Tropical Race Research Articles

Shared Core Microbiome and Functionality of Key Taxa Suppressive to Banana Fusarium Wilt.

Microbial contributions to natural soil suppressiveness have been reported for a range of plant pathogens and cropping systems. To disentangle the mechanisms underlying suppression of banana Panama disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4), we used amplicon sequencing to analyze the composition of the soil microbiome from six separate locations, each comprised of paired orchards, one potentially suppressive and one conducive to the disease. Functional potentials of the microbiomes from one site were further examined by shotgun metagenomic sequencing after soil suppressiveness was confirmed by greenhouse experiments. Potential key antagonists involved in disease suppression were also isolated, and their activities were validated by a combination of microcosm and pot experiments. We found that potentially suppressive soils shared a common core community with relatively low levels of F. oxysporum and relatively high proportions of Myxococcales, Pseudomonadales, and Xanthomonadales, with five genera, Anaeromyxobacter, Kofleria, Plesiocystis, Pseudomonas, and Rhodanobacter being significantly enriched. Further, Pseudomonas was identified as a potential key taxon linked to pathogen suppression. Metagenomic analysis showed that, compared to the conducive soil, the microbiome in the disease suppressive soil displayed a significantly greater incidence of genes related to quorum sensing, biofilm formation, and synthesis of antimicrobial compounds potentially active against Foc4. We also recovered a higher frequency of antagonistic Pseudomonas isolates from disease suppressive experimental field sites, and their protective effects against banana Fusarium wilt disease were demonstrated under greenhouse conditions. Despite differences in location and soil conditions, separately located suppressive soils shared common characteristics, including enrichment of Myxococcales, Pseudomonadales, and Xanthomonadales, and enrichment of specific Pseudomonas populations with antagonistic activity against the pathogen. Moreover, changes in functional capacity toward an increase in quorum sensing, biofilm formation, and antimicrobial compound synthesizing involve in disease suppression.

Biological Control of Fusarium oxysporum f. sp. cubense Tropical Race 4 in Banana Plantlets Using Newly Isolated Streptomyces sp. WHL7 from Marine Soft Coral.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense is a disastrous fungal disease. Foc tropical race 4 (Foc TR4) infects almost all banana cultivars. Use of chemical fungicides caused serious environment pollution. Biological control with antagonistic microbes is a promising strategy for controlling Foc TR4. Here, strain WHL7 isolated from marine soft coral exhibited a high antifungal activity against Foc TR4. Based on the morphological and physicochemical profiles as well as the phylogenetic tree, the strain was assigned to Streptomyces sp. Fermentation broth of Streptomyces sp. WHL7 significantly increased the resistance of banana plantlets to Foc TR4 in the pot experiment. Analysis of antifungal mechanism showed that strain WHL7 extracts inhibited spore germination and mycelial growth of Foc TR4, and destroyed cell integrity and ultrastructure. Hence, Streptomyces sp. WHL7 is an important bioresource for exploring novel natural products and biofertilizer to manage Foc TR4.

Pest categorisation of Fusarium oxysporum f. sp. cubense Tropical Race 4.

The EFSA Plant Health Panel performed a pest categorisation of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), an ascomycete fungus causing Fusarium wilt (Panama disease) on Musa spp. Foc TR4 is pathogenic to the commercial banana varieties including those of the ‘Cavendish’ group and is considered as the most destructive among Foc haplotypes. Uncertainty exists on the host range of Foc TR4, since it has not been demonstrated whether it can infect plant species other than Musa spp., which were previously reported as hosts of other Foc races. Foc TR4 is morphologically and physiologically identical to other representatives of the Fusarium oxysporum Species Complex (FOSC), but all Foc TR4 isolates belong to a single clonal lineage within the vegetative compatibility groups 01213‐01216. Several PCR protocols are described in the literature, but their specificity has been questioned as they may generate false positives. The pathogen is not included in EU Commission Implementing Regulation 2019/2072 and is not reported as present in the EU territory. Several potential entry pathways and means of spread were identified, including host plants for planting other than vitroplants, fresh fruits and leaves of host plants, soil and other substrates originating in infested third countries. Host availability and climate suitability occurring in some areas of the EU are favourable for the establishment of Foc TR4. Being a soil‐borne pathogen, eradication of Foc TR4 once it enters a new area is very difficult. Therefore, effective quarantine measures are essential in pathogen‐free areas. Although not specifically targeting against Foc TR4, phytosanitary measures are currently available to prevent the introduction of the pathogen into the EU. Considering that banana‐growing EU countries account for over 12% of the EU banana supply, it is expected that the economic impact of Foc TR4 on the European banana production areas would be devastating. Foc TR4 satisfies the criteria that are within the remit of EFSA to assess for this pathogen to be regarded as a potential Union quarantine pest.

