Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: The selection of tissue culture-derived somaclonal variants of Giant Cavendish banana (Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4-resistant cultivars: 'Tai-Chiao No. 5' (TC5), 'Tai-Chiao No. 7' (TC7), and 'Formosana' (FM), as well as their susceptible progenitor 'Pei-Chiao' (PC), to investigate the sequence variations among them and develop cultivar-specific markers. RESULTS: A group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4-resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7. CONCLUSIONS: This work demonstrates that reliable SNV markers of tissue culture-derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders' rights.

The Population of Fusarium oxysporum f. sp. cubense in Brazil Is Not Structured by Vegetative Compatibility Group or by Geographic Origin.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most destructive diseases of bananas in Brazil. In this study, a collection of 194 monosporic isolates from several banana-producing regions located in different climatic zones along a south-to-north transect in Brazil was formed to assess the genetic structure of the population of Foc. The isolates underwent pathogenicity tests, PCR diagnosis for the detection of tropical race 4, and screening of SIX homolog genes that produce putative effector proteins. The vegetative compatibility group (VCG) of 119 isolates was determined by pairing against 17 internationally known VCG-tester strains. A group of 158 isolates was selected for simple sequence repeat (SSR) genotyping. There was moderate diversity of Foc in Brazil. Eight VCGs were identified: 0120, 0122, 0124, 0125, 0128, 01215, 01220, and 01222, of which 78% of isolates belong to a single VCG, whereas 22% of isolates are assigned to multiple VCGs, belonging to complexes of VCGs. The distribution of VCGs is uneven and independent of the banana genotype. The isolates of a VCG shared a similar profile of SIX homologs, but there was no association with geographic region. Four SSR loci were polymorphic, and, on average, 7.5 alleles were detected per locus. Thirty-five multilocus genotypes (MLGs) were identified. There was no association between VCG and MLGs, and no genetic structure of the population of Foc in Brazil was detected.

Antagonism of volatile organic compounds of the Bacillus sp. against Fusarium kalimantanense.

Fusarium kalimantanense is a genetic lineage of Fusarium oxysporum f. sp. cubense (Foc) and belongs to the Fusarium oxysporum species complex (FOSC). This pathogen is a causative agent of Panama disease, an infection that has caused damage to the banana crop worldwide. Bacillus sp. (LPPC170) showed preliminary antagonist activity against F. kalimantanense (LPPC130) in vitro tests from the cultivation of axenic culture and co-culture with inhibition of mycelial growth of phytopathogen of 41.23%. According to these findings, volatile organic compounds (VOCs) emitted from Bacillus sp. were obtained by solid-phase microextraction and identified by gas chromatography coupled with a mass spectrometer (GC-MS). The multivariate data analysis tool (PLS-DA and Heatmap) identified short-chain organic acids as the main antagonistic VOCs responsible for inhibiting the mycelial growth of LPPC130. Acetic acid, propanoic acid, butanoic acid, valeric acid, and isovaleric acid exhibited a strong inhibitory effect on the mycelial growth of LPPC130, with inhibition of 20.68%, 33.30%, 26.87%, 43.71%, and 53.10%, respectively. Scanning electron microscopy revealed that VOCs caused damage to the vegetative and reproductive structures of the fungus. These results suggest Bacillus LPPC170 as an excellent biocontrol tool against the phytopathogen causative agents of Panama disease.

A Thermotolerant Marine Bacillus amyloliquefaciens S185 Producing Iturin A5 for Antifungal Activity against Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana (also known as Panama disease), is a severe fungal disease caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc). In recent years, biocontrol strategies using antifungal microorganisms from various niches and their related bioactive compounds have been used to prevent and control Panama disease. Here, a thermotolerant marine strain S185 was identified as Bacillus amyloliquefaciens, displaying strong antifungal activity against Foc. The strain S185 possesses multiple plant growth-promoting (PGP) and biocontrol utility properties, such as producing indole acetic acid (IAA) and ammonia, assimilating various carbon sources, tolerating pH of 4 to 9, temperature of 20 to 50 °C, and salt stress of 1 to 5%. Inoculation of S185 colonized the banana plants effectively and was mainly located in leaf and root tissues. To further investigate the antifungal components, compounds were extracted, fractionated, and purified. One compound, inhibiting Foc with minimum inhibitory concentrations (MICs) of 25 µg/disk, was identified as iturin A5 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). The isolated iturin, A5, resulted in severe morphological changes during spore germination and hyphae growth of Foc. These results specify that B. amyloliquefaciens S185 plays a key role in preventing the Foc pathogen by producing the antifungal compound iturin A5, and possesses potential as a cost-effective and sustainable biocontrol strain for Panama disease in the future. This is the first report of isolation of the antifungal compound iturin A5 from thermotolerant marine B. amyloliquefaciens S185.

