Pathogenic and morphological variability among the isolates of Fusarium udum (Butler) causing wilt of pigeonpea (Cajanus cajan)

Wilt is the most serious disease causing irreversible losses and lethal damage to the crop in Bihar, Jharkhand, Orissa and the West Bengal states of Eastern India. Because of the existence of high pathogen variability, management of the disease using resistant cultivars will be a challenging task. For breeding resistant varieties, knowledge of pathogen variability in the particular crop area is essential. In present study, fifteen isolates viz., Fu-9, Fu-10, Fu-27,Fu-32, Fu-42, Fu-49, Fu-63, Fu-72, Fu-73, Fu-74,Fu-75,Fu-84, Fu-86, Fu-87, Fu-97of Fusarium udum obtained from wilt infected pigeonpea plants of Bihar, Jharkhand, Orissa and West Bengal of Eastern India were assessed for the phenotypic variation. The isolates exhibited considerable variations in cultural and morphological characters viz., radial growth, mycelial color, sporulation, size and shape of macroconidia. Pathogenic variability on soil inoculated pot grown plants of pigeonpea resulted in 12.0 to 57.1 percent wilt incidence. The isolates also exhibited significant variations in symptoms like drooping of shoots, latent period, timing of fungal invasion, vascular clogging, drooping of shoots, and wilt establishment etc. Based on the wilt incidence, the fifteen isolates were distinguished into pathogenic groups. Fu- 10, Fu-27, Fu-32 and Fu-49 were found highly pathogenic and predominantly causing the typical wilt symptoms of more than one 31% wilt incidence. The present study indicates the existence of variability among isolates of vascular wilt pathogen collected from Eastern region of India.

Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.

Improving Pathogen Resistance by Exploiting Plant Susceptibility Genes in Coffee (Coffea spp.)

Coffee (Coffea spp.) is an economically important crop widely cultivated in (sub) tropical countries worldwide. Commercial coffee production relies mainly on two related species, namely C. arabica and C. canephora. Due to their perennial growth habit, cultivation practices, and narrow genetic diversity, coffees are constantly exposed to many diseases and pests. Coffee leaf rust (Hemileia vastatrix Berk. et Br.), coffee berry disease (Colletotrichum kahawae Bridge and Waller), and coffee wilt disease (Gibberella xylarioides Heim and Saccas/Fusarium xylarioides) are the top fungal diseases affecting C. arabica and C. canephora production areas worldwide. In many regions, chemical-based control measures are widely used and are the only way to control the diseases. Developing resistant cultivars is one of the prerequisites for increasing sustainable market demand and agriculture. However, desired and required resistance traits are not always available in the gene pool. Furthermore, from other crops it is clear that dominant resistance genes introduced into varieties are not durable because of pathogen variability and the emergence of new races of the different pathogens. Utilization of altered susceptibility genes (S genes) offers a novel and alternative strategy for the breeding of durable and broad-spectrum resistance. The S gene encodes a host factor that facilitates a compatible interaction with the pathogen, and impairment of S genes leads to loss-of-susceptibility. In this review, guidelines for effective identification, characterization, and utilization of dysfunctional S genes are proposed to aid breeding activities in order to introduce durable resistance in Coffea spp. Several candidate S genes likely contributing to the susceptibility of Colletotrichum spp., Fusarium spp., and Meloidogyne spp. are discussed. With the rapid development of genetic engineering techniques, including CRISPR-associated systems, we now have the potential to accelerate the application of S genes to achieve durable resistance in coffee.

A diagnostic marker kit for Fusarium wilt and sterility mosaic diseases resistance in pigeonpea

Fusarium wilt (FW) and sterility mosaic diseases (SMD) are key biotic constraints to pigeonpea production. Occurrence of these two diseases in congenial conditions is reported to cause complete yield loss in susceptible pigeonpea cultivars. Various studies to elucidate genomic architecture of the two traits have revealed significant marker–trait associations for use in breeding programs. However, these DNA markers could not be used effectively in genomics-assisted breeding for developing FW and SMD resistant varieties primarily due to pathogen variability, location or background specificity, lesser phenotypic variance explained by the reported QTL and cost-inefficiency of the genotyping assays. Therefore, in the present study, a novel approach has been used to develop a diagnostic kit for identification of suitable FW and SMD resistant lines. This kit was developed with 10 markers each for FW and SMD resistance. Investigation of the diversity of these loci has shown the role of different alleles in different resistant genotypes. Two genes (C.cajan_03691 and C.cajan_18888) for FW resistance and four genes (C.cajan_07858, C.cajan_20995, C.cajan_21801 and C.cajan_17341) for SMD resistance have been identified. More importantly, we developed a customized and cost-effective Kompetitive allele-specific PCR genotyping assay for the identified genes in order to encourage their downstream applications in pigeonpea breeding programs. The diagnostic marker kit developed here will offer great strength to pigeonpea varietal development program, since the resistance against these two diseases is essentially required for nominating an improved line in varietal release pipeline.

Anti-Helicobacter pylori vaccine: mith or reality?

