Streptomyces-mediated growth enhancement and Bacterial Panicle Blight disease suppression in rice plants under greenhouse conditions.

Streptomyces corchorusii TKR8, Streptomyces corchorusii JAS2 and Streptomyces misionensis TBS5 were previously obtained from rice fields and have been studied as a biocontrol agent against the causal agent of Bacterial Panicle Blight (BPB) disease on rice, Burkholderia glumae, and rice plant growth promoter. This study evaluated the potential of plant growth-promoting Streptomyces (PGPS) to control B. glumae and promote rice plants' growth under greenhouse conditions. PGPS were further characterized based on their phenotypic and biochemical differences. Multilocus sequence analysis (MLSA) by amplifying gyrB, rpoB and trpB using PCR was conducted to identify the PGPS further. The antimicrobial activity of PGPS against B. glumae was investigated using a survival assay and microscopic analysis. Result indicates that JAS2 (61.2 %) utilized the highest number of carbohydrates tested, followed by TKR8 (53.1 %) and TBS5 (40.8 %) as analyzed using API 50 CH. Based on MLSA analysis of the concatenated partial sequences (1520 bp) from three housekeeping genes, the neighbor-joining tree identified JAS2 and TKR8 as S. corchorusii. Meanwhile, TBS5 as S. misionensis. Antimicrobial activity of PGPS against B. glumae has found that the supernatant of Streptomyces reduced the survival viability of B. glumae up to 50.7-70.3 %. SEM images showed that substantial morphological changes happened in cell membranes of B. glumae after the Streptomyces treatment. The highest vigor index of inoculated seedlings was determined when rice seeds were treated with a spore suspension of 1 × 107 spore/mL (for JAS2 and TKR8) and 1 × 106 spore/mL (for TBS5). Under greenhouse conditions, Streptomyces-treated plants showed improvement in rice plants' growth and grain yield and reduced the BPB disease severity. Results suggest that the S. corchorusii TKR8, S. corchorusii JAS2 and S. misionensis TBS5 should be promoted as biocontrol agents against B. glumae and bioformulations for rice crops.

Identification of non-ribosomal peptide synthetase in Ganoderma boninense Pat. that was expressed during the interaction with oil palm.

Basal stem rot (BSR) of oil palm is a disastrous disease caused by a white-rot fungus Ganoderma boninense Pat. Non-ribosomal peptides (NRPs) synthesized by non-ribosomal peptide synthetases (NRPSs) are a group of secondary metabolites that act as fungal virulent factors during pathogenesis in the host. In this study, we aimed to isolate NRPS gene of G. boninense strain UPMGB001 and investigate the role of this gene during G. boninense-oil palm interaction. The isolated NRPS DNA fragment of 8322 bp was used to predict the putative peptide sequence of different domains and showed similarity with G. sinense (85%) at conserved motifs of three main NRPS domains. Phylogenetic analysis of NRPS peptide sequences demonstrated that NRPS of G. boninense belongs to the type VI siderophore family. The roots of 6-month-old oil palm seedlings were artificially inoculated for studying NRPS gene expression and disease severity in the greenhouse. The correlation between high disease severity (50%) and high expression (67-fold) of G. boninense NRPS gene at 4 months after inoculation and above indicated that this gene played a significant role in the advancement of BSR disease. Overall, these findings increase our knowledge on the gene structure of NRPS in G. boninense and its involvement in BSR pathogenesis as an effector gene.

Fumonisin B1 Production by Fusarium Species and Mycotoxigenic Effect on Larval Zebrafish.