Phylogeny and genetic diversity of the banana Fusarium wilt pathogen Fusarium oxysporum f. sp. cubense in the Indonesian centre of origin.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt or Panama disease on banana, is one of the major constraints in banana production worldwide. Indonesia is the centre of origin for wild and cultivated bananas, which likely co-evolved with Foc. This study explored the widest possible genetic diversity of Foc by sampling across Indonesia at 34 geographically and environmentally different locations in 15 provinces at six islands. This resulted in a comprehensive collection of ∼200 isolates from 40 different local banana varieties. Isolates were identified and assessed using sequence analysis of the translation elongation factor-1alpha (tef1), the RNA polymerase II largest subunit (rpb1), and the RNA polymerase II second largest subunit (rpb2). Phylogenetic analyses of these genes allowed the identification of 180 isolates of Fusarium oxysporum f. sp. cubense (Foc), and 20 isolates of the Fusarium fujikuroi species complex (FFSC), the Fusarium incarnatum-equiseti species complex (FIESC), and the Fusarium sambucinum species complex (FSSC). Further analyses, incorporating a worldwide collection of Foc strains, revealed nine independent genetic lineages for Foc, and one novel clade in the Fusarium oxysporum species complex (FOSC). Selected isolates from each lineage were tested on the banana varieties Gros Michel and Cavendish to characterise their pathogenicity profiles. More than 65 % of the isolates were diagnosed as Tropical Race 4 (Foc-TR4) due to their pathogenicity to Cavendish banana, which supports the hypothesis that Foc-TR4 is of Indonesian origin. Nine independent genetic lineages for Foc are formally described in this study. This biodiversity has not been studied since the initial description of Foc in 1919. This study provides a detailed overview of the complexity of Fusarium wilt on banana and its diversity and distribution across Indonesia.

Microbial-Based Biofungicides Mitigate the Damage Caused by Fusarium oxysporum f. sp. cubense Race 1 and Improve the Physiological Performance in Banana.

Fusarium wilt of banana (FWB) is the most limiting disease in this crop. The phytosanitary emergency caused by FWB since 2019 in Colombia has required the development of ecofriendly control methods. The aim of this study was to test the effectiveness of microbial-based biofungicides against FWB caused by Fusarium oxysporum f. sp. cubense race 1 (Foc R1) and correlate such effect with plant physiological parameters. Five Trichoderma (T1 to T4 and T9) and four Bacillus (T5 to T8)-based biofungicides were evaluated in pot experiments. In vitro, dual confrontation tests were also carried out to test whether the in vitro effects on Foc growth were consistent with the in vivo effects. While Trichoderma-based T3, T4, and T9, and Bacillus-based T8, significantly reduced the growth of Foc R1 in vitro, Trichoderma-based T1, T3, T4, and T9 temporarily reduced the Foc population in the soil. However, the incidence progress of FWB was significantly reduced by Bacterial-based T7 (74% efficacy) and Trichoderma-based T2 (50% efficacy). The molecular analysis showed that T7 prevented the inner tissue colonization by Foc R1 in 80% of inoculated plants. The T2, T4, T7, and T9 treatments mitigated the negative effects caused by Foc R1 on plant physiology and growth. Our data allowed us to identify three promising treatments to control FWB, reducing the progress of the disease, delaying the colonization of inner tissue, and mitigating physiological damages. Further studies should be addressed to determine the modes of action of the biocontrol agents against Foc and validate the utilization in the field.

Biodiversity of Trichoderma species of healthy and Fusarium wilt-infected banana rhizosphere soils in Tenerife (Canary Islands, Spain).