Roving Survey of Panama Wilt Disease (Fusarium oxysporum f. sp. cubense) of Banana in Bihar

A survey of Panama wilt of banana was conducted in different districts of Bihar i.e. Muzaffarpur, Vaishali, Samastipur, Darbhanga, Katihar, Purnea, Bhagalpur, Kishanganj and Saharsa. This disease was present in all the districts with different levels of percent wilt index. In the Vaishali belt (Zone–I), the maximum percent wilt index was recorded in the Vaishali district (30-45%) in cv. Malbhog (AAB) having sucker planting material in the year 2019-20. In the Koshi belt (Zone-II), the percent wilt index (External symptom) was highest in Purnea district (34-42%) in cv. Robusta (AAA) followed by district Saharsa (28-40%) and Kishanganj (25-38%). The lowest percent wilt index was found in districts Bhagalpur (22-38%) and Katihar (24-38%) in the year 2019-20. Panama wilt of banana caused by Fusarium oxysporum f. sp. cubense TR4 was recorded in the Koshi belt (Zone-II) having cv. Grand naine (AAA) and cv. Robusta (AAA) in Bihar only. While in Vaishali belt (Zone-I) was free from incidence of Fusarium wilt of banana incited by Fusarium oxysporum f. sp. cubense TR4.

Exploration of Klebsiella pneumoniae M6 for paclobutrazol degradation, plant growth attributes, and biocontrol action under subtropical ecosystem.

In recent times, injudicious use of paclobutrazol (PBZ) in mango orchards deteriorates the soil quality and fertility by persistence nature and causes a serious ecosystem imbalance. In this study, a new Klebsiella pneumoniae strain M6 (MW228061) was isolated from mango rhizosphere and characterized as a potent plant growth promoter, biocontrol, and PBZ degrading agent. The strain M6 efficiently utilizes PBZ as carbon, energy and nitrogen source and degrades up to 98.28% (50 mgL-1 initial conc.) of PBZ at 15th day of incubation in MS medium. In the soil system first order degradation kinetics and linear model suggested 4.5 days was the theoretical half-life (t1/2 value) of PBZ with strain M6. Box Behnken design (BBD) model of Response surface methodology (RSM) showed pH 7.0, 31°C temperature, and 2.0 ml inoculum size (8 x 109 CFU mL-1) was optimized condition for maximum PBZ degradation with strain M6. Plant growth promoting attributes such as Zn, K, PO4 solubilization IAA, HCN and NH3 production of strain M6 showed positive results and were assessed quantitatively. The relation between plant growth promotion and PBZ degradation was analyzed by heat map, principal component analysis (PCA) and, clustal correlation analysis (CCA). Strain M6 was also showing a significant biocontrol activity against pathogenic fungi such as Fusarium oxysporum (MTCC–284), Colletotrichum gloeosporioides (MTCC– 2190), Pythium aphanidermatum (MTCC– 1024), Tropical race 1 (TR -1), and Tropical race 4 (TR -4). Hence, results of the study suggested that strain M6 can be utilized as an effective bio-agent to restore degraded land affected by persistent use of paclobutrazol.

Phenolics mediate suppression of Fusarium oxysporum f. sp. cubense TR4 by legume root exudates

