QTL-seq and marker development for resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in cultivated watermelon

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.     

Biochemical and genetic analysis of a unique poly(ADP-ribosyl) glycohydrolase (PARG) of the pathogenic fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

The genome sequence of the plant pathogen Fusarium oxysporum f. sp. lycopersici contains a single gene encoding a predicted poly(ADP-ribose) glycohydrolase (FOXG_05947.2, PARG). Here, we assessed whether this gene has a role as a global regulator of DNA repair or in virulence as an ADP ribosylating toxin homologue of bacteria. The PARG protein was purified after expressing its encoding gene in Escherichia coli. Its inhibition by 6,9-diamino-2-ethoxyacridine lactate monohydrate and tannins was similar to its human orthologue that is involved in DNA repair. A deletion strain of F. oxysporum f. sp. lycopersici showed no growth defects and was not affected in pathogenicity. Together, our results indicate that the PARG protein of F. oxysporum f. sp. lycopersici is involved in DNA repair and does not act in pathogenicity as an effector.     

Construction of a genome-anchored,high-density genetic map for melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and identification of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis> race 1 resistance QTL

Key message

Four QTLs and an epistatic interaction were associated with disease severity in response to inoculation with Fusarium oxysporum f. sp. melonis race 1 in a recombinant inbred line population of melon.

Abstract

The USDA Cucumis melo inbred line, MR-1, harbors a wealth of alleles associated with resistance to several major diseases of melon, including powdery mildew, downy mildew, Alternaria leaf blight, and Fusarium wilt. MR-1 was crossed to an Israeli cultivar, Ananas Yok’neam, which is susceptible to all of these diseases, to generate a recombinant inbred line (RIL) population of 172 lines. In this study, the RIL population was genotyped to construct an ultra-dense genetic linkage map with 5663 binned SNPs anchored to the C. melo genome and exhibits the overall high quality of the assembly. The utility of the densely genotyped population was demonstrated through QTL mapping of a well-studied trait, resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) race 1. A major QTL co-located with the previously validated resistance gene Fom-2. In addition, three minor QTLs and an epistatic interaction contributing to Fom race 1 resistance were identified. The MR-1 × AY RIL population provides a valuable resource for future QTL mapping studies and marker-assisted selection of disease resistance in melon.

     

Assay development and marker validation for marker assisted selection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in watermelon

Fusarium wilt, Fusarium oxysporum f. sp. niveum (FON), of watermelon (Citrullus lanatus) is a fungal pathogen that causes significant yield losses in the US watermelon industry. FON damages watermelon through invasion of the root system and remains a difficult pathogen to manage due to its long-lasting survival spores which persist in the soil. Chemical control options for this pathogen are lacking, making development of genetic resistance the best option. There are four known races of FON (0, 1, 2, and 3) which are distinguished based on their pathogenicity of differential cultivars. Most modern cultivar releases have FON race 1 (FON-1) resistance, which has been mapped on the end of chromosome 1. Application of marker assisted selection (MAS) would improve the efficiency of FON-1 resistance breeding. In order to identify markers for selection in the FON-1 region, the QTL-seq method was utilized on an F₂ population segregating for FON-1 resistance. Single nucleotide polymorphism (SNP) markers in the region were developed into Kompetitive allele-specific PCR (KASP?) assays and tested for trait association on the segregating F_2:3 population. Marker validation was done using an F₂ population from a cross between FON-1 susceptible “New Hampshire Midget” and FON-1-resistant “Calhoun Gray.” Further validation on a panel of susceptible and resistant cultivars and Plant Introductions identified SNP marker UGA1_502161 as a useful marker for selection of FON-1 resistance from Calhoun Gray.     

Detection of pea wilt pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> using DNA-based markers

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1₃₈₀, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.     

Combined use of biocontrol agents and zeolite as a management strategy against <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Verticillium</Emphasis> wilt

Vascular wilt pathogens, like Fusarium oxysporum and Verticillium dahliae, cause heavy economic loses to a range of crops. The lack of chemical control intensifies the problem. In the present study, the initial in vitro activity of 134 bacterial isolates, originating from various stages of the composting process of cotton residues, against F. oxysporum f. sp. melonis (FOM) and V. dahliae was evaluated. The most efficient strains, named SP10 and C20 M, belong to Bacillus sp. Both strains significantly reduced Fusarium and Vertilicillium wilt in melon and aubergine respectively. Furthermore, zeolite was tested alone or in combination with SP10 against V. dahliae and FOM. It was shown that the combination of zeolite and SP10 in the transplant soil plug was the most disease suppressive treatment. Interestingly the single application of zeolite was also plant-protective. The positive effect of zeolite on plant health could be linked with the recorded up-regulation of plant defense genes.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

A search for microscopic fungi with directed hydroxylase activity for the synthesis of steroid drugs