Banana (Musa acuminata) is the most important crop in the Canary Islands (38.9% of the total cultivated area). The main pathogen affecting this crop is the soil fungal Fusarium oxysporum f. sp. cubense subtropical race 4 (Foc-STR4), for which there is no effective control method under field conditions. Therefore, the use of native biological control agents may be an effective and sustainable alternative. This study aims to: (i) investigate the diversity and distribution of Trichoderma species in the rhizosphere of different banana agroecosystems affected by Foc-STR4 in Tenerife (the island with the greatest bioclimatic diversity and cultivated area), (ii) develop and preserve a culture collection of native Trichoderma species, and (iii) evaluate the influence of soil chemical properties on the Trichoderma community. A total of 131 Trichoderma isolates were obtained from 84 soil samples collected from 14 farms located in different agroecosystems on the northern (cooler and wetter) and southern (warmer and drier) slopes of Tenerife. Ten Trichoderma species, including T. afroharzianum, T. asperellum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hamatum, T. harzianum, T. hirsutum, T. longibrachiatum, and T. virens, and two putative novel species, named T. aff. harzianum and T. aff. hortense, were identified based on the tef1-α sequences. Trichoderma virens (35.89% relative abundance) and T. aff. harzianum (27.48%) were the most abundant and dominant species on both slopes, while other species were observed only on one slope (north or south). Biodiversity indices (Margalef, Shannon, Simpson, and Pielou) showed that species diversity and evenness were highest in the healthy soils of the northern slope. The Spearman analysis showed significant correlations between Trichoderma species and soil chemistry parameters (mainly with phosphorus and soil pH). To the best of our knowledge, six species are reported for the first time in the Canary Islands (T. afroharzianum, T. asperellum, T. atrobrunneum, T. guizhouense, T. hamatum, T. hirsutum) and in the rhizosphere of banana soils (T. afroharzianum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hirsutum, T. virens). This study provides essential information on the diversity/distribution of native Trichoderma species for the benefit of future applications in the control of Foc-STR4.

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.

Banana Cultivar Field Screening for Resistance to Fusarium oxysporum f.sp. cubense Tropical Race 4 in the Northern Territory.

Fusarium oxysporum f.sp. cubense, causal agent of Panama disease, is one of the biggest threats to global banana production, particularly the Cavendish competent tropical race 4 (Foc TR4). It continues to spread globally with detections occurring in regions of the Middle East and new continents such as Africa and South America in the last decade. As the search was on for new management strategies and resistant cultivars to combat the disease, a banana cultivar-screening trial took place in the Northern Territory of Australia, which examined the responses of 24 banana cultivars to the soil borne fungus. These cultivars included material from TBRI, FHIA and selections from Thailand, Indonesia and Australia and evaluated for their resistance to tropical race 4 for two cropping cycles. Several cultivars displayed considerable resistance to Foc TR4, including several FHIA parental lines and hybrids, the Cavendish (AAA) selections GCTCV 215 and GCTCV 247 from TBRI and an Indonesian selection CJ19 showed either very little to no plant death due to the disease.

Phytotoxic Metabolites Produce by Fusarium oxysporum f. sp. cubense Race 2.

Banana is a major tropical fruit crop but banana production worldwide is seriously threatened due to Fusarium wilt. Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt of banana (also referred as Panama disease) is an asexual, soil inhabiting facultative parasite. Foc isolates can be classified into three races that are not defined genetically, but for their pathogenicity to different banana cultivars. Despite mycotoxins being some of the best studied virulence factors of phytopathogenic fungi and these have been useful for the prediction of Foc virulence on banana plants, toxins produced by Foc race 2 strains have not been previously identified. The aim of this contribution was to identify the phytotoxic metabolites closely related to banana wilt caused by a Foc race 2 strain. We used an in vitro bioassay on detached banana leaves to evaluate the specificity of the microbial culture filtrates before a partial purification and further identification of Foc race 2 phytotoxins. A 29-day-old host-specific culture filtrate was obtained but specificity of culture filtrate was unrecovered after partial purification. The non-specific phytotoxins were characterized as fusaric acid, beauvericin, and enniatin A. Whereas some, if not all, of these phytotoxins are important virulence factors, a proteinaceous fraction from the specific 29-day-old culture filtrate protected the leaves of the resistant banana cultivar from damage caused by such phytotoxic metabolites.