Fusarium oxysporum Schlecht f. sp. cubense (E.F. Smith) Snyder & H.N. Hansen (Foc) is a notorious soil-borne fungal pathogen that causes Fusarium wilt disease of bananas (Musa spp. L.). Foc, especially Tropical Race 4 (TR4), is particularly devastating due its ability to infect diverse banana cultivars including Cavendish bananas. Fusarium wilt is difficult to manage, but intercropping has been reported to efficiently suppress Fusarium wilt through exudation of root metabolites with antagonistic effects on Foc. Yet, the metabolites that inhibit Foc and the underlying mode of action remain unclear. Hydroponic culture and targeted metabolite profiling were used to investigate the potential of root-exuded phenolic acids and flavonoids of two legume species, Desmodium uncinatum (Jacq.) DC. and Mucuna pruriens (L.) DC., to inhibit growth and the biosynthesis of the phytotoxins beauvericin and fusaric acid (FA) in Foc TR4. Of the twelve metabolites targeted, four phenolic acids (benzoic, t-cinnamic, p-coumaric, p-hydroxybenzoic) were common in root exudates of both legumes, whereas vanillin and the flavonoid quercetin were only detected in root exudates of M. pruriens. Bioassays using synthetic benzoic-, t-cinnamic-, or p-hydroxybenzoic acid, or a mixture thereof, showed a concentration-dependent suppressive effect on Foc TR4. Low concentrations of phenolic acids (0.01 and 0.1 mM) inhibited chlamydospore germination and the production of macro- and micro-conidia, as well as the biosynthesis of FA. Mycelial growth of Foc TR4 was only inhibited at high concentrations (1 mM) of benzoic acid, t-cinnamic acid, and a mixture of the three phenolics. This study demonstrates that phenolics in root exudates of D. uncinatum and M. pruriens suppress Foc TR4 by directly inhibiting the early stages of pathogen development. Intercropping banana with D. uncinatum and M. pruriens may thus be considered as a valuable option for suppression of Foc TR4.

Antifungal activity of volatile compounds generated by endophytic fungi Sarocladium brachiariae HND5 against Fusarium oxysporum f. sp. cubense.

The soil-born filamentous fungal pathogen Fusarium oxysporum f. sp. cubense (FOC), which causes vascular wilt disease in banana plants, is one of the most economically important Fusarium species. Biocontrol using endophytic microorganisms is among the most effective methods for controlling banana Fusarium wilt. In this study, volatile organic compounds (VOCs) showed strong antifungal activity against FOC. Seventeen compounds were identified from the VOCs produced by endophytic fungi Sarocladium brachiariae HND5, and three (2-methoxy-4-vinylphenol, 3,4-dimethoxystyrol and caryophyllene) showed antifungal activity against FOC with 50% effective concentrations of 36, 60 and 2900 μL/L headspace, respectively. Transmission electron microscopy (TEM) and double fluorescence staining revealed that 2-methoxy-4-vinylphenol and 3,4-dimethoxystyrol damaged the plasma membranes, resulting in cell death. 3,4-dimethoxystyrol also could induce expression of chitin synthases genes and altered the cell walls of FOC hyphae. Dichloro-dihydro-fluorescein diacetate staining indicated the caryophyllene induced accumulation of reactive oxygen species (ROS) in FOC hyphae. FOC secondary metabolism also responded to active VOC challenge by producing less fusaric acid and expressions of genes related to fusaric acid production were interrupted at sublethal concentrations. These findings indicate the potential of S. brachiariae HND5 as a biocontrol agent against FOC and the antifungal VOCs as fumigants.

The Vulnerability of Bananas to Globally Emerging Disease Threats.

Banana cultivation has increased significantly over the last century to meet the growing demand for this popular fruit. Originating in Southeast Asia, bananas are now produced in >135 different countries in tropical and subtropical regions of the world. Most of this expansion of production is based on a single banana variety, Cavendish, which makes up almost all the export trade grown in large-scale monocultures and a large part of the local trade and represents >40% of all bananas grown globally. Over the last century several major diseases of the banana have emerged and widely expanded their geographic ranges. Cultivars within the Cavendish variety are highly susceptible to these diseases, including yellow Sigatoka, black leaf streak, Eumusae leaf spot, freckle, Fusarium wilt tropical race 4, banana bunchy top, and the bacterial wilts Moko, Xanthomonas wilt, and banana Blood disease. This review graphically illustrates the emergence and rapid intercontinental spread of these diseases and discusses several major disease epidemics in bananas. Evidently, the large-scale monoculture based on the single variety Cavendish has resulted in an extreme level of genetic vulnerability. The resistance to diversification in the Cavendish production chain and the lack of investment in genetics and plant breeding in the recent past means that currently limited genetic solutions are available to replace the Cavendish banana with a set of market acceptable resistant varieties from a range of different genetic backgrounds.