Fumonisin B1 (FB1) is a common mycotoxin produced by Fusarium species particularly F. proliferatum and F. verticillioides. The toxin produced can cause adverse effects on humans and animals. The objectives of this study were to detect the production of FB1 based on the amplification of FUM1 gene, to quantify FB1 produced by the isolates using Ultra-fast Liquid Chromatography (UFLC) analysis, to examine the embryotoxicity effect of FB1 and to determine EC50 toward the larvae of zebrafish (Danio rerio). Fifty isolates of Fusarium species were isolated from different hosts throughout Malaysia. Successful amplification of the FUM1 gene showed the presence of this gene (800 bp) in the genome of 48 out of 50 isolates. The highest level of FB1 produced by F. proliferatum isolate B2433 was 6677.32 ppm meanwhile F. verticillioides isolate J1363 was 954.01 ppm. From the assessment of embryotoxicity test of FB1 on larvae of zebrafish, five concentrations of FB1 (0.43 ppm, 0.58 ppm, 0.72 ppm, 0.87 ppm and 1.00 ppm) were tested. Morphological changes of the FB1 exposed-larvae were observed at 24 to 168 hpf. The mortality rate and abnormality of zebrafish larvae were significantly increased at 144 hpf exposure. Meanwhile, the spontaneous tail coiling showed a significant difference. There were no significant differences in the heartbeat rate. As a conclusion, the presence of FUM1 in every isolate can be detected by FUM1 gene analysis and both of the species produced different concentrations of FB1. This is the first report of FB1 produced by Fusarium species gave a significant effect on zebrafish development.

Morphological and molecular characterization, sexual reproduction, and pathogenicity of Setosphaeria rostrata isolates from rice leaf spot.

Setosphaeria rostrata, a common plant pathogen causing leaf spot disease, affects a wide range of plant species, mainly grasses. Fungi were isolated from brown spots on rice leaves throughout Peninsular Malaysia, and 45 isolates were identified as Setosphaeria rostrata The isolates were then characterized using morphological and molecular approaches. The mating type was determined using PCR amplification of the mating type alleles, and isolates of opposite mating types were crossed to examine sexual reproduction. Based on nuclear ribosomal DNA ITS1-5.8S-ITS2 region (ITS) and beta-tubulin (BT2) sequences, two phylogenetic trees were constructed using the maximum likelihood method; S. rostrata was clustered in one well-supported clade. Pathogenicity tests showed that S. rostrata isolates are pathogenic, suggesting that it is the cause of the symptoms. Mating-type analyses indicated that three isolates carried the MAT1-1 allele, and the other 42 isolates carried MAT1-2 After isolates with opposite mating types were crossed on Sach's medium and incubated for 3 wk, six crosses produced pseudothecia that contained eight mature ascospores, and 12 other crosses produced numerous pseudothecia with no ascospores. To our knowledge, this is the first report on S. rostrata isolated from leaf spots on rice.

Comparative chemical screening and genetic analysis reveal tentoxin as a new virulence factor in Cochliobolus miyabeanus, the causal agent of brown spot disease on rice.

Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.

Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4'-Phosphopantetheinyl Transferase Ppt1.

The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

Tolerance and biosorption of copper (Cu) and lead (Pb) by filamentous fungi isolated from a freshwater ecosystem.

Filamentous fungi are able to accumulate significant amount of metals from their environment. The potential of fungal biomass as agents for biosorption of heavy metals from contaminated sediments is currently receiving attention. In the present study, a total of 41 isolates of filamentous fungi obtained from the sediment of the Langat River, Selangor, Malaysia were screened for their tolerance and uptake capability of copper (Cu) and lead (Pb). The isolates were identified as Aspergillus niger, A. fumigatus, Trichoderma asperellum, Penicillium simplicissimum and P. janthinellum. A. niger and P. simplicissimum, were able to survive at 1000 mg/L of Cu(II) concentration on Potato Dextrose Agar (PDA) while for Pb, only A. niger survived at 5000 mg/L concentration. The results showed that A. niger, P. simplicissimum and T. asperellum have a better uptake capacity for Pb compared to Cu and the findings indicated promising biosorption of Cu and Pb by these filamentous fungi from aqueous solution. The present study was also determined the maximum removal of Cu(II) and Pb(II) that was performed by A. niger. The metal removal which occurred at Cu(II) 200 mg/L was (20.910 +/- 0.581) mg/g and at 250 mg/L of Pb(II) was (54.046 +/- 0.328) mg/g.