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 variety succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial variety worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to Cavendish and other commonly grown varieties. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana varieties. 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, TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.Peer reviewe

Effector Profiles of Endophytic <i>Fusarium</i> Associated with Asymptomatic Banana (<i>Musa</i> sp.) Hosts

During the infection of a host, plant pathogenic fungi secrete small proteins called effectors, which then modulate the defence response of the host. In the Fusarium oxysporum species complex (FOSC), the secreted in xylem (SIX) gene effectors are important for host-specific pathogenicity, and are also useful markers for identifying the various host-specific lineages. While the presence and diversity of the SIX genes has been explored in many of the pathogenic lineages of F. oxysporum, there is a limited understanding of these genes in non-pathogenic, endophytic isolates of F. oxysporum. In this study, universal primers for each of the known SIX genes are designed and used to screen a panel of endophytically-associated Fusarium species isolated from healthy, asymptomatic banana tissue. SIX gene orthologues are identified in the majority of the Fusarium isolates screened in this study. Furthermore, the SIX gene profiles of these endophytic isolates do not overlap with the SIX genes present in the pathogenic lineages of F. oxysporum that are assessed in this study. SIX gene orthologues have not been commonly identified in Fusarium species outside of the FOSC nor in non-pathogenic isolates of F. oxysporum. The results of this study indicate that the SIX gene effectors may be more broadly distributed throughout the Fusarium genus than previously thought. This has important implications for understanding the evolution of pathogenicity in the FOSC

Small RNA profiling of Cavendish banana roots inoculated with Fusarium oxysporum f. sp. cubense race 1 and tropical race 4

Fusarium wilt, caused by the soil-borne fungal pathogen, Fusarium oxysporum f. sp. cubense (Foc), is considered as one of the most threatening diseases of banana. The Cavendish variety, resistant to Foc race 1 (R1), is susceptible to tropical race 4 (TR4), an aggressive race of the pathogen which is of increasing concern worldwide. Previous studies have revealed that plant small RNAs (sRNAs) play crucial roles in the host response to pathogen infection. To investigate the roles of sRNAs involved in the interaction of the banana-Foc pathosystem, small RNA profiles of Cavendish banana roots inoculated with Foc TR4 and Foc R1 were obtained and analyzed in the present study using Next-Generation Sequencing (NGS) technology. A total of 112 discrete mature microRNAs (miRNAs) belonging to 22 known miRNA families were found across all constructed sRNA libraries. The expression of miR166, miR159 and miR156 was up-regulated in TR4-inoculated samples as compared to mock-inoculated samples, while the expression of these miRNAs was approximately the same in R1-inoculated and mock-inoculated samples. Consistent with the sequencing data, qRT-PCR results demonstrated up-regulation of these miRNAs and down-regulation of their target genes in TR4-inoculated samples, but not in R1-inoculated samples. Considering Cavendish banana is resistant to R1 and susceptible to TR4, it is possible that these sRNAs and their target genes are involved in particular plant defence pathways such as salicylic acid-based defence. The findings will pave way for future investigations of the defence mechanism and potential approaches of resistance improvement

Image-based quantification of Fusarium wilt severity in banana

The severity of Fusarium wilt of bananas has long been classified based on visual assessment of necrosis in rhizome or pseudostem cross-sections. The improved method proposed here uses digital image analysis to quantify the proportion of rhizome tissue that is necrotic. It agrees well with visual classification, but provides greater reproducibility, precision and statistical power

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea.

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides