Isolation and identification of Metarhizium anisopliae from Chilo venosatus (Lepidoptera: Pyralidae) cadaver

Sugarcane stem borer, Chilo venosatus Walker (Lepidoptera: Pyralidae) is a significant sugarcane pest in South China. Conidia or mycelia collected from the surface of sugarcane stem borer cadavers were cultured. The colony morphology, mycelia and conidial yield were observed with three-agar culture media: potato dextrose agar medium (PDA), potato dextrose with 1% (w/v) peptone agar medium (PPDA), and oatmeal agar medium (OMA). 16 different isolates were identified as Metarhizium anisopliae (Metschnikoff) based on macromorphological, micromorphological, and molecular characteristics, and PPDA was the better culture medium for vegetative growth and conidial yield (109 conidia/ml) than PDA (108 conidia/ml) and OMA (108 conidia/ml). To confirm whether these isolates were pathogenic to  C. venosatus, their virulence to the sugarcane stem borer was tested in the laboratory. Both HS (10 isolates) and LY (6 isolates) strains were pathogenic to C. venosatus. Several highly virulent strains were screened in vitro (the mortalities of the eight isolates HS1, HS6, HS7, LY2, LY3, LY6, HS3 and HS9 were from 96 to 100%), and tests for controlling the sugarcane stem borer were preliminarily performed in vivo. The results show that significant (p=0.01) reductions in adult population were caused by the strains. So, M. anisopliae isolated from the cadavers of C. venosatus Walker is a potential biocontrol agent against this pest in South China.Keywords: Metarhizium anisopliae, isolation, identification, Chilo venosatus, culture medium, biological contro

The Effect of Banana Rhizosphere Chemotaxis and Chemoattractants on Bacillus velezensis LG14-3 Root Colonization and Suppression of Banana Fusarium Wilt Disease

Fusarium oxysporum f. sp. cubense (Foc) causes banana Fusarium wilt disease, which is a destructive soil-borne disease. Many plants can recruit rhizosphere microorganisms using their root exudates, thereby shaping the rhizosphere microbiome to resist pathogen infection. Therefore, this study was conducted to explore the role of root exudates in the process of biocontrol strain colonization and resistance to pathogens. In this study, the banana root exudates used as chemoattractants were obtained by hydroponics. Bacillus velezensis strain LG14-3 was isolated from the infected area of the root system of banana and showed significant chemotaxis to banana root exudates and strong inhibition of Fusarium oxysporum f. sp. cubense. Further analysis found that LG14-3 showed chemotaxis toward the components of banana root exudates, such as citric acid, succinic acid, glycine, D-galactose and D-maltose, and glycine and citric acid, which resulted in more significant chemotaxis of LG14-3. Moreover, banana root exudates enhanced the swarming motility and biofilm formation of LG14-3. Pot experiments showed that glycine and citric acid enhanced the colonization ability of Bacillus velezensis LG14-3 in the banana rhizosphere and reduced the disease severity index of banana fusarium wilt. Glycine and citric acid enhanced the growth-promoting ability of LG14-3 under pathogen stress. Our results showed that the addition of chemotactic substances enhanced the biocontrol potential of Bacillus velezensis LG14-3 to prevent banana Fusarium wilt

Genome and Transcriptome Analysis of the Fungal Pathogen <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> Causing Banana Vascular Wilt Disease

<div><p>Background</p><p>The asexual fungus <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (Foc) causing vascular wilt disease is one of the most devastating pathogens of banana (<i>Musa</i> spp.). To understand the molecular underpinning of pathogenicity in Foc, the genomes and transcriptomes of two Foc isolates were sequenced.</p><p>Methodology/Principal Findings</p><p>Genome analysis revealed that the genome structures of race 1 and race 4 isolates were highly syntenic with those of <i>F. oxysporum</i> f. sp. <i>lycopersici</i> strain Fol4287. A large number of putative virulence associated genes were identified in both Foc genomes, including genes putatively involved in root attachment, cell degradation, detoxification of toxin, transport, secondary metabolites biosynthesis and signal transductions. Importantly, relative to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with some expanded gene families of transporters and transcription factors for transport of toxins and nutrients that may facilitate its ability to adapt to host environments and contribute to pathogenicity to banana. Transcriptome analysis disclosed a significant difference in transcriptional responses between Foc1 and Foc4 at 48 h post inoculation to the banana ‘Brazil’ in comparison with the vegetative growth stage. Of particular note, more virulence-associated genes were up regulated in Foc4 than in Foc1. Several signaling pathways like the mitogen-activated protein kinase Fmk1 mediated invasion growth pathway, the FGA1-mediated G protein signaling pathway and a pathogenicity associated two-component system were activated in Foc4 rather than in Foc1. Together, these differences in gene content and transcription response between Foc1 and Foc4 might account for variation in their virulence during infection of the banana variety ‘Brazil’.</p><p>Conclusions/Significance</p><p>Foc genome sequences will facilitate us to identify pathogenicity mechanism involved in the banana vascular wilt disease development. These will thus advance us develop effective methods for managing the banana vascular wilt disease, including improvement of disease resistance in banana.</p></div