A Medium-Term Field Experiment to Study the Effect of Managing Soil Chemical Properties on Fusarium Wilt in Banana (Musa AAA).

Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungus causing Fusarium wilt (FW) in banana. It is practically impossible to eradicate Foc in soils. Our understanding of soil-Foc-banana interactions is hampered by inconsistent research results caused by agro-ecological variability and the complexity of the soil system. This study aimed to evaluate the options to manage soil chemical properties to reduce disease expression and maintain banana production. The expression of FW (Foc Race 1) and the agronomic performance of the Gros Michel (Musa AAA) banana were evaluated in two medium-term factorial field experiments at representative locations in the Costa Rican banana region. In the experiments, five soil chemical properties (pH, N, Ca, Mg, and Mn) were managed to achieve a low and a high level. Plant mortality caused by FW, soil fertility, plant nutrition, and agronomic performance were monitored during four crop cycles. After the first crop cycle, the treatments started to present differences in plant mortality. There was a significant rise of plant mortality after the second crop cycle resulting in a cumulative plant mortality exceeding 60% in both experiments. A lower soil pH consistently resulted in significantly higher plant mortality. The interactions between soil properties (pH-N, pH-CaMg, pH-Mn, N-Mn, and CaMg-Mn) also influenced plant mortality. Soil N was the most significant treatment affecting leaf nutrient concentrations, bunch weight, and clusters per bunch. The experiments confirmed the potential role of soil management in FW expression in banana. Our results suggest that the management of soil chemical properties in the conditions here studied may help to reduce the expression rate of FW, but not to control the disease in the long run.

Incidence, Spatial Pattern and Temporal Progress of Fusarium Wilt of Bananas.

The effective management of Fusarium wilt of bananas (FWB) depends on the knowledge of the disease dynamics in time and space. The objectives of this work were: to estimate disease intensity and impact, and to investigate the spatial and temporal dynamics of FWB. Fields planted with Silk (n = 10), Pome (n = 17), or Cavendish (n = 3) banana subgroups were surveyed in Brazil, totaling 95 ha. In each field, all plants were visually assessed, and diseased plants were georeferenced. The incidence of FWB and the impact of the disease on the yield on a regional scale were estimated. Spatial patterns were analyzed using quadrat- and distance-based methods. FWB incidence ranged from 0.09% to 41.42%, being higher in Silk fields (median = 14.26%). Impacts of epidemics on yield ranged from 18.4 to 8192.5 kg ha-1 year-1, with an average of 1856.7 kg ha-1 year-1. The higher economic impact of the disease was observed on Silk cultivar with an average loss of USD 1974.2 ha-1 year-1. Overall, estimated losses increased on average by USD 109.8 ha-1 year-1 at each 1% of incidence. Aggregation of FWB was detected by all analytical methods in 13 fields (1 of Cavendish, 11 of Pome, and 1 of Silk). In the other 17 fields, at least one analytical method did not reject the null hypothesis of randomness. One field (5 ha), composed of six plots, was selected for spatial and temporal studies during two years with bi-monthly assessments. A sigmoidal curve represented the FWB progress and the Gompertz model best-fitted disease progress. The level of aggregation varied over time, and evidence of secondary infection to neighboring and distant plants was detected. FWB is a widespread problem in Brazil and yield losses can be of high magnitude. Epidemiology-based management strategies can now be better established.

Weevil Borers Affect the Spatio-Temporal Dynamics of Banana Fusarium Wilt.

Dispersal of propagules of a pathogen has remarkable effects on the development of epidemics. Previous studies suggested that insect pests play a role in the development of Fusarium wilt (FW) epidemics in banana fields. We provided complementary evidence for the involvement of two insect pests of banana, the weevil borer (Cosmopolites sordidus L., WB) and the false weevil borer (Metamasius hemipterus L., FWB), in the dispersal of Fusarium oxysporum f. sp. cubense (Foc) using a comparative epidemiology approach under field conditions. Two banana plots located in a field with historical records of FW epidemics were used; one was managed with Beauveria bassiana to reduce the population of weevils, and the other was left without B. bassiana applications. The number of WB and FWB was monitored biweekly and the FW incidence was quantified bimonthly during two years. The population of WB and the incidence (6.7%) of FW in the plot managed with B. bassiana were lower than in the plot left unmanaged (13%). The monomolecular model best fitted the FW disease progress data, and as expected, the average estimated disease progress rate was lower in the plot managed with the entomopathogenic fungus (r = 0.002) compared to the unmanaged plot (r = 0.006). Aggregation of FW was higher in the field with WB management. WB affected the spatial and temporal dynamics of FW epidemics under field conditions. Management of the insects may reduce yield loss due to FW.

