Comparative Genomic Analysis Reveals Genetic Variation and Adaptive Evolution in the Pathogenicity-Related Genes of Phytophthora capsici

Phytophthora capsici is an oomycete pathogen responsible for damping off, root rot, fruit rot, and foliar blight in popular vegetable and legume crops. The existence of distinct aggressiveness levels and physiological races among the P. capsici population is a major constraint to developing resistant varieties of host crops. In the present study, we compared the genomes of three P. capsici isolates with different aggressiveness levels to reveal their genomic differences. We obtained genome sequences using short-read and long-read technologies, which yielded an average genome size of 76 Mbp comprising 514 contigs and 15,076 predicted genes. A comparative genomic analysis uncovered the signatures of accelerated evolution, gene family expansions in the pathogenicity-related genes among the three isolates. Resequencing two additional P. capsici isolates enabled the identification of average 1,023,437 SNPs, revealing the frequent accumulation of non-synonymous substitutions in pathogenicity-related gene families. Furthermore, pathogenicity-related gene families, cytoplasmic effectors and ATP binding cassette (ABC) transporters, showed expansion signals in the more aggressive isolates, with a greater number of non-synonymous SNPs. This genomic information explains the plasticity, difference in aggressiveness levels, and genome structural variation among the P. capsici isolates, providing insight into the genomic features related to the evolution and pathogenicity of this oomycete pathogen.

First Report of Rhizoctonia solani AG-2-2 IIIB Causing Foliar Blight on Bletilla striata in China

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae), a perennial plant, is a traditional Chinese herb (known as baiji) used to treat hemorrhage, scalding injuries, gastric ulcers, pulmonary diseases, and inflammation (Zu et al. 2019). In May 2019, foliar blight symptoms were observed on approximately 25% of B. striata (cv. Guiji No.1) plants in three plantations (∼4.5 hectares in total) in Ziyuan County, Guangxi Province, China. Initial symptoms were light brown, irregular, water-soaked spots on the plant leaves. Several spots often merged, forming large, irregular, lesions that extended onto the stem after a week and led to leaf abscission, and even plant death. To determine the causal agent, 5-mm squares cut from the margin of 6 infected leaves were surface disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C (12-h light-dark cycle) for 3 days. The emerging hyphal tip of a single mycelium was transferred to PDA to obtain pure cultures of the isolates. Twenty isolates were obtained, and 10 isolates (50%) were initially white before turning light brown (∼4 days). Septate hyphae were 4.29 to 10.75 μm (average 6.42 μm) in diameter and branched at right angles with a constriction at the origin of the branch point. Staining with 1% safranin O and 3% KOH solution (Bandoni 1979) revealed multinucleated cells (3 to 9 nuclei per cell, n = 142). This morphology was typical of Rhizoctonia solani Kühn (Meyer et al. 1990). For species confirmation by molecular identification, three isolates (BJ101.6, BJ101.11, and BJ102.2) were cultured on PDA for 4 days, then DNA was extracted from the mycelium using the CTAB method (Guo et al. 2000), and the ribosomal ITS1-5.8S-ITS2 region was amplified by PCR using the universal fungal primers ITS1 and ITS4 (White et al. 1990). Internal transcribed spacer (ITS) sequences of strains BJ101.6, BJ101.11, and BJ102 (deposited in GenBank under accession nos MT406271, MT892815, and MT892814, respectively) had over 99% similarity with those of R. solani AG-2-2 IIIB in GenBank (accession nos JX913810 and AB054858) (Carling et al. 2002; Hong et al. 2012). Phylogenetic analysis using ITS sequences showed that the isolates clustered monophyletically with strains of R. solani AG-2-2 IIIB. The AG of the isolates was confirmed by their ability to grow well on PDA at 35°C, which separates AG-2-2 IIIB from AG-2-2 IV (Inokuti et al. 2019). Based on morphological characteristics and nucleotide sequence analysis, the isolates were identified as R. solani AG-2-2 IIIB. Pathogenicity was tested using 1.5-year-old B. striata (cv. Guiji No.1) plants grown in a perlite and peat moss mixture (1:3) in 7-cm pots. Healthy leaves on plants were inoculated with an aqueous suspension (approximately 1 × 105 hyphal fragments/mL, 100 μL) prepared from cultures of strains BJ101.6, BJ101.11, and BJ102.2, each isolate was inoculated onto three plants; three other plants with sterile water served as controls. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C for 14 days (12-h photoperiod). Three days post-inoculation, leaves exposed to the mycelial fragments had symptoms similar to those originally observed in the field. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch’s postulates, R. solani AG-2-2 IIIB was re-isolated on PDA from symptomatic leaves and confirmed by sequencing, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of R. solani AG-2-2 IIIB causing foliar blight on B. striata in China, and these findings will be useful for further control strategies and research.