The hydroxylase activities of new strains such as Curvularia lunata, C. geniculata, C. eragrostidis, C. prasadii, Ulocladium botrytis, Alternaria tenuis, and Fusarium oxysporum toward three steroid substrates, namely, androstenedione (AD), cortexolone (S), and dehydroepiandrosterone acetate (DAA), were characterized. The 9α-hydroxylase activity of C. lunata 1011 cells against S to form 9α-hydroxy-S was shown for the first time. It was found that C. geniculata 837 and F. oxysporum 11dn1 strains can hydroxylate substrates to form pharmacologically promising 7α-hydroxysteroids. C. geniculata 837 cells selectively hydroxylate AD, resulting in 7α-hydroxytestosterone, whereas F. oxysporum 11dn1 leads to the transformation of DAA to 7α-hydroxydehydroepiandrosterone.     

Controlled biosynthesis of AgCl nanoparticles by a thermotolerant <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in the L-Tryptophan supplemented media: Characterization and antimicrobial activity

A simple and green method was developed for the extracellular biosynthesis of silver chloride nanoparticles, free from silver nanoparticles, using cell-free filtrate of a thermotolerant fungal strain Aspergillus terreus 8. The synthesized silver chloride nanoparticles exhibited characteristic absorption maximum at 275 nm. As-fabricated AgCl-NPs were characterized by UV-vis spectroscopy, XRD, SEM-EDX, and FT-IR. The biosynthesized silver chloride nanoparticles exhibited strong antimicrobial activity towards pathogenic microorganisms such as Fusarium oxysporum f. sp. vasinfectum and Verticillium dahliae. The synthesized silver chloride nanoparticles can be exploited as a promising new biocide bionanocomposite against pathogenic microorganisms.     

Phylogeny,antimicrobial, antioxidant and enzyme-producing potential of fungal endophytes found in <Emphasis Type="Italic">Viola odorata</Emphasis>

Viola odorata, a medicinal plant, is traditionally used to treat common cold, congestion and cough. Given its medicinal properties and occurrence in the northwestern Himalayas, we isolated and characterized endophytic fungi from this plant morphologically, microscopically and by internal transcribed spacer-based rDNA sequencing. In total, we isolated 27 morphotypes of endophytes belonging to phyla Ascomycota and Basidiomycota. The roots showed the highest diversity of endophyte as well as fungal dominance, followed by leaves and leaf nodes. The fungal extract of VOR16 (Fusarium oxysporum) displayed potent antimicrobial activity against Salmonella typhimurium, Klebsiella pneumoniae and Escherichia coli, with a minimum inhibitory concentration of 0.78, 0.78 and 1.56 μg/mL, respectively, while fungal extract VOLF4 (Aspergillus sp.) exhibited promising antioxidant activity (IC₅₀ of 17.4 μg/mL). To identify the components responsible for various bioactivities, we analyzed the content of penicillin G in the extract of bioactive endophytes. The results suggested that the expression of penicillin G under the fermentation conditions applied was too low to display antimicrobial effects. Thus, the activity may be contributed by a different, novel secondary metabolite. The antioxidant activity of VOLF4 may be attributed to its high content of flavonoids. Of the endophytic fungi assessed, 27% were found to be enzyme producers. The highest zone of clearance was observed in VOLN5 (Colletotrichum siamense) for protease production. Only VOR5 (Fusarium nematophilum) was found to be a producer of cellulase, glutenase, amylase and protease. In summary, this is the first report of the isolation of endophytes, namely Fusarium nematophilum, Colletotrichum trifolii, C. destructivum, C. siamense and Peniophora sp., from V. odorata and their bioactive and enzyme-producing potential.     

The role of strigolactones during plant interactions with the pathogenic fungus <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis>

Main conclusion

Strigolactones (SLs) do not influence spore germination or hyphal growth of Fusarium oxysporum. Mutant studies revealed no role for SLs but a role for ethylene signalling in defence against this pathogen in pea. Strigolactones (SLs) play important roles both inside the plant as a hormone and outside the plant as a rhizosphere signal in interactions with mycorrhizal fungi and parasitic weeds. What is less well understood is any potential role SLs may play in interactions with disease causing microbes such as pathogenic fungi. In this paper we investigate the influence of SLs on the hemibiotrophic pathogen Fusarium oxysporum f.sp. pisi both directly via their effects on fungal growth and inside the plant through the use of a mutant deficient in SL. Given that various stereoisomers of synthetic and naturally occuring SLs can display different biological activities, we used (+)-GR24, (?)-GR24 and the naturally occurring SL, (+)-strigol, as well as a racemic mixture of 5-deoxystrigol. As a positive control, we examined the influence of a plant mutant with altered ethylene signalling, ein2, on disease development. We found no evidence that SLs influence spore germination or hyphal growth of Fusarium oxysporum and that, while ethylene signalling influences pea susceptibility to this pathogen, SLs do not.