Phloem Delivery of Fludioxonil by Plant Amino Acid Transporter-Mediated Polysuccinimide Nanocarriers for Controlling Fusarium Wilt in Banana.

Fusarium wilt disease poses a serious threat to the global production of bananas. The targeted delivery of fungicides to banana phloem tissues may offer new hope for controlling this hard-to-treat vascular disease. In this study, fludioxonil (FLU)-loaded glycine methyl ester-conjugated polysuccinimide nanoparticles (PGA) were prepared with a loading efficiency (LE) of 27.9%. The obtained nanoparticles (FLU@PGA) exhibited pH-sensitive controlled release, specifically under an alkaline pH in plant phloem. In vivo experiments in potted bananas demonstrated that FLU@PGA can achieve the downward delivery of FLU to banana rhizomes and roots after foliar application, reducing disease severity by 50.4%. The phloem transport studies showed that the phloem loading of FLU@PGA was involved in an active transport mechanism at the organ level (castor bean seedlings). The observation of fluorescein-5-isothiocyanate cadaverine-labeled PGA nanocarriers showed that they could be absorbed by mesophyll cells and loaded into vascular tissues through the symplastic pathway. Furthermore, the interaction of FLU@PGA with the plant amino acid transporter AtLHT1 was observed to enhance transmembrane uptake at the cellular level (Xenopus oocytes). These results suggested that the phloem-targeted delivery of fungicide by transporter-mediated nanocarriers could be a promising new strategy for the management of Fusarium wilt in bananas.

A MADS-box transcription factor FoRlm1 regulates aerial hyphal growth, oxidative stress, cell wall biosynthesis and virulence in Fusarium oxysporum f. sp. cubense.

The fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt that affects banana plants. However, the detailed molecular mechanisms of Foc virulence determinants have not been elucidated. In this study, we identified the MADS-box transcription factor FoRlm1 that is conserved among mitogen-activated protein kinases. Our data revealed that FoRlm1 is essential for aerial hyphal growth and virulence. Transcriptional analysis revealed that FoRlm1 deletion altered the expression of anti-oxidant enzymes, chitin synthases, fusaric acid (FA), and beauvericin biosynthesis genes. Furthermore, FoRlm1 deletion promoted tolerance to Congo red and increased sensitivity to hydrogen peroxide. Transcriptome analysis of ΔFoRlm1 mutant and wild-type strain indicated that the expression of many genes associated with fungal physiology and virulence was up- or down-regulated. Overall, these results suggested that FoRlm1 plays a critical role in the regulation of hyphal growth, anti-oxidation mechanisms, cell wall biosynthesis, transcription of mycotoxin biosynthetic genes encoding FA and beauvericin, and virulence in Foc.

Molecular Diagnostics of Banana Fusarium Wilt Targeting Secreted-in-Xylem Genes.

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Corrigendum: Biological Control Agents Against Fusarium Wilt of Banana.

[This corrects the article DOI: 10.3389/fmicb.2019.00616.].

Biological Control Agents Against Fusarium Wilt of Banana.