English

The study aimed at understanding the spatial distribution of Fusarium wilt (FW) in different banana growing regions, ascertain the effect of management practices and plantation age on FW incidence, as well as investigate farmers’ knowledge regarding the symptoms and spread of FW in Uganda. Individual interviews were conducted in 119 farms using a pre-tested questionnaire and field observations during a survey in major banana growing regions. Results indicate that FW is widely distributed across the banana growing areas with more occurrences (70%) in Kapchorwa district and majority of respondents (63.4%) reported increasing disease prevalence. A chi-square test performed revealed significant association between FW incidence and plantation age with more incidences (51.6%) recorded in older plantations (>20 years of establishment) than newly (1-5 years) established ones (11.1%). FW incidence was significantly associated with plantation management with higher incidences (86.9%) recorded in well managed plantations. Half of interviewed farmers could explain and distinguish symptoms associated with FW from other diseases, but only 38.4% of these could tell how the disease spreads; thus, a need for more concerted efforts in building the capacity of farmers to identify the symptoms and spread of FW for effective management program. We identified preliminary evidence that field abandonment is sometimes used as a last option for coping up with FW. Understanding the mechanism behind this requires more detailed research as well as establishing how farmers are managing FW culturally.   Key words: Fusarium wilt, farmers’ knowledge, spatial distribution, Uganda.

The histone acetyltransferase FocGCN5 regulates growth, conidiation, and pathogenicity of the banana wilt disease causal agent Fusarium oxysporum f.sp. cubense tropical race 4

Chromatin structure modifications by histone acetyltransferase are involved in multiple biological processes in eukaryotes. In the present study, the GCN5 homologue FocGCN5 was identified in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The coding gene was then knocked out to investigate the roles of FocGNC5. The mutant ΔFocGCN5 was found significantly reduced in growth rate and conidiation, and almost completely lost pathogenicity to banana plantlets. The RNA-seq analysis provide an insight into the underlying mechanism. Firstly, transcription of the genes involved in carbohydrate metabolism and fungal cell wall synthesis was reduced in ΔFocGCN5, leading to the impairment of apical deposition of cell-wall material. Secondly, FocabaA, one of the pivotal regulators of conidiation, was significantly reduced in expression in ΔFocGCN5, which might be the main cause of the conidiation reduction. Thirdly, the pathogenicity-associated factors, including effectors and plant cell wall degrading enzymes, were almost all down-regulated in ΔFocGCN5, which accounts for the decrease of pathogenicity. In addition, the stress tolerance to salt, heat, and cell wall inhibitors was slightly increased in ΔFocGCN5. Taken together, our studies clarify the roles of FocGCN5 in growth, conidiation, and pathogenicity of Foc TR4, and explore the possible mechanism behind its biological functions.

Suppression of Banana Fusarium Wilt Induced by Soil Microbiome Reconstruction Through Green Manure Intercropping

Fusarium wilt of banana, also known as Panama disease, caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases and currently threatens banana production around the world. In this study, pot and field experiments were conducted to assess the control effect of banana intercropping with five intercropping green manures (crotalaria, sesban, melilotus officinalis, vicia villosa and trefoil) on banana Panama disease in high disease incidence soil. Soil microbiota was analysed using the MiSeq Illumina sequencing platform. The results showed that compared with banana monoculture, five intercropping treatments significantly reduced the incidence of banana Fusarium wilt and decreased the richness and diversity of soil fungi, vicia villosa and trefoil intercropping treatment showed greater effect in disease suppression than the other treatments. The abundance of Aspergillus was increased, while the abundance of Fusarium was decreased in all five intercropping treatments compared with the banana monoculture treatment. A structural equation model showed that the changes in soil organic matter and NH4 +-N content caused by intercropping may play a key role in the change of the soil fungal community and the reduction of banana Fusarium wilt incidence. In summary, the reduction in the incidence of banana Panama disease caused by banana intercropping with green manures can be attributed to the decrease in harmful microorganisms such as Fusarium and the increase in beneficial microorganisms such as Aspergillus, which may establish a beneficial soil microbial community.

Spent Pleurotus ostreatus Substrate Has Potential for Managing Fusarium Wilt of Banana.

