Genotypic and Phenotypic Characterization of Lettuce Bacterial Pathogen Xanthomonas hortorum pv. vitians Populations Collected in Quebec, Canada

Bacterial leaf spot of lettuce, caused by Xanthomonas hortorum pv. vitians, is an economically important disease worldwide. For instance, it caused around 4 million CAD in losses in only a few months during the winter of 1992 in Florida. Because only one pesticide is registered to control this disease in Canada, the development of lettuce cultivars tolerant to bacterial leaf spot remains the most promising approach to reduce the incidence and severity of the disease in lettuce fields. The lack of information about the genetic diversity of the pathogen, however, impairs breeding programs, especially when disease resistance is tested on newly developed lettuce germplasm lines. To evaluate the diversity of X. hortorum pv. vitians, a multilocus sequence analysis was performed on 694 isolates collected in Eastern Canada through the summers of 2014 to 2017 and two isolates in 1996 and 2007. All isolates tested were clustered into five phylogroups. Six pathotypes were identified following pathogenicity tests conducted in greenhouses, but when phylogroups were compared with pathotypes, no correlation could be drawn. However, in vitro production of xanthan and xanthomonadins was investigated, and isolates with higher production of xanthomonadins were generally causing less severe symptoms on the tolerant cultivar Little Gem. Whole-genome sequencing was undertaken for 95 isolates belonging to the pathotypes identified, and de novo assembly made with reads unmapped to the reference strain’s genome sequence resulted in 694 contigs ranging from 128 to 120,795 bp. Variant calling was performed prior to genome-wide association studies computed with single-nucleotide polymorphisms (SNPs), copy-number variants and gaps. Polymorphisms with significant p-values were only found on the cultivar Little Gem. Our results allowed molecular identification of isolates likely to cause bacterial leaf spot of lettuce, using two SNPs identified through genome-wide association study.

First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander fields in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

Incorporation of Engineered Nanoparticles of Biochar and Fly Ash Against Bacterial Leaf Spot of Pepper

In agriculture, the search for higher net profit is the main challenge in the economy of the producers and nano biochar attracts increasing interest in recent years due to its unique environmental behaviour and increasing the productivity of plants by inducing resistance against phyto-pathogens. The effect of rice straw biochar and fly ash nanoparticles (RSBNPs and FNPs, respectively) in combination with compost soil on bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria was investigated both in vitro and in vivo. The application of nanoparticles as soil amendment significantly improved the chili pepper plant growth. However, RSBNPs were more effective in enhancing the above and belowground plant biomass production. Moreover, both RSBNPs and FNPs, significantly reduced (30.5 and 22.5 %, respectively), while RSBNPs had shown in vitro growth inhibition of X. campestris pv. vesicatoria by more than 50%. The X-ray diffractometry of RSBNPs and FNPs highlighted the unique composition of nano forms which possibly contributed in enhancing the plant defence against invading X. campestris pv. vesicatoria. On the basis of our findings, it is suggested that biochar and fly ash nanoparticles can be used for reclaiming the problem soil and enhance the crop productivity depending upon the nature of soil and the pathosystem under investigation.

Suppression of Bacterial Leaf Spot by Green Synthesized Silica Nanoparticles and Antagonistic Yeast Improves Growth, Productivity and Quality of Sweet Pepper

Plants are challenged with many kinds of biotic stresses caused by different living organisms, which result in various types of diseases, infections, and damage to crop plants and ultimately affect crop productivity. Plant disease management strategies based on current approaches are necessary for sustainable agriculture. A pot experiment was carried out under greenhouse conditions to evaluate the potential of green synthesized silica nanoparticles (SiO2-NPs) and antagonistic yeast (Saccharomyces cerevisiae) against pepper bacterial leaf spot disease, caused by Xanthomonas vesicatoria. In addition, to assess their efficacy and suppressive effects in reducing disease severity and improving sweet pepper growth, productivity, and quality. Results revealed that the combination of BCA (5%) and SiO2-NPs (150 ppm) was the most effective treatment for reducing disease severity and improving vegetative growth characters, mineral contents (N, P, K, Ca, Mg, and Si in leaves), as well as stimulating polyphenol oxidase (PPO) activity of sweet pepper leaves at 90 days from transplanting, while also at harvesting time enhancing sweet pepper fruit yield quality parameters significantly. In conclusion, green synthesized silica nanoparticles combined with antagonistic yeast have the potential to suppress a bacterial leaf spot disease with ecologically-sound management, while also boosting sweet pepper growth, productivity, and quality.

First Report of Pseudomonas cichorii causing Bacterial Leaf Spot on Romaine lettuce (Lactuca sativa var. longifolia) and escarole (Cichorium endivia) in New Jersey.

