Secreted in Xylem 6 (SIX6) Mediates Fusarium oxysporum f. sp. fragariae Race 1 Avirulence on FW1-Resistant Strawberry Cultivars.

Fusarium oxysporum f. sp. fragariae (Fof) race 1 is avirulent on cultivars with the dominant resistance gene FW1, while Fof race 2 is virulent on FW1-resistant cultivars. We hypothesized there was a gene-for-gene interaction between a gene at the FW1 locus and an avirulence gene (AvrFW1) in Fof race 1. To identify a candidate AvrFW1, we compared genomes of 24 Fof race 1 and three Fof race 2 isolates. We found one candidate gene that was present in race 1, was absent in race 2, was highly expressed in planta, and was homologous to a known effector, secreted in xylem 6 (SIX6). We knocked out SIX6 in two Fof race 1 isolates by homologous recombination. All SIX6 knockout transformants (ΔSIX6) gained virulence on FW1/fw1 cultivars, whereas ectopic transformants and the wildtype isolates remained avirulent. ΔSIX6 isolates were quantitatively less virulent on FW1/fw1 cultivars Fronteras and San Andreas than fw1/fw1 cultivars. Seedlings from an FW1/fw1 × fw1/fw1 population were genotyped for FW1 and tested for susceptibility to a SIX6 knockout isolate. Results suggested that additional minor-effect quantitative resistance genes could be present at the FW1 locus. This work demonstrates that SIX6 acts as an avirulence factor interacting with a resistance gene at the FW1 locus. The identification of AvrFW1 enables surveillance for Fof race 2 and provides insight into the mechanisms of FW1-mediated resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First report of Fusarium oxysporum f. sp. fragariae race 2 causing Fusarium wilt of strawberry (Fragaria × ananassa) in California.

In California, Fusarium wilt of strawberry is widespread and causes significant yield losses. Resistant cultivars with the FW1 gene were protected against Fusarium wilt because all strains of Fusarium oxysporum f. sp. fragariae (Fof) in California were race 1 (i.e., avirulent to FW1-resistant cultivars) (Henry et al. 2017; Pincot, et al. 2018; Henry et al. 2021). In the fall of 2022, severe wilt disease was observed in an organic, summer-planted strawberry field in Oxnard, California. Fusarium wilt symptoms were common and included wilted foliage, deformed and highly chlorotic leaflets, and crown discoloration. The field was planted with Portola, a cultivar with the FW1 gene that is resistant to Fof race 1 (Pincot et al. 2018; Henry et al. 2021). Two samples, each consisting of four plants, were collected from two different locations within the field. Crown extracts from each sample were tested for Fof, Macrophomina phaseolina, Verticillium dahliae, and Phytophthora spp. by recombinase polymerase amplification (RPA) (Steele et al. 2022). Petioles were surface sterilized in 1% sodium hypochlorite for 2 minutes and plated on Komada's medium to select for Fusarium spp. (Henry et al. 2021; Komada, 1975). The RPA results were positive for M. phaseolina in one sample and negative for all four pathogens in the other sample. Salmon-colored, fluffy mycelia grew profusely from petioles of both samples. Colony morphology and non-septate, ellipsoidal microconidia (6.0-13 µm × 2.8-4.0 µm) borne on monophialides resembled F. oxysporum. Single hyphal tip isolation of fourteen cultures (P1-P14) was done to purify single genotypes. None of these pure cultures amplified with Fof-specific qPCR (Burkhardt et al. 2019), confirming the negative result obtained with RPA. Translation elongation factor 1-alpha (EF1α) was amplified using EF1/EF2 primers (O'Donnell et al. 1998) from three isolates. Amplicons were sequenced (GenBank OQ183721) and found through BLAST search to have 100% identity with an isolate of Fusarium oxysporum f. sp. melongenae (GenBank FJ985297). There was at least one nucleotide difference when compared to all known strains of Fof race 1 (Henry et al. 2021). Five isolates (P2, P3, P6, P12, and P13) and an Fof race 1 control isolate (GL1315) were tested for pathogenicity on Fronteras (FW1) and Monterey (fw1; susceptible to race 1). Five plants per isolate × cultivar combination were inoculated by dipping roots in 5 × 106 conidia per mL of 0.1% water agar, or in sterile 0.1% water agar for the negative control, and grown as described by Jenner and Henry (2022). After six weeks, all non-inoculated control plants remained healthy while plants of both cultivars inoculated with the five isolates were severely wilted. Petiole assays yielded colonies identical in appearance to the inoculated isolates. For Fof race 1-inoculated plants, wilt symptoms were observed in Monterey but not in Fronteras. This experiment was repeated with P2, P3, P12, and P13 on another FW1 cultivar, San Andreas, and the same results were observed. To our knowledge, this is the first report of F. oxysporum f. sp. fragariae race 2 in California. Losses to Fusarium wilt are likely to increase until genetic resistance to this strain of Fof race 2 is deployed in commercially viable cultivars.

