Antifungal Potential of Melaleuca alternifolia against Fungal Pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of bananas caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) poses the most serious threat to banana production globally. The disease has been managed using chemical fungicides, yet the control levels are still unsatisfactory. This study investigated the antifungal activities of tea tree (Melaleuca alternifolia) essential oil (TTO) and hydrosol (TTH) against Foc TR4 and their bioactive components. The potential of TTO and TTH in inhibiting the growth of Foc TR4 was evaluated in vitro using agar well diffusion and spore germination assays. Compared to the chemical fungicide, TTO effectively suppressed the mycelial growth of Foc TR4 at 69%. Both the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TTO and TTH were established at 0.2 µg/µL and 50% v/v, respectively, suggesting the fungicidal nature of the plant extracts. The disease control efficacies were also demonstrated by a (p ≤ 0.05) delayed Fusarium wilt symptom development in the susceptible banana plants with reduced LSI dan RDI scores from 70% to around 20-30%. A GC/MS analysis of TTO identified terpinen-4-ol, eucalyptol, and α-terpineol as the major components. In contrast, an LC/MS analysis of TTH identified different compounds, including dihydro-jasmonic acid and methyl ester. Our findings indicate the potential of tea tree extracts as natural alternatives to chemical fungicides to control Foc TR4.

Synergism: biocontrol agents and biostimulants in reducing abiotic and biotic stresses in crop.

In today's fast-shifting climate change scenario, crops are exposed to environmental pressures, abiotic and biotic stress. Hence, these will affect the production of agricultural products and give rise to a worldwide economic crisis. The increase in world population has exacerbated the situation with increasing food demand. The use of chemical agents is no longer recommended due to adverse effects towards the environment and health. Biocontrol agents (BCAs) and biostimulants, are feasible options for dealing with yield losses induced by plant stresses, which are becoming more intense due to climate change. BCAs and biostimulants have been recommended due to their dual action in reducing both stresses simultaneously. Although protection against biotic stresses falls outside the generally accepted definition of biostimulant, some microbial and non-microbial biostimulants possess the biocontrol function, which helps reduce biotic pressure on crops. The application of synergisms using BCAs and biostimulants to control crop stresses is rarely explored. Currently, a combined application using both agents offer a great alternative to increase the yield and growth of crops while managing stresses. This article provides an overview of crop stresses and plant stress responses, a general knowledge on synergism, mathematical modelling used for synergy evaluation and type of in vitro and in vivo synergy testing, as well as the application of synergism using BCAs and biostimulants in reducing crop stresses. This review will facilitate an understanding of the combined effect of both agents on improving crop yield and growth and reducing stress while also providing an eco-friendly alternative to agroecosystems.

Association of soil fungal community composition with incidence of Fusarium wilt of banana in Malaysia.

Banana (Musa spp.), an important food crop in many parts of the world, is threatened by a deadly wilt disease caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4). Increasing evidence indicates that plant actively recruits beneficial microbes in the rhizosphere to suppress soil-borne pathogens. Hence, studies on the composition and diversity of the root-associated microbial communities are important for banana health. Research on beneficial microbial communities has focused on bacteria, although fungi can also influence soil-borne disease. Here, high-throughput sequencing targeting the fungal internal transcribed spacer (ITS) was employed to systematically characterize the difference in the soil fungal community associated with Fusarium wilt (FW) of banana. The community structure of fungi in the healthy and TR4-infected rhizospheres was significantly different compared with that of bulk soil within the same farm. The rhizosphere soils of infected plants exhibited higher richness and diversity compared with healthy plants, with significant abundance of Fusarium genus at 14%. In the healthy rhizosphere soil, Penicillium spp. were more abundant at 7% and positively correlated with magnesium. This study produced a detailed description of fungal community structure in healthy and TR4-infected banana soils in Malaysia and identified candidate biomarker taxa that may be associated with FW disease promotion and suppression. The findings also expand the global inventory of fungal communities associated with the components of asymptomatic and symptomatic banana plants infected by TR4.

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.

Analysis of soil bacterial communities and physicochemical properties associated with Fusarium wilt disease of banana in Malaysia.

Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4) is a soil-borne disease that infects bananas, causing severe economic losses worldwide. To reveal the relationship between bacterial populations and FW, the bacterial communities of healthy and TR4-infected rhizosphere and bulk soils were compared using 16S rRNA gene sequencing. Soil physicochemical properties associated with FW were also analyzed. We found the community structure of bacteria in the healthy and TR4 infected rhizosphere was significantly different compared to bulk soil within the same farm. The rhizosphere soils of infected plants exhibited higher richness and diversity than healthy plant with significant abundance of Proteobacteria. In the healthy rhizosphere soil, beneficial bacteria such as Burkholderia and Streptomyces spp. were more abundant. Compared to the infected rhizosphere soil, healthy rhizosphere soil was associated with RNA metabolism and transporters pathways and a high level of magnesium and cation exchange capacity. Overall, we reported changes in the key taxa of rhizospheric bacterial communities and soil physicochemical properties of healthy and FW-infected plants, suggesting their potential role as indicators for plant health.

