Biological control of the native endophytic fungus Pochonia chlamydosporia from the root nodule of Dolichos lablab on Fusarium wilt of banana TR4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense, Tropical Race 4 (TR4) is a soil-borne disease, and it is devastating. At present, the biological control using antagonistic microorganisms to mitigate TR4 is one of the best strategies as a safe and green way. Yunnan has abundant and diverse microbial resources. Using the dual-culture method, the antagonistic endophytic fungi against TR4 were isolated and screened from the root nodule of Dolichos lablab. The effect of the highest antagonistic activity strain on the morphology of the TR4 mycelium was observed using the scanning electron microscope. According to morphological characteristics and sequence analysis, the strain was identified. The biocontrol effect and plant growth promotion were investigated by greenhouse pot experiment. Using the confocal laser scanning microscope and the real-time fluorescence quantitative PCR, the dynamics of TR4 infestation and the TR4 content in banana plant roots and corms would also be detected. In this study, 18 native endophytic fungi were isolated from a root nodule sample of Dolichos lablab in the mulch for banana fields in Yuxi, Yunnan Province, China. The YNF2217 strain showed a high antagonistic activity against TR4 in plate confrontation experiments, and the inhibition rate of YNF2217 is 77.63%. After TR4 culture with YNF2217 for 7 days in plate confrontation experiments, the morphology of the TR4 mycelium appeared deformed and swollen when observed under a scanning electron microscope. According to morphological characteristics and sequence analysis, the strain YNF2217 was identified as Pochonia chlamydosporia. In the greenhouse pot experiment, the biocontrol effect of YNF2217 fermentation solution on TR4 was 70.97% and 96.87% on banana plant leaves and corms, respectively. Furthermore, YNF2217 significantly promoted the growth of banana plants, such as plant height, leaf length, leaf width, leaf number, pseudostem girth, and both the aboveground and underground fresh weight. Observations of TR4 infestation dynamics in banana roots and corms, along with real-time fluorescence quantitative PCR, verified that YNF2217 inoculation could significantly reduce the TR4 content. Therefore, YNF2217 as P. chlamydosporia, which was found first time in China and reported here, is expected to be an important new fungal resource for the green control of Fusarium wilt of banana in the future.

Banana disease-suppressive soil drives Bacillus assembled to defense Fusarium wilt of banana.

Fusarium wilt of banana (FWB) caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), poses a serious problem for sustainable banana production. Biological control is one of the effective measures to control this destructive disease. High-throughput sequencing of soil microorganisms could significantly improve the efficiency and accuracy of biocontrol strain screening. In this study, the soil microbial diversity of six main banana-producing areas in Yunnan was sequenced by Illumina Miseq platform. The outcome of this study showed the genus of Chujaibacter, Bacillus, and Sphingomonas were significantly enriched in microorganism community composition. Further correlation analysis with soil pathogen (Foc TR4) content showed that Bacillus was significantly negatively correlated with pathogen content. Therefore, we isolated and identified Bacillus from the disease-suppressive soils, and obtained a B. velezensis strain YN1910. In vitro and pot experiments showed that YN1910 had a significant control effect (78.43-81.76%) on banana Fusarium wilt and had a significant growth promotion effect on banana plants.

Effect of natural weed and Siratro cover crop on soil fungal diversity in a banana cropping system in southwestern China.

Introduction: Natural weed cover and a legume cover crop were examined to determine if they could impact soil fungal diversity as an indicator of soil quality in banana production. Methods: Banana in Yunnan Province, China, was grown under three treatments: conventional tillage (bare soil), natural weed cover (primarily goosegrass (Eleusine indica (L.) Gaerth)), or a cover crop (Siratro (Macroptilium atropurpureum (DC.) Urb.)). Analysis of the soil fungal communities between 2017 and 2020 was done by Illumina Miseq high-throughput sequencing. Results: Most significant effects were in the intercropping area for the treatments, whereas it was rarely observed in the furrow planted with banana. Based on the Shannon and Simpson diversity indices, soil fungal diversity in the intercropping area significantly decreased following planting banana in 2017 with all three treatments. However, both the Shannon and Simpson diversity indices showed that there were significant increases in fungal soil diversity in 2019 and 2020 with natural weed cover or Siratro compared to bare soil. At the end of the experiment, significant increases in fungal genera with Siratro compared to bare soil were observed with Mortierella, Acremonium, Plectophaerella, Metarhizium and Acrocalymma, and significant decreases were observed with Fusicolla, Myrothecium, Exserohilum, Micropsalliota and Nigrospora. Siratro resulted in higher stability of the soil fungal microbiome by increasing the modularity and the proportion of negative co-occurrences compared to bare soil. For fungal guilds, Siratro significantly increased saprotrophs_symbiotrophs in 2019 and 2020 and significantly decreased pathogens_saprotrophs in 2020 compared to bare soil. Discussion: Using Siratro as a cover crop in the intercropping area of banana helped maintain soil fungal diversity, which would be beneficial for soil health with more symbiotrophs and less pathogens in the soil. However, further research is needed to determine the long-term impact of weed or Siratro cover crop on the fungal soil ecosystem and growth of banana.

