Assessing performance of simplified bioassays for soil-borne pathogens in smallholder systems of western Kenya.

Introduction: Soil-borne pathogens cause considerable crop losses and food insecurity in smallholder systems of sub-Saharan Africa. Soil and crop testing is critical for estimating pathogen inoculum levels and potential for disease development, understanding pathogen interactions with soil nutrient and water limitations, as well as for developing informed soil health and disease management decisions. However, formal laboratory analyses and diagnostic services for pathogens are often out of reach for smallholder farmers due to the high cost of testing and a lack of local laboratories. Methods: To address this challenge, we assessed the performance of a suite of simplified soil bioassays to screen for plant parasitic nematodes (e.g., Meloidogyne, Pratylenchus) and other key soil-borne pathogens (Pythium and Fusarium). We sampled soils from on-farm trials in western Kenya examining the impact of distinct nutrient inputs (organic vs. synthetic) on bean production. Key soil health parameters and common soil-borne pathogens were evaluated using both simple bioassays and formal laboratory methods across eleven farms, each with three nutrient input treatments (66 samples in total). Results and Discussion: The soil bioassays, which involved counting galls on lettuce roots and lesions on soybean were well correlated with the abundance of gall forming (Meloidogyne) and root lesion nematodes (e.g., Pratylenchus) recovered in standard laboratory-based extractions. Effectiveness of a Fusarium bioassay, involving the counting of lesions on buried bean stems, was verified via sequencing and a pathogenicity test of cultured Fusarium strains. Finally, a Pythium soil bioassay using selective media clearly distinguished pathogen infestation of soils and infected seeds. When examining management impact on nematode communities, soils amended with manure had fewer plant parasites and considerably more bacterivore and fungivore nematodes compared to soils amended with synthetic N and P. Similarly, Pythium presence was 35% lower in soils amended with manure, while the Fusarium assays indicated 23% higher Fusarium infection in plots with amended manure. Our findings suggest that relatively simple bioassays can be used to help farmers assess soil-borne pathogens in a timely manner, with minimal costs, thus enabling them to make informed decisions on soil health and pathogen management.

Genomic and Transcriptomic Comparisons of the Twig Blight Pathogen, Passalora sequoiae, with Mycosphaerellaceae Foliar and Conifer Pathogens.

Passalora sequoiae is a foliar pathogen to conifer tree species. In this study, we conducted whole-genome and transcriptome analyses on isolates of P. sequoiae collected from symptomatic Leyland cypress leaves from a Christmas tree farm in Mississippi. The objectives for this research were to elucidate the pathogenicity mechanisms of P. sequoiae by characterizing the genome and transcriptome and possibly identify unique and shared predicted genes in comparison with non-conifer/canker and foliar pathogens in the family Mycosphaerellaceae. P. sequoiae was found to be similar to other foliar Mycosphaerellaceae pathogens and likely represents a hemibiotrophic lifestyle based on comparisons across pathogens. The genome and in planta transcriptome highlighted some unique features of P. sequoiae: the significant presence of chitin synthases and fructose-degrading carbohydrate-degrading enzymes, trans-AT PKS genes, and antibiotic gene clusters that were unique to P. sequoiae compared with the other Mycosphaerellaceae species genomes. Several transcripts that were highly expressed in planta were identified as effectors, yet the functions were not characterized. These targets provide ample resources to continue to characterize pathogen-conifer host interactions in conifer foliar pathogens. Furthermore, this research helps build genomic resources for an important plant pathogen on Leyland cypress that will further our ability to develop novel management practices that could begin with breeding for resistance.

First draft genome and transcriptome of Cercosporidium personatum, causal agent of late leaf spot disease of peanut.

OBJECTIVE: Two main fungal leaf spot diseases occur in peanut, namely early leaf spot (ELS) and late leaf spot (LLS), these cause a yearly average of $44 million losses. Limited genetic information, 3534 bp of sequencing, exists about the causal agent of LLS, Cercosporidium personatum (syn. Nothopassalora personata, syn. Phaeoisariopsis personata). The extremely slow growth of this fungus, approximately 1 cm colony in 6 months, and challenges in nucleic acid extractions have hindered research on LLS. Our goal in this work is to provide a reference genome for research on this pathogen. RESULTS: Whole genome and transcriptome sequencing of the LLS fungus were obtained. A total of 233,542,110 reads of the genome were de novo assembled resulting in 1061 scaffolds, and estimated genome size 27,597,787 bp. RNA sequencing resulted in 11,848,198 reads that were de novo assembled into 13,343 contigs. Genome annotation resulted in 10,703 putative genes. BUSCO analysis of the genome and annotation resulted in 91.1% and 89.5% completeness, respectively. Phylogenetic dendrograms for 5442 bp and 4401 bp of RNA Polymerase II largest and second largest subunits, and for 5474 bp of the ribosomal RNA cistron of C. personatum are presented in relation to closely related fungi.

Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring.

BACKGROUND: Development and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs. RESULTS: This study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa. CONCLUSIONS: These results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.