A range of basidiomycetes including the edible mushroom Pleurotus ostreatus (Po) can suppress plant pathogens such as Fusarium spp. With the current increase in production and consumption of Po in Uganda, the spent Po substrate (SPoS) could be an alternative to manage Fusarium wilt of banana (FWB), caused by the soil borne pathogen Fusarium oxysporum f. sp. cubense, race 1 (Foc). This study determined the potential of SPoS to inhibit Foc in vitro and in potted plants. In vitro studies confirmed suppression of Foc in pure co-culture (Po vs. Foc) assays and media amended with different concentrations (0% to 50% w/v) of un-sterilized SPoS filtrates. Foc growth in the sterile SPoS filtrate was comparable to the water control, suggesting possible roles of biotic or thermolabile components of the SPoS. To further verify the suppressive effects of SPoS, pot experiments were carried out with a resistant (‘Mbwazirume’, AAA) and susceptible (‘Sukali Ndizi’, AAB) banana cultivar using both artificially and naturally infested soils. Independent of the inoculation method, SPoS significantly reduced the severity of FWB in pot experiments. Susceptible cultivar ‘Sukali Ndizi’ growing in substrates amended with SPoS showed lower (1.25) corm damage (Scale 0–5) than the un-amended control (3.75). No corm damage was observed in uninoculated controls. The resistant cultivar ‘Mbwazirume’, showed slight (0.25) corm damage only in the Foc-inoculated plants without SPoS. These findings suggest that SPoS could be used as part of the management practices to reduce the impact of FWB.

Identification and Antifungal Mechanism of a Novel Actinobacterium Streptomyces huiliensis sp. nov. Against Fusarium oxysporum f. sp. cubense Tropical Race 4 of Banana.

Banana is an important fruit crop. Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) seriously threatens the global banana industry. It is difficult to control the disease spread using chemical measures. In addition, commercial resistant cultivars are also lacking. Biological control is considered as a promising strategy using antagonistic microbes. Actinomycetes, especially Streptomyces, are potential sources of producing novel bioactive secondary metabolites. Here, strain SCA2-4T with strong antifungal activity against Foc TR4 was isolated from the rhizospheric soil of Opuntia stricta in a dry hot valley. The morphological, physiological and chemotaxonomic characteristics of the strain were consistent with the genus Streptomyces. Based on the homology alignment and phylogenetic trees of 16S rRNA gene, the taxonomic status of strain SCA2-4T exhibited a paradoxical result and low bootstrap value using different algorithms in the MEGA software. It prompted us to further discriminate this strain from the closely related species by the multilocus sequence analysis (MLSA) using five house-keeping gene alleles (atpD, gyrB, recA, rpoB, and trpB). The MLSA trees calculated by three algorithms demonstrated that strain SCA2-4T formed a distinct clade with Streptomyces mobaraensis NBRC 13819T. The MLSA distance was above 0.007 of the species cut-off. Average nucleotide identity (ANI) values between strain SCA2-4T genome and two standard strain genomes were below 95-96% of the novel species threshold. Strain SCA2-4T was assigned to a novel species of the genus Streptomyces and named as Streptomyces huiliensis sp. nov. The sequenced complete genome of SCA2-4T encoded 51 putative biosynthetic gene clusters of secondary metabolites. Genome alignment revealed that ten gene clusters were involved in the biosynthesis of antimicrobial metabolites. It was supported that strain SCA2-4T showed strong antifungal activities against the pathogens of banana fungal diseases. Extracts abstracted from the culture filtrate of strain SCA2-4T seriously destroyed cell structure of Foc TR4 and inhibited mycelial growth and spore germination. These results implied that strain SCA2-4T could be a promising candidate for biological control of banana Fusarium wilt.

Aureoverticillactam, a Potent Antifungal Macrocyclic Lactam from Streptomyces aureoverticillatus HN6, Generates Calcium Dyshomeostasis-Induced Cell Apoptosis via the Phospholipase C Pathway in Fusarium oxysporum f. sp. cubense Race 4.