Here we review the data on the current studies aimed at developing anti-Helicobacter pylori vaccines. Unfortunately, no vaccines recommended for use in human are available now, despite a more than 30-year history of their development and a great body of evidence on vaccine efficiency in animals. Mechanisms underlying vaccine-related effects in animals and human are poorly determined and expect to be further clarified. Moreover, side effects related to vaccines have not investigated in detail. A long-lasting stay of H. pylori in the gastric lumen restricts potential protective effects of host cellular immunity (an effect is mainly associated with antibodies and antimicrobial peptides), that results in low efficacy of systemic immunization and weak immune response. In addition, further complications in developing natural and artificial (vaccination) immune response may be due to the high pathogen variability and low immunogenicity of related antigens. A choice of antigen is crucial upon generating any vaccine. The data on the main pathogen-derived antigens is of high importance while generating both mono- and multicomponent H. pylori vaccines. A number of various antigens was proposed for immunization against H. pylori, some of which are involved in the pathogenetic mechanisms of Helicobacter pylori infection: VacA, CagA, NapA, BabA, SabA and urease. Such vaccines turned out to be efficient in preventing experimental infection in animals. The use of purified microbial antigens successfully induces protective mechanisms to fight against infection, as demonstrated in animal studies (preventive and therapeutic protocols). Compared to using a single antigen, an association of two or three antigens can trigger stronger immune response. Currently, bacterial urease is considered as the most promising candidate antigen, which has been proved to be a valuable a vaccine antigen in numerous studies with mice, ferrets and primates. It remains unclear which route of administration for Helicobacter pylori vaccine would be superior compared to the remainder. Comparing various routes of vaccine administration demonstrated that that mice immunized intranasally and intrarectally resulted in markedly higher protection against Helicobacter pylori infection compared to oral vaccination. Development of H. pylori vaccine faced substantial obstacles due to the pathophysiological, immunological and technological challenges noted above, still remaining an issue so far. At present, a promising approach in advancing H. pylori vaccines is based on using mucosal adjuvants and generation of recombinant probiotics expressing H. pylori-derived antigens for triggering specific immune response upon vaccination.

Phenotypic and Genetic Characterization of Botrytis cinerea Population from Kiwifruit in Sichuan Province, China

Botrytis cinerea (anamorph of Botryotinia fuckeliana) causes gray mold on numerous plants, including kiwifruit. The primary aim of this study was to investigate the phenotypic and genetic characteristics of the Botrytis cinerea population from kiwifruit in Sichuan Province, China. In all, 176 isolates were collected from kiwifruit orchards from eight geographic regions in Sichuan. All isolates were identified as B. cinerea sensu stricto based on the combined datasets, including morphological criteria, determination of the Bc-hch allele, and phylogenetic analysis of the genes RPB2, G3PDH, and HSP60. Three colony types (i.e., sclerotial, mycelial, and conidial) were observed on potato dextrose agar after 2 weeks, with sclerotial isolates, the predominant category, accounting for 40.91%. No obvious differences in microscopic characteristics were observed among the three types. Three genotypes of transposable elements were identified in the B. cinerea population: boty, flipper, and transposa types. The most prevalent genotype from different geographic populations of B. cinerea was transposa; in contrast, the flipper genotype accounted for only 3.98% of the total population, whereas the vacuma genotype was absent. According to MAT locus amplification, 87 and 89 isolates are MAT1-1 and MAT1-2 type, respectively, and the two mating types were found to be balanced overall in the population. Forty-eight representative isolates were all able to cause gray mold to some extent, and disease severities were significantly different between the cultivars Hongyang and Hort16A (P < 0.01). Disease severity was significantly greater on young leaves than on mature leaves (P < 0.01). No significant relationship was found between pathogenicity and geographical region, colony type, or transposon distribution. The results obtained in the present study suggest a relatively uniform species diversity of Botrytis but rich phenotypic and genetic differentiation within the B. cinerea population on kiwifruit in China. Utilizing resistant cultivars and rain-shelter cultivation instead of fungicides may be an effective approach to delaying pathogen variability.

Epidemiological significance of genome variations in Pseudomonas aeruginosa causing chronic lung infection in patients with cystic fibrosis

Objective. To present the data on the main mechanism of molecular variation in P. aeruginosa causing chronic lung infection in patients with cystic fibrosis. Materials and Methods. A total of 1800 throat swabs and sputum samples from cystic fibrosis patients were included in the study over the 10-year period. P. aeruginosa isolates were primarily identified by the biochemical method using the API 20NE test strips (bioMerieux, France). Antimicrobial susceptibility testing was performed by disc diffusion method. Genotyping was conducted by RAPD-PCR and MLST. Whole genome sequencing of three typical P. aeruginosa isolates was performed on an Ion PGM Torrent platform with Ion Sequencing Kit and 316v1 chips (Life Technologies Thermo Fisher, US) according to the manufacturer’s protocol. The RAST web application was used for initial annotation. Results. There were three main variants of the pathogen variability found: population heterogeneity, pathogen microevolution, and replacement by another genotype of the same species. The variation of the pathogen’s genome is due to the acquisition of mobile genetic elements (plasmids), mutations in the chromosomal genes responsible for antibiotic resistance, bacterial viability and survival during persistence in a host, and changes in the prophage regions of the pathogen. Conclusions. Epidemiological significance of the molecular mechanisms of pathogen variation is primarily due to the ability of strains to form epidemiologically significant clone. This requires control measures aimed to limit emergence and distribution of such clones to be developed.