Thrips Settling, Oviposition and IYSV Distribution on Onion Foliage

Thrips tabaci Lindeman (Thysanoptera: Thripidae) adult and larval settling and oviposition on onion (Allium cepa L.) foliage were investigated in relation to leaf position and leaf length at prebulb plant growth stages under controlled conditions. In the laboratory, four and six adult females of T. tabaci were released on onion plants at three-leaf stage and six- to eight-leaf stage, respectively, and thrips egg, nymph, and adult count data were collected on each of the three inner most leaves at every 2-cm leaf segment. Thrips settling and oviposition parameters were quantified during the light period on the above ground portion of onion plants from the distal end of the bulb or leaf sheath “neck” through the tips of the foliage. Results from studies confirmed that distribution of thrips adults, nymphs, and eggs were skewed toward the base of the plant. The settling distributions of thrips adults and nymphs differed slightly from the egg distribution in that oviposition occurred all the way to the tip of the leaf while adults and nymphs were typically not observed near the tip. In a field study, the foliage was divided into three equal partitions, i.e., top, middle, basal thirds, and thrips adults by species, primarily Frankliniella fusca (Hinds) and T. tabaci, were collected from each partition to determine if there was a similar bias of all adult thrips toward the base of the plant. The results suggested that adults of different species appear to segregate along leaf length. Finally, thrips oviposition on 2-cm segments and Iris yellow spot virus positive leaf segments were quantified in the field, irrespective of thrips species. Both variables demonstrated a very similar pattern of bias toward the base of the plant and were significantly correlated

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics

A semiselective agar medium for the identification and isolation of (Xanthomonas campestris pv. vesicatoria) from tomato and pepper seeds.

A semiselective agar medium was developed for the identification and isolation of Xanthomonas campestris pv. vesicatoria,the causal agent of bacterial spot of pepper and tomato. The new medium,designated as CKTM,contained pourite,soy peptone,bacto tryptone,dextrose,L-glutamine,L-histidine, ammonium phosphate,potassium phosphate,magnesium sulfate,calcium chloride,and agar. Selectivity was afforded through the use of cycloheximide,bacitracin,neomycin,cephalexin,5-fluorouracil,tobramycin and tween 80. All strains of X.c. pv. vesicatoria could easily be distinguished from other pathovars of X. campestris by the formation of clear ring around their colonies,1-2 days after colony transfer or 3-4 days after dilution spread on CKTM. The clear ring usually followed by the formation of minute tan to white crystals of various intensity,depending on X.c. pv. vesicatoria strains. Detection of X.c. pv. vesicatoria was positive on 15 of 22 tomato seed lots tested,and none was detected on pepper seeds. Recovery of X.c. pv. vesicatoria ranged from 17.7 to 100% on CKTM,while 6.3 to 44.4% on Tween B medium. CKTM medium was qualitatively and quantitatively superior than Tween B medium. Reduction of contamination microflora was also achieved

First report of Pantoea agglomeranscausing a leaf blight and bulb rot of onions in Georgia