In the last century, the banana crop and industry experienced dramatic losses due to an epidemic of Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f.sp. cubense (Foc) race 1. An even more dramatic menace is now feared due to the spread of Foc tropical race 4. Plant genetic resistance is generally considered as the most plausible strategy for controlling effectively such a devastating disease, as occurred for the first round of FWB epidemic. Nevertheless, with at least 182 articles published since 1970, biological control represents a large body of knowledge on FWB. Remarkably, many studies deal with biological control agents (BCAs) that reached the field-testing stage and even refer to high effectiveness. Some selected BCAs have been repeatedly assayed in independent trials, suggesting their promising value. Overall under field conditions, FWB has been controlled up to 79% by using Pseudomonas spp. strains, and up to 70% by several endophytes and Trichoderma spp. strains. Lower biocontrol efficacy (42-55%) has been obtained with arbuscular mycorrhizal fungi, Bacillus spp., and non-pathogenic Fusarium strains. Studies on Streptomyces spp. have been mostly limited to in vitro conditions so far, with very few pot-experiments, and none conducted in the field. The BCAs have been applied with diverse procedures (e.g., spore suspension, organic amendments, bioformulations, etc.) and at different stages of plant development (i.e., in vitro, nursery, at transplanting, post-transplanting), but there has been no evidence for a protocol better than another. Nonetheless, new bioformulation technologies (e.g., nanotechnology, formulation of microbial consortia and/or their metabolites, etc.) and tailor-made consortia of microbial strains should be encouraged. In conclusion, the literature offers many examples of promising BCAs, suggesting that biocontrol can greatly contribute to limit the damage caused by FWB. More efforts should be done to further validate the currently available outcomes, to deepen the knowledge on the most valuable BCAs, and to improve their efficacy by setting up effective formulations, application protocols, and integrated strategies.

Genetic Diversity in FUB Genes of Fusarium oxysporum f. sp. cubense Suggests Horizontal Gene Transfer.

Fusaric acid (FA) is an important secondary metabolite of many Fusarium species and involved in the wilt symptoms caused in banana by Fusarium oxysporum f. sp. cubense (Foc). To investigate the evolution characteristics of the 12 Foc FA biosynthetic genes (FUB), coding sequences of the 12 FUB genes and three housekeeping genes, EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II), were subjected to genetic diversity analysis, phylogenetic analysis, recombination detection, and selective pressure analysis. The results of selective pressure analysis showed that the 15 genes were mainly subjected to negative selection. However, a significantly higher number of silent mutations, which could not be simply explained by selective pressure difference, were observed in the 12 FUB genes in Foc than in the three housekeeping genes. Infraspecies phylogeny and recombination detection analysis showed that significantly more horizontal gene transfer (HGT) events (normalized) had occurred in the FUB genes than in the three housekeeping genes. In addition, many of these events involved outgroup isolates and significantly increased the genetic diversity of FUB genes in Foc. The infraspecies phylogenetic analysis suggested that the polyphyletic phylogeny proposed for Foc requires further discussion, and the divergence of race 1, race 4, and the common ancestor of several F. oxysporum (Fo) isolates pathogenic to nonbanana plants should have diverged over a short period. Finally, our results suggest that the FUB genes in Fo should have benefited from HGT to gain a relatively high genetic diversity to respond to different host plants and environments despite mainly being subject to negative selection.

Comparative transcriptome analysis reveals resistance-related genes and pathways in Musa acuminata banana 'Guijiao 9' in response to Fusarium wilt.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases in bananas resulting in significant loss of Cavendish bananas production worldwide. Here we show the agronomic traits and the resistance of 'Guijiao 9' in the field trials from 2012 to 2017. And then we dissect and compare the transcriptome response from these two cultivars (cv. 'Guijiao 9' and cv. Williams) in an attempt to understand the molecular basis that contribute to the enhanced Foc tropical race 4 (Foc-TR4) resistance. 'Guijiao 9' is a Cavendish cultivar with strong resistance to Foc-TR4, which was reflected in a lower disease severity and incidence in glasshouse and field trails, when compared to the susceptible cultivar Williams. Gene expression profiles of 'Guijiao 9' and Williams were captured by performing RNA-Seq analysis on 16 biological samples collected over a six day period post inoculation with Foc-TR4. Transcriptional reprogramming in response to Foc-TR4 was detected in both genotypes but the response was more drastic in 'Guijiao 9' than in Williams. Specific genes involved in plant-pathogen interaction and defense signaling including MAPK, calcium, salicylic acid, jasmonic acid and ethylene pathways were analyzed and compared between 'Guijiao 9' and Williams. Genes associated with defense-related metabolites synthesis such as NB-LRR proteins, calmodulin-binding protein and phenylpropanoids biosynthesis genes were significantly up-regulated in 'Guijiao 9' resistant to Foc-TR4 infection. Taken together, this study highlights the important roles of plant hormone regulation and defense gene activation in mediating resistance in 'Guijiao 9'.