Disentangling the resistant mechanism of Fusarium wilt TR4 interactions with different cultivars and its elicitor application.

Fusarium wilt of banana, especially Tropical Race 4 (TR4) is a major factor restricting banana production. Developing a resistant cultivar and inducing plant defenses by elicitor application are currently two of the best options to control this disease. Isotianil is a monocarboxylic acid amide that has been used as a fungicide to control rice blast and could potentially induce systemic acquired resistance in plants. To determine the control effect of elicitor isotianil on TR4 in different resistant cultivars, a greenhouse pot experiment was conducted and its results showed that isotianil could significantly alleviate the symptoms of TR4, provide enhanced disease control on the cultivars 'Baxi' and 'Yunjiao No.1' with control effect 50.14% and 56.14%, respectively. We compared the infection processes in 'Baxi' (susceptible cultivars) and 'Yunjiao No.1' (resistant cultivars) two cultivars inoculated with pathogen TR4. The results showed that TR4 hyphae could rapidly penetrate the cortex into the root vascular bundle for colonization, and the colonization capacity in 'Baxi' was significantly higher than that in 'Yunjiao No.1'. The accumulation of a large number of starch grains was observed in corms cells, and further analysis showed that the starch content in 'Yunjiao No. 1' as resistant cultivar was significantly higher than that in 'Baxi' as susceptible cultivar, and isotianil application could significantly increase the starch content in 'Baxi'. Besides, a mass of tyloses were observed in the roots and corms and these tyloses increased after application with isotianil. Furthermore, the total starch and tyloses contents and the control effect in the corms of 'Yunjiao No.1' was higher than that in the 'Baxi'. Moreover, the expression levels of key genes for plant resistance induction and starch synthesis were analyzed, and the results suggested that these genes were significantly upregulated at different time points after the application of isotianil. These results suggest that there are significant differences between cultivars in response to TR4 invasion and plant reactions with respect to starch accumulation, tyloses formation and the expression of plant resistance induction and starch synthesis related genes. Results also indicate that isotianil application may contribute to disease control by inducing host plant defense against TR4 infection and could be potentially used together with resistant cultivar as integrated approach to manage this destructive disease. Further research under field conditions should be included in the next phases of study.

A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers' Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in Bacillus.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), especially Tropical Race 4 (TR4), seriously threatens banana production worldwide. There is no single effective control measure, although certain Bacillus strains secrete antibiotics as promising disease-biocontrol agents. This study identified five Bacillus strains displaying strong antibiotic activity against TR4, using a systemic assessment for presence/absence of genetic markers at genome level, and expression profiles at transcriptome level. A conventional PCR with 13 specific primer pairs detected biocontrol-related genes. An accurate, quantitative real-time PCR protocol with novel designed specific primers was developed to characterise strain-specific gene expression, that optimises strain-culturing and RNA-isolation methodologies. Six genes responsible for synthesising non-ribosomal peptide synthetase biocontrol metabolites were detected in all five strains. Three genes were involved in synthesising three Polyketide synthetase metabolites in all five strains, but the macrolactin synthase gene mln was only detected in WBN06 and YN1282-2. All five Bacillus strains have the genes dhb and bioA, essential for synthesising bacillibactin and biotin. However, the gene sboA, involved in subtilisin synthesis, is absent in all five strains. These genes' expression patterns were significantly different among these strains, suggesting different mechanisms involved in TR4 biocontrol. Results will help elucidate functional genes' biocontrol mechanisms.