Extensive efforts have been made to discover new biofungicides of high efficiency for control of Fusarium oxysporum f. sp. cubense race 4, a catastrophic soilborne phytopathogen causing banana Fusarium wilt worldwide. We confirmed for the first time that aureoverticillactam (YY3) has potent antifungal activity against F. oxysporum f. sp. cubense race 4, with effective dose for 50% inhibition (EC50) of 20.80 μg/ml against hyphal growth and 12.62 μg/ml against spore germination. To investigate its mechanism of action, we observed the cellular ultrastructures of F. oxysporum f. sp. cubense race 4 with YY3 treatment and found that YY3 led to cell wall thinning, mitochondrial deformities, apoptotic degradation of the subcellular fractions, and entocyte leakage. Consistent with these variations, increased permeability of cell membrane and mitochondrial membrane also occurred after YY3 treatment. On the enzymatic level, the activity of mitochondrial complex III, as well as the ATP synthase, was significantly suppressed by YY3 at a concentration >12.50 μg/ml. Moreover, YY3 elevated the cytosolic Ca2+ level to promote mitochondrial reactive oxygen species (ROS) production. Cell apoptosis also occurred as expected. On the transcriptome level, key genes involved in the phosphatidylinositol signaling pathway were significantly affected, with the expression level of Plc1 increased approximately fourfold. The expression levels of two apoptotic genes, casA1 and casA2, were also significantly increased by YY3. Of note, phospholipase C activation was observed with YY3 treatment in F. oxysporum f. sp. cubense race 4. These findings indicate that YY3 exerts its antifungal activity by activating the phospholipase C calcium-dependent ROS signaling pathway, which makes it a promising biofungicide.

English

Information on the genetic control of resistance to Fusarium oxysporum f.sp.cubense (FOC) race (1) and fruit quality traits in Sukali Ndizi cultivar of banana is key to its genetic improvement. The mode of resistance to Fusarium wilt in Sukali Ndizi was analyzed in 536 hybrids and 5 parental lines and quality attributes in 137 hybrids and two parental lines, grown in same environment. Fruit quality was assessed by physical measurements and fruit composition whereas resistance to Fusarium wilt was assessed by corm discoloration symptoms. All the assessed traits were quantitatively inherited.  Flavor attributes, and pulp texture showed a predominance of additive inheritance with complementary gene action whereas total soluble solutes showed non-additive gene with dominant gene action. Fruit acidity had incomplete dominancy with the genetic model explained by both single gene and certain multiple genes. Resistance to Fusarium wilt showed dominant gene action and polygenic effects. Involvement of a few genes governing wilt resistance suggested the ease of breeding for this trait. Pedigree breeding method could be recommended for incorporating various traits in (cv. Sukali Ndizi) of banana.  Key words: Sukali Ndizi, quantitative inheritance, fruit quality, Fusarium wilt resistance.

Isolation and Evaluation of Rhizosphere Actinomycetes With Potential Application for Biocontrolling Fusarium Wilt of Banana Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) is globally one of the most destructive soil-borne fungal diseases. Biological control using environmental microorganisms is considered as an alternative and sustainable strategy. Actinomycetes have the potential to explore biocontrol agents due to their production of diverse metabolites. The isolation and identification of high-efficiency and broad-spectrum antagonistic actinomycetes are the key for the application of biocontrol agents. In the present study, 60 actinomycetes were obtained from the rhizosphere soil of Machilus pingii in the primitive ecological natural reserve of Hainan province, China. Seventeen isolates and their extracts exhibited significant antifungal activity against F. oxysporum TR4. Particularly, strain BITDG-11 with the strongest inhibition ability had a broad-spectrum antifungal activity. The assay of its physiological and biochemical profiles showed that strain BITDG-11 had the ability to produce IAA and siderophores and had a positive response to gelatin liquefaction and nitrate reduction. Enzyme activities of chitinase, β-1,3-glucanase, lipase, and urease were also detected. Average nucleotide identity calculated by comparison with the standard strain genome of Streptomyces albospinus JCM3399 was 86.55% below the novel species threshold, suggesting that the strain could be a novel species. In addition, Streptomyces BITDG-11 obviously reduced the disease index of banana plantlets and promoted plant growth at 45 days post inoculation. The higher and lasting expression levels of defense genes and activities of antioxidant enzymes were induced in the roots of banana. Genome sequencing revealed that the Streptomyces BITDG-11 chromosome contained large numbers of conserved biosynthesis gene clusters encoding terpenes, non-ribosomal peptides, polyketides, siderophores, and ectoines. Fifteen bioactive secondary metabolites were further identified from Streptomyces BITDG-11 extract by gas chromatography–mass spectrometry. Dibutyl phthalate demonstrating a strong antifungal activity was the major compound with the highest peak area. Hence, Streptomyces sp. BITDG-11 has a great potential to become an essential constituent of modern agricultural practice as biofertilizers and biocontrol agents.