Differential Analysis of Mycelial Proteins and Metabolites From Rigidoporus Microporus During In Vitro Interaction With Hevea Brasiliensis.

Rigidoporus microporus is the fungus accountable for the white root rot disease that is detrimental to the rubber tree, Hevea brasiliensis. The pathogenicity mechanism of R. microporus and the identity of the fungal proteins and metabolites involved during the infection process remain unclear. In this study, the protein and metabolite profiles of two R. microporus isolates, Segamat (SEG) and Ayer Molek (AM), were investigated during an in vitro interaction with H. brasiliensis. The isolates were used to inoculate H. brasiliensis clone RRIM 2025, and mycelia adhering to the roots of the plant were collected for analysis. Transmission electron microscope (TEM) images acquired confirms the hyphae attachment and colonization of the mycelia on the root of the H. brasiliensis clones after 4 days of inoculation. The protein samples were subjected to 2-DE analysis and analyzed using MALDI-ToF MS/MS, while the metabolites were extracted using methanol and analyzed using LC/MS-QTOF. Based on the differential analyses, upregulation of proteins that are essential for fungal evolution such as malate dehydrogenase, fructose 1,6-biphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase hints an indirect role in fungal pathogenicity, while metabolomic analysis suggests an increase in acidic compounds which may lead to increased cell wall degrading enzyme activity. Bioinformatics analyses revealed that the carbohydrate and amino acid metabolisms were prominently affected in response to the fungal pathogenicity. In addition to that, other pathways that were significantly affected include "Protein Ubiquitination Pathway," Unfolded Protein Response," "HIFα Signaling," and "Sirtuin Signaling Pathway." The identification of responsive proteins and metabolites from this study promotes a better understanding of mechanisms underlying R. microporus pathogenesis and provides a list of potential biological markers for early recognition of the white root rot disease.

A Consortium of Pseudomonas aeruginosa and Trichoderma harzianum for Improving Growth and Induced Biochemical Changes in Fusarium Wilt Infected Bananas.

Fusarium wilt of banana cannot be effectively controlled by current control strategies. The most virulent form that caused major losses in the banana production is Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc-TR4). Biocontrol of Foc-TR4 using microbial antagonists offers a sustainable and eco-friendly alternative. A consortium of biocontrol agents (BCAs), Pseudomonas aeruginosa DRB1 and Trichoderma harzianum CBF2 was formulated into pesta granules, talc powder, alginate beads and liquid bioformulations. Previous study indicated bioformulations containing both BCAs successfully reduced the disease severity of Foc-TR4. To date, the biocontrol mechanism and plant growth promoting (PGP) traits of a consortium of BCAs on infected bananas have not been explored. Therefore, the study was undertaken to investigate the effect of a consortium of DRB1 and CBF2 in the growth and biochemical changes of Foc-TR4 infected bananas. Results indicated pesta granules formulation produced bananas with higher biomass (fresh weight: 388.67 g), taller plants (80.95 cm) and larger leaves (length: 39.40 cm, width: 17.70 cm) than other bioformulations. Applying bioformulations generally produced plants with higher chlorophyll (392.59 µg/g FW-699.88 µg/g FW) and carotenoid contents (81.30 µg/g FW-120.01 µg/g FW) compared to pathogen treatment (chlorophyll: 325.96 µg/g FW, carotenoid: 71.98 µg/g FW) which indicated improved vegetative growth. Bioformulation-treated plants showed higher phenolic (49.58-93.85 µg/g FW) and proline contents (54.63 µg/g FW-89.61 µg/g FW) than Foc-TR4 treatment (phenolic: 46.45 µg/g FW, proline: 28.65 µg/g FW). The malondialdehylde (MDA) content was lower in bioformulation treatments (0.49 Nm/g FW-1.19 Nm/g FW) than Foc-TR4 treatment (3.66 Nm/g FW). The biochemical changes revealed that applying bioformulations has induced host defense response by increasing phenolic and proline contents which reduced root damage caused by Foc-TR4 resulting in lower MDA content. In conclusion, applying bioformulations containing microbial consortium is a promising method to improve growth and induce significant biochemical changes in bananas leading to the suppression of Foc-TR4.

Plant Growth-Promoting Bacteria as an Emerging Tool to Manage Bacterial Rice Pathogens.

As a major food crop, rice (Oryza sativa) is produced and consumed by nearly 90% of the population in Asia with less than 9% produced outside Asia. Hence, reports on large scale grain losses were alarming and resulted in a heightened awareness on the importance of rice plants' health and increased interest against phytopathogens in rice. To serve this interest, this review will provide a summary on bacterial rice pathogens, which can potentially be controlled by plant growth-promoting bacteria (PGPB). Additionally, this review highlights PGPB-mediated functional traits, including biocontrol of bacterial rice pathogens and enhancement of rice plant's growth. Currently, a plethora of recent studies address the use of PGPB to combat bacterial rice pathogens in an attempt to replace existing methods of chemical fertilizers and pesticides that often lead to environmental pollutions. As a tool to combat bacterial rice pathogens, PGPB presented itself as a promising alternative in improving rice plants' health and simultaneously controlling bacterial rice pathogens in vitro and in the field/greenhouse studies. PGPB, such as Bacillus, Pseudomonas, Enterobacter, Streptomyces, are now very well-known. Applications of PGPB as bioformulations are found to be effective in improving rice productivity and provide an eco-friendly alternative to agroecosystems.