Identification and Fine Mapping of Pi69(t), a New Gene Conferring Broad-Spectrum Resistance Against Magnaporthe oryzae From Oryza glaberrima Steud.

The discovery and deployment of new broad-spectrum resistance (R) genes from cultivated rice and its wild relatives is a strategy to broaden the genetic basis of modern rice cultivars to combat rice blast disease. Oryza glaberrima possessing many valuable traits for tolerance to biotic and abiotic stresses, is an elite gene pool for improvement of Asian cultivated rice. An introgression line IL106 derived from O. glaberrima (Acc. IRGC100137) confers complete resistance to Magnaporthe oryzae in blast nursery. Genetic analysis using 2185 BC6F2 progenies derived from a cross between IL106 and the recurrent parent Dianjingyou 1 showed that IL106 harbors a single dominant resistance gene against M. oryzae strain 09BSH-10-5A. This gene was preliminarily mapped on the long arm of chromosome 6 of rice in a region of ca. 0.9 cM delimited by two SSR markers (RM20650 and RM20701). In order to finely map this gene, 17,100 additional progenies were further analyzed. As a result, this gene was further narrowed down to a region flanked by two molecular markers STS69-15 and STS69-7, and co-segregated with 3 molecular markers, RM20676, STS69-21 and STS69-22 on the long arm of chromosome 6. Based on reference genome sequences, this R gene was mapped in silico in 76.1-Kb and 67.7-Kb physical intervals, and containing 4 and 3 NBS-LRR candidate genes in O. sativa cultivar Nipponbare and O. glaberrima cultivar CG14, respectively. Because no blast resistance gene was finely mapped in this physical interval before, this R gene was considered as not described yet and designated as Pi69(t), which is the first identified and finely mapped blast R gene from O. glaberrima, as far as we know. Evaluation of IL106 with 151 blast strains collected from 6 countries in Asia showed that 148 strains are avirulent on IL106, suggesting that Pi69(t) is a broad-spectrum blast R gene, and a promising resistant resource for improvement of Asian cultivated rice.

Complete mitochondrial genome of banana new pest Basilepta fulvipes (Coleoptera: Eumolpinae) and phylogenetic analysis.

Basilepta fulvipes (Motschulsky, 1860) is a banana new pest and mainly distributed in Eastern Asia. The complete mitogenome of B. fulvipes (GenBank accession number MT627597) is 15,762 bp in size, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding D-loop region. The D-loop region is located between 12S rRNA and tRNAIle . The base composition of the whole B. fulvipes mitogenome is 41.66% for A, 8.89% for G, 34.32% for T and 15.12% for C, with a high AT bias of 75.98%. The present data could contribute to further detailed phylogeographic analysis and comprehensive control of this banaba new pest.

New Geographical Insights of the Latest Expansion of Fusarium oxysporum f.sp. cubense Tropical Race 4 Into the Greater Mekong Subregion.

Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.

Fine mapping of Pi57(t) conferring broad spectrum resistance against Magnaporthe oryzae in introgression line IL-E1454 derived from Oryza longistaminata.

Wild species of the genus Oryza are excellent gene pools for improvement of agronomic traits of Asian cultivated rice. The blast resistance gene Pi57(t) in the introgression line IL-E1454 derived from Oryza longistaminata was previously mapped on rice chromosome 12. Inoculation with 322 Magnaporthe oryzae isolates collected from 6 countries indicated that Pi57(t) conferred broad spectrum resistance against M. oryzae. Two mapping populations consisting of 29070 and 10375 F2 plants derived from the crosses of resistant donor IL-E1454 with susceptible parents RD23 and Lijiangxintuanheigu respectively, were used for fine mapping of Pi57(t) locus. Based on genotyping and phenotyping results of recombinants screened from the two crosses, Pi57(t) was finally mapped to a 51.7-kb region flanked by two molecular markers (STS57-320 and STS57-372) on the short arm and close to the centromere of chromosome 12. Six candidate resistance genes were predicted in the target region according to the reference sequence of Nipponbare. These results could facilitate both marker-assisted selection for disease-resistant breeding and gene cloning.

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus.

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.