Phytophthora Root Rot Resistance and Its Correlation with Fruit Rot Resistance in Capsicum annuum

Phytophthora capsici causes root and fruit rot and foliar blight of pepper. Multiple sources of resistance to Phytophthora root rot have previously been identified, but most display only partial resistance. One source, CM334, has broad spectrum root rot resistance to multiple pathogen isolates but has only low to moderate fruit rot resistance. This study evaluated previously identified pepper lines for resistance to two P. capsici isolates, OP97 and 12889, and compared root rot resistance to fruit rot resistance and genetic structure. CM334 was confirmed as a broad spectrum resistance genotype, whereas all other sources of resistance evaluated were susceptible to infection by one or both isolates evaluated. Although not completely resistant, PI 566811 displayed moderate resistance to fruit and root rot to both P. capsici isolates. Fruit rot resistance had a significant but small to moderate positive correlation (r = 0.26–0.63) with root rot resistance depending on the isolate and length of exposure. Pepper accessions with resistance to Phytophthora root and fruit rot belonging to different genetic subpopulations were identified and could serve as candidates for partial-resistance loci to incorporate into pepper breeding programs.

FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF WHEAT RHIZOSPHERE BACTERIA AND THEIR ANTAGONISTIC ACTIVITY AGAINST WHEAT FOLIAR BLIGHT PATHOGENS

The current study aimed to explore the wheat rhizospheric bacterial community for in vitro plant growth-promoting (PGP) traits and antagonistic activity against foliar blight disease of wheat caused by Alternaria triticina and Bipolaris sorokiniana. The soil samples from the wheat fields across four Indian states namely Uttarakhand (2 sites), Uttar Pradesh, Madhya Pradesh, and Maharashtra were analyzed for their physicochemical properties, enzymatic activities, and bacterial population density (CFU g-1). Amongst the tested soil samples, field soil from Uttar Pradesh has the highest bacterial population density (2.5x107 CFU g 1) while the Uttarakhand (Almora) soil has the lowest (8.5x 105 CFU g 1). A total of 45 bacterial isolates recovered from all the sites were morphologically identified and screened for in-vitro solubilization of phosphate & zinc, production of ammonia, siderophore, chitinase, protease, cellulase, amylase, lipase, and antagonistic activity. All isolates were found positive for one or more tested functional traits. Amongst 45 isolates, six showed >50% inhibition of Alternaria triticina and Bipolaris sorokiniana mycelium, and three isolates P10, UP11 & MH13 exhibited antagonistic activity against both the tested phytopathogens. Through 16S rDNA sequencing six putative biocontrol isolates, P10, UP11, MH13, MP17, MH12, and MP14 were identified as Bacillus methylotrophicus (MN099430.1), Bacillus subtilis (MN099431.1), Bacillus sp. (MN099432.1), Streptomyces sp. (MN099435.1), Lysinibacillus sp. (MN099433.1), and Staphylococcus epidermidis (MN099434.1). The selected wheat rhizobacteria exhibited PGP traits and biocontrol potential hence, may serve as putative biocontrol agents, for the management of foliar blight disease of wheat.

Integrated Management of Foliar Blight of Medicinal Crop (Turmeric) Caused by Alternaria alternata

The infected samples were collected from three districts viz; Kathua, Samba and Jammu for the study of the disease. The causal fungus of the disease was isolated and identified as Alternaria alternata on the basis of morphological characteristics. The cultivars PH-1 and local cultivar of turmeric were sown with three replications in the year 2014 and 2015 respectively, each to study the disease development and its management. During in vitro studies, the five groups of fungicide (Mancozeb, Carbendazim, Copper Oxychloride, Hexaconazole and Propiconazole) were evaluated against Altenaria alternata causing disease, best results were obtained with hexaconazole. The antagonistic activity of two fungal and two bacterial biocontrol agents were studied by dual culture thus Trichoderma viride and Pseudomonas flurescens portrayed better results. Under compatibility of fungal bioagents and bacterial bioagents best compatibility was observed between copper oxychloride and Trichoderma viride and hexaconazole and Pseudomonas flurescens. Under the field conditions the best results among all fifteen treatments, with minimum disease intensity was observed in terms of integration i.e (hexaconazole + Trichoderma viride) (4.2%) in PH-1 cultivar and (6.7%) in local cultivar.

Limiting Fungal Foliar Diseases on Carrots for Organic and Conventional Markets

Michigan ranks fourth in carrot production for the combined fresh and processing markets. Fungal foliar diseases caused by Alternaria dauci and Cercospora carotae occur annually in the state, causing blighted and weakened leaves and petioles. Our objective was to update current disease management strategies for both organic and conventional production by testing Organic Materials Review Institute (OMRI)–approved and conventional fungicides against C. carotae and A. dauci. Field trials conducted in 2015 and 2016 found that the copper-based fungicides (copper hydroxide and copper hydroxide/copper oxychloride) were the only OMRI-approved products that, as indicated by relative area under the disease progress curve (rAUDPC) data, consistently limited foliar blight. In field trials of conventional fungicides, all treatments limited symptomatic foliar area and protected petiole health compared with the control in both years with one exception: propiconazole was similar to the control in 2016 for petiole health and in 2015 for rAUDPC values. During 2016 when disease pressure was high, pyraclostrobin/fluxapyroxad outperformed iprodione, pyraclostrobin, azoxystrobin/propiconazole, and cyprodinil/fludioxonil for the control of foliar blight. Yields differed significantly among conventional treatments only in 2016. In plots sprayed with pyraclostrobin/boscalid, pyraclostrobin/fluxapyroxad, chlorothalonil, and boscalid had higher yields than penthiopyrad, iprodione, and propiconazole. These results can contribute to management strategies for C. carotae and A. dauci in organic or conventional production systems.