Between June and September of 2013, sweet onions (Allium cepa L.) in six different counties in Michigan displayed symptoms similar to those caused by infection from Pantoea agglomerans or P. ananatis (Edens et al. 2006; Schwartz and Mohan 2008). Symptoms included leaf and stalk water-soaked or necrotic lesions. In scouted fields, disease incidence progressed throughout the season and reached an incidence of up to 80% or more in some fields. Symptomatic samples of six cultivars were collected from each county for disease identification. Isolations were made using the semiselective onion extract medium (OEM) (Zaid et al. 2012) and single colonies were transferred and maintained on nutrient broth yeast extract (NBY) medium. The colony morphology of the bacterium on OEM medium was pale yellow with a slight greenish pigment in the center, circular to irregular shape, raised elevation, smooth margin, and mucoid texture. On NBY, the colony color was yellow with slightly darker yellow in the center, circular to occasionally slightly irregular, moderately umbonate elevation, with slightly curled margins. Fourteen representative strains were biochemically analyzed through BIOLOG (Hayward, CA) and identified as P. agglomerans (98 to 99% probability). The 16s rDNA was amplified for the same 14 strains using previously described universal primers (De Baere et al. 2004) and sequenced. Sequences were compared with the NCBI database using BLASTn and confirmed as P. agglomerans (97 to 99% identity). To test pathogenicity, five representative strains were tested on onion bulbs in the laboratory and plants in the greenhouse. Fifty microliters of an aqueous bacterial suspension of 108 CFU ml−1 were injected into three 10-week-old plant replicates of onion leaves (n = 15) and 0.5 ml were injected into bulbs (n = 15) using a 12.7 mm-long hypodermic needle and 1-ml gauge syringe. Control bulbs and leaves were injected with sterile water. Inoculated bulbs were maintained at room temperature (23°C) under constant light, whereas plants were maintained in the greenhouse. After 2 weeks of incubation, onion bulbs injected with putative P. agglomerans strains showed rot symptoms at the injection sites, while the water controls had no symptoms. For onion leaves, severe blight occurred 1 week after inoculation. Foliar symptoms were similar to those previously observed in the field. The pathogen was recovered from symptomatic bulbs and leaves, and confirmed as P. agglomerans using morphological characteristics on OEM. The pathogen has been reported to infect onion in Cuba, Israel, and South Africa (Hattingh and Walters 1981). In the United States, it was first reported in 2006 as a causal agent of leaf blight and bulb rot of onions in Georgia (Edens et al. 2006). To the best our knowledge, this is the first official report of P. agglomerans infected onion, as part of the leaf necrotic and “bulb rot complex” in Michigan. The bacterium is one of several bacterial diseases causing major loses of onion industries in Michigan

First report ofColletotrichum gloeosporioides causing ‘twister disease’ of onion (Allium cepa L.) in Georgia,USA

In the fall of 2007, onion seedlings with twisted and distorted leaves were observed in seedbeds in multiple fields in the Vidalia onion region of Georgia. Tests for viruses and bacteria were negative and chemical injury was deemed improbable because of disease distribution in the fields. Upon further investigation, fungal fruiting bodies were observed on the outside sheath of a few of the seedlings. Symptomatic plants were cut into 1-cm segments and surface sterilized in 70% ethanol for 3 min. After rinsing in sterile water, the segments were placed onto potato dextrose agar amended with tetracycline. The fungus isolated from symptomatic plants fit the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. Conidia were aseptate, cylindrical, and hyaline. Sequencing of the internal transcribed spacer region and a BLAST search in GenBank (99% sequence similarity to C. gloeosporioides accessions) confirmed the identification. Ten onion seedlings were spray inoculated with a suspension of 1 × 107 spores/ml until runoff, and four seedlings were inoculated with water as negative controls. Plants were bagged for 12 h to maintain high relative humidity. Five plants were placed in the greenhouse and five plants placed in a growth chamber at 22°C. All plants inoculated with C. gloeosporioides developed distorted and twisted leaves 3 weeks after inoculation in the growth chamber and 5 weeks after inoculation in the greenhouse. Night time temperatures in the greenhouse (15°C) were lower than those in the growth chamber (22°C). Seedlings inoculated with water showed no symptoms. The fungus was reisolated from symptomatic plants. C. gloeosporioides has been reported to cause a disease called twister on onion in tropical regions (1). The fall of 2007 was unusually warm with maximum temperatures reaching 26°C during the day. The pathogen is present on many crops in the United States, but to our knowledge, this is the first report of C. gloeosporioides causing twister disease of onion in the United States. In Nigeria and Brazil, yield losses as much as 100% were observed in fields with infected onions (1). The impact of infection on the growth of the transplants and subsequent yield in Vidalia onions is currently unknown

Use of fatty acid methyl ester profiles to compare copper-tolerant andcopper-sensitive strains of Pantoea ananatis

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 (ED2) 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 μg/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 μg/ml) and maneb (40 μg/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