight to severe defects were each passed through the inspection machine 50 times, respectively, with orientation not controlled to ascertain consistency in defect detection. The machine passed onions with no to slight defect presence (based on subsequent internal visual evaluation of onion halves) nearly 100% of the time. Onions with severe defects were rejected 100% of the time. In 2004, a center rot disease (caused by Pantoea ananztis) study showed that 80% of bulbs that had passed a routine surface inspection and had been deemed to be diseased by the machine in fact exhibited the disorder on halving. False positives were in the 10% to 15% range. In the 2002 and 2004 studies, the machine detected bulbs with disease that passed human visual inspection (HVI) and (in 2004 only) individual tactile grading. These accuracy and false positive rates are very close to the 90% and 10% levels generally accepted for these respective statistics. With appropriate addition of multiple lanes, commercially viable throughputs are possible.

First Report of Phakopsora pachyrhizi, the Causal Agent of Asian Soybean Rust, on Florida Beggarweed in the United States

Phakopsora pachyrhizi Syd. & P. Syd., which causes Asian soybean rust (SBR), was observed on Florida beggarweed, Desmodium tortuosum (Sw) DC., in Attapulgus, GA during late October and early November 2005. Tan to brown lesions (<1.0 mm in diameter) consistent with symptoms of SBR (2) were observed on older leaves of several plants collected near an SBR-infected soybean trial. Dissection (40 to 60×) and compound microscopy (×200 to 400) revealed conical pustules and ellipsoid, echinulate urediniospores (average size 15 × 20 μm) on the abaxial leaf surface. Polymerase chain reaction (PCR) (primers Ppm1 and Ppa2) (1) was conducted on four samples to confirm identification of P. pachyrhizi or P. meibomiae. Three were positive for P. pachyrhizi, and one was negative for both species. Using morphology and real-time PCR, SBR was confirmed as P. pachyrhizi by the USDA/APHIS in Beltsville, MD. Six noninfected Florida beggarweed plants were transplanted to pots during December 2005 and grown at 22 to 24°C in a greenhouse. On 11 January 2006, a water suspension of urediniospores collected from SBR-infected soybeans (1 × 105 spores per ml) was spray inoculated on all leaves to almost runoff and incubated for 48 h in a plastic humidity chamber. Lesions, pustules, and urediniospores consistent with SBR (2) were observed on 3 February 2006. A PCR assay was conducted on six samples from the infected greenhouse plants and all were positive for P. pachyrhizi. Florida beggarweed is widespread in the southern United States and may serve as an additional overwintering source for P. pachyrhizi and a potential inoculum source for the soybean crop. References: (1) R. D. Fredrick et al. Phytopathology 92:217, 2002. (2) J. B. Sinclair and G. L. Hartman. Soybean rust. Pages 25-26 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999.

Use of Fatty Acid Methyl Ester Profiles to Compare Copper-Tolerant and Copper-Sensitive Strains of Pantoea ananatis

ABSTRACT A survey was conducted to evaluate differences in fatty acid methyl ester (FAME) profiles among strains of Pantoea ananatis, causal agent of center rot of onion (Allium cepa), isolated from 15 different onion cultivars in three different sites in Georgia. Differences in FAME composition were determined by plotting principal components (PCs) in two-dimensional plots. Euclidean distance squared (ED(2)) values indicated a high degree of similarity among strains. Plotting of PCs calculated from P. ananatis strains capable of growing on media amended with copper sulfate pentahydrate (200 mug/ml) indicated that copper-tolerant strains grouped into tight clusters separate from clusters formed by wild-type strains. However, unlike copper-sensitive strains, the copper-tolerant strains tended to cluster by location. A total of 80, 60, and 73% of the strains from Tift1, Tift2, and Tattnall, respectively, exhibited either confluent growth or partial growth on copper-amended medium. However, all strains were sensitive to a mixture of copper sulfate pentahydrate (200 mug/ml) and maneb (40 mug/ml). When copper-tolerant clones were analyzed and compared with their wild-type parents, in all cases the plotting of PCs developed from copper-tolerant clones formed tight clusters separate from clusters formed by the parents. Eigenvalues generated from these tests indicated that two components provided a good summary of the data, accounting for 98, 98, and 96% of the standardized variance for strains Pna 1-15B, Pna 1-12B, and Pna 2-5A, respectively. Furthermore, feature 4 (cis-9-hexadecenoic acid/2-hydroxy-13-methyltetradecanoic acid) and feature 7 (cis-9/trans-12/cis-7-octadecenoic acid) were the highest or second highest absolute values for PC1 in all three strains of the parents versus copper-tolerant clones, and hexadecanoic acid was the highest absolute value for PC2 in all three strains. Along with those fatty acids, dodecanoic acid and feature 3 (3-hydroxytetradecanoic acid/14-methylpentadecenoic acid) also had an impact on the differences observed between copper-sensitive parents and copper-resistant mutants. Finding these changes in bacterial fatty acid composition could lead to the development of a laboratory assay to identify copper-tolerant strains using gas chromatography as well as providing clues to further elucidate the mode of action of copper tolerance.