Protein classification of Foc1 and Foc4.

<p>Protein classification of Foc1 and Foc4.</p

Gene Ontology (GO) functional annotation of differentially expressed genes (DEGs) in <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> race 1 (Foc1).

<p>(<b>A</b>) Up-regulated genes in Foc1 could be only enriched into 2 groups. (<b>B</b>) Down-regulated genes in Foc1 could be enriched into three main GO categories and 23 groups on GO.level2 and GO.level3, 10 groups in biological process, 8 in cellular component, and 4 in molecular function. The X- axis represents the number of genes in a functional group.</p

Differential gene expression profiling for the orthologous genes encoding histidine kinase from <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> race 1 (Foc1) and race 4 (Foc4) infecting the banana ‘Brazil’.

<p>The Heat Map figures were generated using the log2 ratio of corresponding Foc1 (or Foc4) gene transcription data at 48 h post inoculation (48h_Reads Per Kilo bases per Million reads, 48h_RPKM) against Foc1 (or Foc4) 0h_RPKM data at vegetative growth stage, i.e. Log2 (48h-RPKM/0h_RPKM_). The figures show HK genes that are up regulated (red) and down regulated (green) relative to that at vegetative growth stage.</p

The orthologs of <i>SIX</i>-genes in Foc1 and Foc4.

<p>The orthologs of <i>SIX</i>-genes in Foc1 and Foc4.</p

Schematic representation of signaling pathways activated in <i>F. oxysporum</i> f. sp. <i>cubense</i> race 4 isolate (Foc4) during infection of the banana variety ‘Brazil’.

<p>FGA1 (Gα) mediated G protein signaling, the FMK1-controlled mitogen-activated protein kinase signaling pathway and the pathogenicity associated two-component signal transduction system might be activated in Foc4. GPCR, G protein coupled receptor; Gα, G protein alpha subunit; Gβ & Gγ, G protein beta and gamma subunits; GTP, guanosine triphosphate; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; ATP, adenosine triphosphate; PKA, protein kinase A; MAPKKK, mitogen-activated protein kinase kinase kinase; MAPKK, mitogen-activated protein kinase kinase; FMK1, mitogen-activated protein kinase; Ste12, transcription factor; SLN, histidine kinase; RR, response regulator. These pathways were characterized to associate with pathogenesis in fungal pathogens.</p

Infection cycle of the banana vascular wilt pathogen <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (Foc).

<p>(A) macroconidia. (B) microconidia. (C) chlamydospore produced by the GFP-marked Foc isolate. (D) Attachment of Foc hyphae on banana roots. (E) Colonization of Foc hyphae in vascular bundles of banana roots (the arrow indicated). (F) A longitudinal section of banana roots shows fungal hyphae growing in vascular bundles. (G) The diseased banana plants with the dominant symptoms of yellowing leaves. (H–I) the vascular bundles of pseudostem and rhizome from diseased banana turn dark-reddish brown (the arrow indicated).</p

Comparison of genome structures of the <i>F. oxysporum</i> f. sp. <i>cubense</i> race 1 (Foc1) and race 4 (Foc4) with that of <i>F. oxysporum</i> f. sp. <i>lycopersici</i> (Fol).

<p>The size of window is 100(a) <i>F. oxysporum</i> f. sp. <i>lycopersici</i> (<i>Fol</i>) chromosomes. (b) The average depth of Foc1 reads mapped on Fol chromosomes. (c) The average depth of Foc4 reads mapped on Fol chromosomes. (d) The SNP and InDel number of Foc1. (e) The SNP and InDel number of Foc4. (f) Gene density of Fol. (g) Repetitive sequence density of Fol.</p