The effect of soil properties on the relation between soil management and Fusarium wilt expression in Gros Michel bananas

AimsThis study looks whether the response of soil management (liming and nitrogen fertilization) on the incidence of Fusarium wilt (Foc Race 1) in Gros Michel banana (Musa AAA) varies with different soil properties.MethodsThe effect of inoculation with Foc Race 1 was studied in a factorial greenhouse trial with soil samples from eight representative soil types from the Costa Rican banana region, two pH levels; and three levels of N-fertilization. After an 8-week period, plant biomass and a disease index were measured.ResultsThere were significant effects of soil pH and N, and their interactions on disease expression. Low pH levels and high N-fertilization increased the disease expression. The response to changes in soil pH and N-fertilization differed considerably between the different soils.ConclusionsAlthough soil pH and N influence Fusarium wilt in banana, each soil differs in its response to these soil properties. This complicates the development of standard soil management strategies in terms of e.g., N-fertilization and liming to mitigate or fight the disease.

Molecularly Imprinted Photonic Crystals Based on Fusaric Acid for the Detection of Banana Fusarium Wilt

Banana Fusarium wilt is a soil-borne disease caused by Fusarium oxysporum f. sp. cubense (Foc) in which F. oxysporum f. sp. cubense race 4 (Foc4) could infect all kinds of banana and has caused great damage to the banana planting industry. The most important means to prevent and control banana Fusarium wilt in agricultural production is to control the spread and invasion of pathogens. Among them, the detection and determination of banana Fusarium wilt in the field is essential and thus is the basis of protection. Since fusaric acid (FA) is one of the specific products present during the metabolic processing of Fusarium wilt pathogens, by combining FA with molecularly imprinted photonic crystal (MIPC) technologies, a series of MIPCs based on monodispersed silica, FA, and cross-linked polymers, i.e., MIPC-FA, which could be recycled, were designed and prepared. It was found that the responses of MIPC-FA were affected by the cross-linking degree of the polymer matrix and the pH values. The results showed that MIPC-FA could effectively detect FA in a solution of FA in a minute scale, Foc4-containing medium, and banana plants infected by Foc4, and the response was related to the FA concentration, which makes it an effective and fast method to detect and determine banana Fusarium wilt caused by Foc4 in the field.

Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System.

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

Beneficial Microbes and Basal Fertilization in Antagonism of Banana Fusarium Wilt

Fusarium Wilt Tropical Race 4 (TR4) is spreading rapidly all over the world and threatens banana production, especially the Cavendish variety, which is the main commercial cultivar in Asia. This work aims to use beneficial microbes and fertilizers to control TR4. Both Bacillus mycoides NP02 (BM) and Bacillus amyloliquefaciens BaPD1 (BA) antagonized TR4 growth in plate tests. In addition, basal nutrients (BN) in combination with BM and BA were used to treat 1-month-old banana seedlings infected by Fusarium Wilt; plant growth and nutrients of leaves were investigated after 6-month cultivation in the field. The seedling survival rates for uninfected and infected controls without the microbes were 89.4 ± 14.8% and 50 ± 10.7%, respectively. In contrast, the corresponding rates for 2BM, 5BM, 1BA, F1 (5BM + 4BN + 1BA) and G1 (1BM + 4BN + 1BA) in the infected plants were 80 ± 9.4%, 83 ± 4.3%, 85 ± 14.2%, 96.7 ± 1.9% and 96.7 ± 3.3%, respectively. The 2BM treatment promoted the growth of both uninfected and infected banana plants and the 5BA treatment significantly reduced the severity index by 1.45-fold. Plants infected with TR4 showed significantly reduced contents of nitrogen and potassium, but the contents of iron, copper and zinc were higher than those of healthy plants. Additionally, 1BM treatment stimulated the accumulation of nitrogen and zinc ions in the leaves of uninfected plants. Both the 1BA and 2BN treatments increased the iron (Fe) and nickel (Ni) metal ion levels of TR4 infected plants. The F2 (5BM + 2BN + 1BA) treatment significantly maintained the growth of banana plants under TR4 stress with increased contents of nickel and zinc in banana leaves, suggesting that these ions may play a key role in stimulating the growth of banana plants under the threat of TR4. This work shows the potential of applying BM, BN and BA in the control of Fusarium wilt in field conditions.