Genome-Wide Association Analysis for Resistance to Coniothyrium glycines Causing Red Leaf Blotch Disease in Soybean.

Soybean is a high oil and protein-rich legume with several production constraints. Globally, several fungi, viruses, nematodes, and bacteria cause significant yield losses in soybean. Coniothyrium glycines (CG), the causal pathogen for red leaf blotch disease, is the least researched and causes severe damage to soybean. The identification of resistant soybean genotypes and mapping of genomic regions associated with resistance to CG is critical for developing improved cultivars for sustainable soybean production. This study used single nucleotide polymorphism (SNP) markers generated from a Diversity Arrays Technology (DArT) platform to conduct a genome-wide association (GWAS) analysis of resistance to CG using 279 soybean genotypes grown in three environments. A total of 6395 SNPs was used to perform the GWAS applying a multilocus model Fixed and random model Circulating Probability Unification (FarmCPU) with correction of the population structure and a statistical test p-value threshold of 5%. A total of 19 significant marker-trait associations for resistance to CG were identified on chromosomes 1, 5, 6, 9, 10, 12, 13, 15, 16, 17, 19, and 20. Approximately 113 putative genes associated with significant markers for resistance to red leaf blotch disease were identified across soybean genome. Positional candidate genes associated with significant SNP loci-encoding proteins involved in plant defense responses and that could be associated with soybean defenses against CG infection were identified. The results of this study provide valuable insight for further dissection of the genetic architecture of resistance to CG in soybean. They also highlight SNP variants and genes useful for genomics-informed selection decisions in the breeding process for improving resistance traits in soybean.

Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants.

Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation.

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

Emergence of rice yellow mottle virus in eastern Uganda: Recent and singular interplay between strains in East Africa and in Madagascar.

Epidemics of rice yellow mottle virus (RYMV) have developed recently in eastern Uganda, close to Lake Victoria in East Africa. Unexpectedly, all isolates from the affected area belonged to a single strain (named S4ug), a strain that is different from the S4lv strain that has been prevalent in the Lake Victoria basin for the past five decades. Interestingly, the S4ug strain is most closely related at the genomic level (except ORF1) to the strain present in Madagascar (S4mg), 2000km away. The minor parent of the S4mg recombinant strain could not be detected. Molecular clock dating analysis indicated that the singular sequence of events - that associated the emergence of a new strain (S4ug), a modular recombination between closely related strains (S4mg and S4ug) and a long distance transmission (S4mg) - occurred recently, within the past few decades. This finding is at variance with the process of gradual strain dispersal and diversification over two centuries throughout Africa that was previously established.

Field evaluation of selected cassava genotypes for cassava brown streak disease based on symptom expression and virus load.

BACKGROUND: Production of cassava (Manihot esculenta Crantz), a food security crop in sub-Saharan Africa, is threatened by the spread of cassava brown streak disease (CBSD) which manifests in part as a corky necrosis in the storage root. It is caused by either of two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), resulting in up to 100% yield loss in susceptible varieties. METHODS: This study characterized the response of 11 cassava varieties according to CBSD symptom expression and relative CBSV and UCBSV load in a field trial in Uganda. Relative viral load was measured using quantitative RT-PCR using COX as an internal housekeeping gene. RESULTS: A complex situation was revealed with indications of different resistance mechanisms that restrict virus accumulation and symptom expression. Four response categories were defined. Symptom expression was not always positively correlated with virus load. Substantially different levels of the virus species were found in many genotypes suggesting either resistance to one virus species or the other, or some form of interaction, antagonism or competition between virus species. CONCLUSIONS: A substantial amount of research still needs to be undertaken to fully understand the mechanism and genetic bases of resistance. This information will be useful in informing breeding strategies and restricting virus spread.

In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis.

BACKGROUND: A key challenge for designing RNAi-based crop protection strategies is the identification of effective target genes in the pathogenic organism. In this study, in vitro antifungal activities of a set of synthetic double-stranded RNA molecules on spore germination of two major pathogenic fungi of banana, Fusarium oxysporum Schlecht f. sp. cubense WC Snyder & HN Hans (Foc) and Mycosphaerella fijiensis Morelet (Mf) were evaluated. RESULTS: All the tested synthetic dsRNAs successfully triggered the silencing of target genes and displayed varying degrees of potential to inhibit spore germination of both tested banana pathogens. When Foc dsRNAs were applied to Foc spores, inhibition ranged from 79.8 to 93.0%, and from 19.9 to 57.8% when Foc dsRNAs were applied to Mf spores. However, when Mf dsRNAs were applied on Mf spores, inhibition ranged from 34.4 to 72.3%, and from 89.7 to 95.9% when Mf dsRNAs were applied to Foc spores. CONCLUSION: The dsRNAs for adenylate cyclase, DNA polymerase alpha subunit and DNA polymerase delta subunit showed high levels of spore germination inhibition during both self- and cross-species tests, making them the most promising targets for RNA-mediated resistance in banana against these fungal pathogens. © 2013 Society of Chemical Industry.

High Potassium, Calcium, and Nitrogen Application Reduce Susceptibility to Banana Xanthomonas Wilt Caused by Xanthomonas campestris pv. musacearum.

The effect of exogenous applications of potassium (K), calcium (Ca), and nitrogen (N) on the susceptibility of four banana cultivars to Banana Xanthomonas wilt (BXW) was studied. Murashige and Skoog (MS) medium with normal concentrations of K at 783 mg/liter, Ca at 121 mg/liter, and N at 841 mg/liter was modified to contain various concentrations of K, Ca, and N. Each nutrient was varied singly, each with three replicate experiments. The concentrations were K at 78, 157, 391, 783, 1,565, and 3,913 mg/liter; Ca at 12, 24, 60, 121, 241, and 603 mg/liter; and N at 84, 168, 420, 841, and 1,682 mg/liter. Plantlets were generated in vitro on normal MS medium and later exposed to the nutrient concentrations for a total of 8 weeks. Thereafter, they were artificially inoculated with Xanthomonas campestris pv. musacearum using an insulin syringe. In each nutrient, plantlets exposed to higher nutrient concentrations significantly (P < 0.0001) accumulated more nutrient in their tissues compared with those exposed to lesser nutrient concentrations. Wilt incidences were significantly reduced, and incubation periods (time from inoculation to appearance of first disease symptoms) increased, with increasing nutrient application. The study lays a background for in vivo studies aimed at management of BXW using nutrients, such as fertilizer application.

A Polymerase Chain Reaction Assay for the Detection of Xanthomonas campestris pv. musacearum in Banana.

Polymerase chain reaction (PCR) primers (BXW-1 and BXW-3) for conventional PCR were developed from conserved sequences in the hrpB operon of the hrp gene cluster from Xanthomonas campestris pv. musacearum, the causative agent of banana Xanthomonas wilt (BXW). All 50 strains of X. campestris pv. musacearum, isolated from Uganda, Rwanda, and Tanzania, produced a 214-bp amplicon when whole cells, bacterial ooze from infected tissue, and genomic DNA purified from bacterial ooze or infected tissue were used as template. The BXW primers also detected strains of X. axonopodis pv. vasculorum isolated from sugarcane and maize and strains of X. vasicola pv. holcicola isolated from sorghum. All of the strains of X. campestris pv. musacearum were clonal when compared using enterobacterial repetitive intergenic consensus PCR.