Plant Nitric Oxide Signaling under Drought Stress.

Water deficit caused by drought is a significant threat to crop growth and production. Nitric oxide (NO), a water- and lipid-soluble free radical, plays an important role in cytoprotection. Apart from a few studies supporting the role of NO in drought responses, little is known about this pivotal molecular amendment in the regulation of abiotic stress signaling. In this review, we highlight the knowledge gaps in NO roles under drought stress and the technical challenges underlying NO detection and measurements, and we provide recommendations regarding potential avenues for future investigation. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool with which plant fitness can be improved under adverse growth conditions.

Vacuolar Processing Enzymes Modulating Susceptibility Response to Fusarium oxysporum f. sp. cubense Tropical Race 4 Infections in Banana.

Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) is a destructive necrotrophic fungal pathogen afflicting global banana production. Infection process involves the activation of programmed cell death (PCD). In this study, seven Musa acuminata vacuolar processing enzyme (MaVPE1-MaVPE7) genes associated with PCD were successfully identified. Phylogenetic analysis and tissue-specific expression categorized these MaVPEs into the seed and vegetative types. FocTR4 infection induced the majority of MaVPE expressions in the susceptible cultivar "Berangan" as compared to the resistant cultivar "Jari Buaya." Consistently, upon FocTR4 infection, high caspase-1 activity was detected in the susceptible cultivar, while low level of caspase-1 activity was recorded in the resistant cultivar. Furthermore, inhibition of MaVPE activities via caspase-1 inhibitor in the susceptible cultivar reduced tonoplast rupture, decreased lesion formation, and enhanced stress tolerance against FocTR4 infection. Additionally, the Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infection, indicated by reduced sporulation rate, low levels of H2O2 content, and high levels of cell viability. Comparative proteomic profiling analysis revealed increase in the abundance of cysteine proteinase in the inoculated susceptible cultivar, as opposed to cysteine proteinase inhibitors in the resistant cultivar. In conclusion, the increase in vacuolar processing enzyme (VPE)-mediated PCD played a crucial role in modulating susceptibility response during compatible interaction, which facilitated FocTR4 colonization in the host.

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography.

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.

Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium.

Dieback disease caused by Erwinia mallotivora is a major threat to papaya plantation in Malaysia. The current study was conducted to evaluate the potential of endophytic lactic acid bacteria (LAB) isolated from papaya seeds for disease suppression of papaya dieback. Two hundred and thirty isolates were screened against E. mallotivora BT-MARDI, and the inhibitory activity of the isolates against the pathogen was ranging from 11.7-23.7 mm inhibition zones. The synergistic experiments revealed that combination of W. cibaria PPKSD19 and Lactococcus lactis subsp. lactis PPSSD39 increased antibacterial activity against the pathogen. The antibacterial activity was partially due to the production of bacteriocin-like inhibitory substances (BLIS). The nursery experiment confirmed that the application of bacterial consortium W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 significantly reduced disease severity to 19% and increased biocontrol efficacy to 69% of infected papaya plants after 18 days of treatment. This study showed that W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 are potential candidate as biocontrol agents against papaya dieback disease.

Analysis of MAP kinase MPK4/MEKK1/MKK genes of Carica papaya L. comparative to other plant homologues.

Mitogen-activated protein kinase 4 (MPK4) interacts with the (Mitogen-activated protein kinase kinase kinase 1) MEKK1/ Mitogenactivated protein kinase kinase 1 (MKK1)/ Mitogen-activated protein kinase kinase 2 (MKK2) complex to affect its function in plant development or against pathogen attacks. The KEGG (Kyoto Encyclopedia of Genes and Genomes) network analysis of Arabidopsis thaliana revealed close interactions between those four genes in the same plant-pathogen interaction pathway, which warrants further study of these genes due to their evolutionary conservation in different plant species. Through targeting the signature sequence in MPK4 of papaya using orthologs from Arabidopsis, the predicted sequence of MPK4 was studied using a comparative in silico approach between different plant species and the MAP cascade complex of MEKK1/MKK1/MKK2. This paper reported that MPK4 was highly conserved in papaya with 93% identical across more than 500 bases compared in each species predicted. Slight variations found in the MEKK1/MKK1/MKK2 complex nevertheless still illustrated sequence similarities between most of the species. Localization of each gene in the cascade network was also predicted, potentiating future functional verification of these genes interactions using knock out or/and gene silencing tactics.

Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.