We previously reported an outbreak of bacterial leaf spot (BLS) caused by Pseudomonas cichorii occurring on sweet basil in New Jersey during the summer of 2018 (Patel et al. 2019), a growing season characterized by increased leaf wetness due to high humidity and unusually high levels of summer rains. Leaf spot was also observed, as a one-time event, on older mature leaves on romaine and escarole lettuce during that same year. Symptoms on escarole were observed as grayish brown-to-black concentric lesions on leaf parenchyma tissue, ranging from 1 mm to 1-2 cm in diameter. In more severely diseased samples, lesions coalesced to form larger necrotic areas giving a blight appearance. On occasion, infection was observed in leaf midveins as brownish gray necrosis. Symptoms on romaine lettuce were observed mostly as coalesced blackened lesions on leaf parenchyma tissue near margins with a rotted consistency that spread to the midvein in severe cases. Margins of leaf lesions were excised and macerated in sterile water before streaking onto Nutrient Agar (NA) and King’s medium B agar (KMB) (Schaad et al. 2001). Growth on both NA and KMB were predominantly cream-colored circular bacterial colonies with undulated margins. Colonies on KMB fluoresced blue under 365nm UV light. Two representative colonies isolated from each host were selected for further characterization. All isolated tested negative for levan production, positive for oxidase, negative for potato rot, negative for arginine dihydrolase, and induced a strong hypersensitive response on tobacco within 24 h, consistent with LOPAT descriptions for P. cichorii (Lelliott et al. 1966). A single strain from each host (ESC6F2 and Rom1-1) was further characterized genetically to confirm species. PCR analysis using two primers sets: 16S rRNA gene universal primers 27F/1492R and Hcr1 primers used to amplify a 520 bp region of the pathogenicity gene cluster hrcRST in P. cichorii (Patel et al. 2019; Cottyn et al. 2011). Partial 16S rDNA gene sequences were deposited in the GenBank database for each isolate (ESC6F2: MT974180; and Rom1-1: MT982172). Sequence comparisons of ESC6F2 and Rom1-1 shared 99% identity with each other and several P. cichorii strains within the GenBank database, including strain B5-2-1 isolated from sweet basil in NJ the same year (MK501753). The partial hrcRST locus (strain ESC6F2: MW048775 and Rom1-1: MW048774) shared 100% identity to each other and strain B5-2-1 (MK507764), and 99% identity with P. cichorii strain P18-1 (MH396007) isolated from Ocimum basilicum in Hawaii. Koch’s postulate was performed on escarole var. Full Heart and Romaine lettuce var. Ideal Cos to confirm pathogenicity of the isolated strains. Bacterial suspensions (1x107 cfu/ml) were syringe-injected (0.1 ml) into the leaf midribs, and pressure infiltrated into leaf parenchyma tissue of 3 plants. Control plants were inoculated with sterile water. Blackened necrosis developed within 72 h around bacteria-inoculation points, which expanded beyond inoculation points within a week. In contrast, control plants remained healthy and symptomless. Although significant crop loss occurred due to BLS on escarole and romaine lettuce, P. cichorii has not been isolated from diseased plant material since 2018. This suggests inoculum sources did not persist beyond 2018, or favorable environmental conditions for disease are inconsistent to cause noticeable damage to New Jersey crops. References: Cottyn, B., et al. 2011. Plant Pathol. 60:453. Lelliott, R. A., et al. 1966. J. Appl. Bacteriol. 29:470. Patel, N., et al. 2019. Plant Disease. 103:2666

Field Efficacy of Thiophanate Methyl 44.8% + Kasugamycin 2.6% Sc against Major Foliar Diseases of Tomato

Tomato (Solanum lycopersicum L.) is the important edible solanaceous plant originated from western South and Central America. Despite botanically being a fruit, it’s generally eaten and preferred like a vegetable. Tomatoes are the major dietary source of the antioxidant lycopene, which has been linked to many health benefits, including reduced risk of heart disease and cancer. Early blight caused by Alternaria solani and powdery mildew caused by Erysiphe orontii and bacterial leaf spot caused by Xanthomonas campestris has become a serious problem for successful cultivation of tomato. Therefore, a field experiment was carried out to know the efficacy of Thiophanate methyl 44.8% + Kasugamycin 2.6% Sc on tomato diseases during 2017-18 and 2018-19, at College of Agriculture, Shivamogga. Experimental results revealed that all the treatments significantly reduced the early blight, bacterial leaf spot and powdery mildew disease severity over untreated control. Among all the treatments Thiophanate methyl 44.8% + Kasugamycin 2.6% SC @ 1250 ml/ha recorded significantly less Per cent Disease Index (PDI) of Early blight (Alternaria solani) (7.78 % and 10.19 %), Bacterial leaf spot (Xanthomonas campestris) ( 3.96 and 1.39 %) and Powdery mildew (Erysiphe orontii) ( 1.67 and 2.50 %) with yield of 340.33 and 333.33 q/ha followed by Thiophanate methyl 44.8% + Kasugamycin 2.6% SC @ 1000 ml/ha.