RESUMO
Melon (Cucumis melo L.) is the second most exported fruit in Brazil with an annual production of 27.5 million tons (FAO 2023). From September 2019 through February 2020, 50-day-old melon plants started showing root rot symptoms (dark-brow necrotic zones in their roots that extended to the collar zone) in northeastern Brazil, 30% of the plants in the fields were affected by the disease. The fields are in clay soil where melon, in monoculture, is produced all year long with three cycles of the culture per year. A total of 132 samples from "Yellow" and "Cantaloupe" cultivars were collected from four melon fields (4°59'45.3"S, 37°33'39.7"W; 4°57'10.2"S, 37°31'37.1"W; 5°38'17.9"S, 37°56'27.7"W; and 5°00'25.5"S, 37°23'55.3"W). Small pieces of diseased tissues were surface disinfested in 70% ethanol for 30 sec, in 2% sodium hypochlorite for 1 min, washed in sterilized distilled water, plated on a PDA Petri dishes with tetracycline (0.05g/L), and incubated for seven days at 28 ± 2 ºC. Nine representative isolates were selected for downstream analysis. Colonies were white and later became dark gray, pycnidia and conidia were produced after 30 days ofncubation at 25°C under near-UV light in water-agar medium. Conidia were hyaline when immature and dark brown when mature, ranging from cylindrical subovoid to ellipsoidal and septate to non-septate, and with an average size of 12.54 to 21.97 µm. The colonies were morphologically identified as Lasiodiplodia sp. (Phillips et al. 2013). Total DNA from the isolates was extracted and the ITS, TUB, and TEF-1α genes (Jayawardena et al. 2019) were partially amplified by PCR, Sanger sequenced, and deposited in Genbank: ITS (OM102511 to OM102520), TUB (OR062087 to OR062094 and OR062095), and TEF-1α (OP536826 to OP536835). Blastn analysis of the partial sequences ITS (519bp), TUB (388bp), and TEF-1α (315bp) showed 100% nucleotide similarity of the isolates with sequences of L. brasiliensis and L. theobromae from the GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis method. All nine isolates were grouped into the L. brasiliensis clade with 71% bootstrap support, confirming the isolates's identity. Pathogenicity assays were conducted in a greenhouse using the wooden toothpick inoculation method (Nogueira et al. 2019). "Goldex" Yellow melon seedlings were used in a completely randomized experimental design, with 10 treatments (9 isolates + Mock) and six replicates, with one plant per pot. Plants were inoculated 15 days after sowing, and disease severity was evaluated 50 days after inoculation. All nine isolates caused symptoms in the assessed melon plants. The fungus was reisolated from the lesions and looked morphologically identical to the inoculated fungus, fulfilling Koch's postulates. The pathogenicity test was repeated and yielded similar results. All samples in this study were provided by melon growers who were concerned about the high incidence of root rot disease in their plantations. More research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to melons to develop strategies for disease control to properly assist the growers's concerns. This pathogen has been reported to cause disease in other crops in Brazil, e.g., watermelon (Alves et al. 2023) and apples (Martins et al. 2018). However, to the best of our knowledge, this is the first report of L. brasiliensis causing root rot in melons in Brazil.
RESUMO
Root rot in common bean is a disease that causes serious damage to grain production, particularly in the upland areas of Eastern and Central Africa where significant losses occur in susceptible bean varieties. Pythium spp. and Fusarium spp. are among the soil pathogens causing the disease. In this study, a panel of 228 lines, named RR for root rot disease, was developed and evaluated in the greenhouse for Pythium myriotylum and in a root rot naturally infected field trial for plant vigor, number of plants germinated, and seed weight. The results showed positive and significant correlations between greenhouse and field evaluations, as well as high heritability (0.71-0.94) of evaluated traits. In GWAS analysis no consistent significant marker trait associations for root rot disease traits were observed, indicating the absence of major resistance genes. However, genomic prediction accuracy was found to be high for Pythium, plant vigor and related traits. In addition, good predictions of field phenotypes were obtained using the greenhouse derived data as a training population and vice versa. Genomic predictions were evaluated across and within further published data sets on root rots in other panels. Pythium and Fusarium evaluations carried out in Uganda on the Andean Diversity Panel showed good predictive ability for the root rot response in the RR panel. Genomic prediction is shown to be a promising method to estimate tolerance to Pythium, Fusarium and root rot related traits, indicating a quantitative resistance mechanism. Quantitative analyses could be applied to other disease-related traits to capture more genetic diversity with genetic models.
RESUMO
MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.