Evaluation of Lisianthus as an Indicator Host for Iris yellow spot virus

Iris yellow spot virus (IYSV) can severely affect onion production. IYSV is transmitted by the onion thrips, Thrips tabaci. However, information on IYSV-thrips-onion interactions is limited due to the difficulty associated with infecting onion plants experimentally. Lisianthus (Eustoma russellianum) was used as an indicator host to study mechanical transmission of IYSV, IYSV transmission by T. tabaci, IYSV distribution in the host plant, and the effect of temperature on IYSV symptom expression. Mechanical inoculation tests from IYSV-infected onion plants to noninfected lisianthus plants resulted in a mean transmission rate of 82.5 ± 6.9% (mean ± standard error), and from IYSV-infected lisianthus plants to noninfected lisianthus plants resulted in a mean transmission rate of 89.2 ± 7.1%. T. tabaci adults transmitted IYSV at a rate of 80.0 ± 8.3% from infected onion plants to noninfected lisianthus plants. To assess IYSV distribution in infected lisianthus plants, leaf sections, stems, and roots were tested by enzyme-linked immunosorbent assay (ELISA). All the plant parts tested positive for IYSV, but not on every plant assayed. Alternating night and day temperatures of 18 and 23°C, 25 and 30°C, and 30 and 37°C were evaluated for the effects on IYSV symptom expression. More severe symptoms developed on inoculated plants incubated at the 18 and 23°C or 25 and 30°C temperature regimes than at the 30 and 37°C regime, and symptoms were observed earliest on plants incubated at the 25 and 30°C temperature regime compared to the other temperature regimes.

Assessment of Variation Among Thrips tabaci Populations from Georgia and Peru Based on Polymorphisms in Mitochondrial Cytochrome Oxidase I and Ribosomal ITS2 Sequences

The onion thrips, Thrips tabaci (Lindeman), is the only known vector of Iris yellow spot virus (IYSV). IYSV was detected in Georgia for the first time in 2003. Phylogenetic analysis using nucleotide sequences of the IYSV capsid gene indicated that it may have been accidentally introduced from repackaging of imported Peruvian onions in the Vidalia onion-growing region. The tobacco thrips, Frankliniella fusca (Hinds), has been the dominant thrips species on onions in Georgia. However, in recent years the incidence of T. tabaci on onions has been consistently increasing. Laboratory competition studies indicated that T. tabaci outcompeted F. fusca on onion foliage. This led to speculation that a new biotype of T. tabaci may have been introduced along with IYSV through importation of Peruvian onions. This hypothesis was tested by analyzing variations in the mitochondrial cytochrome oxidase I gene and internal transcribed spacer region 2 of T. tabaci populations from Georgia and Peru. DNA was extracted from T. tabaci samples from Georgia and Peru and subjected to PCR using specific primers. The resulting amplicons were sequenced. Parsimony and Bayesian analysis of the COI sequences indicated that all the Peruvian taxa fell into a single clade along with one Georgia taxon. All the other Georgia taxa were in a separate clade. ITS2 sequence comparisons indicated that Georgia and Peru taxa were found in numerous clades. High variation among taxa from each region indicated that ITS2 may not be suitable to assess intraspecific variation among T. tabaci populations.

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.

Pantoea ananatis Genetic Diversity Analysis Reveals Limited Genomic Diversity as Well as Accessory Genes Correlated with Onion Pathogenicity

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.

Interaction of Onion Cultivar and Growth Stages on Incidence of Pantoea ananatis Bulb Infection

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 × 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 cultivar 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.

A Risk Assessment Model For Bacterial Leaf Spot (BLS) Of Pepper (Capsicum annuum L.), Caused By Xanthomonas euvesicatoria, Based On Concentrations Of Macronutrients and Micronutrients and Micronutrient Ratios

The phytopathogenic bacterium Xanthomonas euvesicatoria causes bacterial leaf spot (BLS) of pepper and has a worldwide distribution. BLS is difficult to control and an integrated management strategy that incorporates crop rotation, use of clean seed and clean plants, weed control, resistant varieties, applications of bactericides, biocontrol agents, and systemic acquired resistance (SAR) inducers is generally recommended. However, even with that arsenal of weapons, BLS can still be responsible for severe losses under favorable environmental conditions. Thus, additional tools need to be added to an overall integrated management strategy to combat BLS. In this article, we developed several models from 2012 to 2014 that were based on how macronutrients, micronutrients, and micronutrient ratios affect BLS severity. Factors used to select a model for validation included highly significant P values, high adjusted R2 values, low variance inflation factor values (<5), root mean square error, Mallows Cp, and high Akaikes information criterion correction values. In addition, salicylic acid (SA) concentrations and relative expression of nonexpresser pathogenesis-related gene1 (NPR1) and pathogenesis-related protein 1 (PR1) in pepper tissues were also considered in model selection. A model (ECGA1) consisting of concentrations of copper, manganese, potassium, and the iron/zinc ratio as independent variables was used for validation in three different commercial pepper fields in Georgia: Colquitt County and Worth County in 2015 and Tift County in 2016. When area under the disease progress curve (AUDPC) values for two field sites (Colquitt and Worth Counties) in 2015 were pulled together and plotted against ECGA1-predicted values for both sites, the resulting relationship was highly significant (P = 0.0001) with an R2 value of 0.92. A significant relationship between observed AUDPC versus predicted values was also observed in Tift County in 2016 (P < 0.001; adjusted R2 = 0.98). Relative gene expression of both NPR1 and PR1 genes was significantly (P < 0.01) higher in pepper grown in predicted low-risk sites compared with pepper from high-risk sites in Colquitt, Worth, and Tift Counties. Although BLS severity will fluctuate depending on environmental conditions, the data indicate that the level of risk at a particular location may be influenced by how macronutrient and micronutrient concentrations affect plant disease resistance genes in the SAR pathway.