Sporodochia Formed by Fusarium oxysporum f. sp. fragariae Produce Airborne Conidia and Are Ubiquitous on Diseased Strawberry Plants in California.

Sporodochia are dense masses of fungal hyphae bearing asexual conidia. For Fusarium oxysporum, sporodochia are known to produce airborne conidia and enhance the dissemination of this otherwise soilborne pathogen. Sporodochia are small and transient, and they are documented for only a few formae speciales of F. oxysporum. This study reports airborne conidia and sporodochia produced by F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry, in the Monterey Bay region of California. Sporodochia were discovered in 21 of 24 Fusarium wilt-diseased fields surveyed for this study and were readily observed on most symptomatic plants in these fields. Only necrotic tissues bore sporodochia, and they were most frequently observed on petioles and peduncles. Sporodochia covered significantly greater lengths of peduncles than petioles, extending from the base of the plant toward the upper part of the canopy. A stolon hosted the longest stretch of sporodochial growth, found covering the stolon's entire 35-cm length and the base of the daughter plant. Macroconidia were produced by all sporodochia samples, and we did not find microconidia on any samples. An initial series of experiments confirmed the potential for conidia produced by sporodochia to disperse with wind over short distances. The prevalence of sporodochia producing airborne spores of F. oxysporum f. sp. fragariae has great importance for disease management and biosecurity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae.

Verticillium dahliae is a soilborne fungus that causes vascular wilt diseases on numerous plant species worldwide. The production of darkly melanized microsclerotia is crucial in the disease cycle of V. dahliae, as these structures allow for long-term survival in soil. Previously, transcriptomic and genomic analysis identified a cluster of genes in V. dahliae that encodes some dihydroxynaphthalene (DHN) melanin biosynthetic pathway homologues found in related fungi. In this study, we explored the roles of cluster-specific transcription factor VdCmr1, as well as two other genes within the cluster encoding a polyketide synthase (VdPKS1) and a laccase (VdLac1), enzymes at initial and endpoint steps in DHN melanin production. The results revealed that VdCmr1 and VdPKS1 are required for melanin production, but neither is required for microsclerotia production. None of the three genes were required for pathogenesis on tobacco and lettuce. Exposure of ΔVdCmr1 and wild-type strains to UV irradiation, or to high temperature (40 °C), revealed an approx. 50 % reduction of survival in the ΔVdCmr1 strain, relative to the wild-type strain, in response to either condition. Expression profiles revealed that expression of some melanin biosynthetic genes are in part dependent on VdCmr1. Combined data indicate VdCmr1 is a key regulator of melanin biosynthesis, and that via regulation of melanogenesis, VdCmr1 affects survival of V. dahliae in response to abiotic threats. We conclude with a model showing regulation of VdCmr1 by a high osmolarity glycerol response (Hog)-type MAP kinase pathway.

Host Genotype and Hypersensitive Reaction Influence Population Levels of Xanthomonas campestris pv. vitians in Lettuce.

Dynamics of population sizes of Xanthomonas campestris pv. vitians inoculated onto or into lettuce leaves were monitored on susceptible and resistant cultivars. In general, population growth was greater for susceptible (Clemente, Salinas 88, Vista Verde) than resistant (Batavia Reine des Glaces, Iceberg, Little Gem) cultivars. When spray-inoculated or infiltrated, population levels of X. campestris pv. vitians were consistently significantly lower on Little Gem than on susceptible cultivars, while differences in the other resistant cultivars were not consistently statistically significant. Populations increased at an intermediate rate on cultivars Iceberg and Batavia Reine des Glaces. There were significant positive correlations between bacterial concentration applied and disease severity for all cultivars, but bacterial titer had a significantly greater influence on disease severity in the susceptible cultivars than in Little Gem and an intermediate influence in Iceberg and Batavia Reine des Glaces. Infiltration of X. campestris pv. vitians strains into leaves of Little Gem resulted in an incompatible reaction, whereas compatible reactions were observed in all other cultivars. It appears that the differences in the relationship between population dynamics for Little Gem and the other cultivars tested were due to the hypersensitive response in cultivar Little Gem. These findings have implications for disease management and lettuce breeding because X. campestris pv. vitians interacts differently with cultivars that differ for resistance mechanisms.

Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ.

The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were developed from 16S rDNA sequences to be useful for the specific detection and quantification of S. suberifaciens. Quantitative PCR (qPCR) protocols specifically amplified DNA from the type strain of S. suberifaciens (LMG 17323) and other members of this species but not from other members of the Sphingomonadaceae. The detection limit was as little as 100 fg DNA (equivalent to 2 × 10(2) cells) in the qPCR. Detection was successful from soils inoculated with as little as 1 × 10(3) CFU/g soil. DNA isolated from naturally infested soils and diseased lettuce roots was amplified and sequenced fragments were identical or nearly identical to 16S rDNA sequences from S. suberifaciens. In growth chamber experiments, there was a positive correlation between disease severity and S. suberifaciens population levels in roots and soil, as detected by qPCR. Detection levels were below population levels of the pathogen necessary for disease development.