Transgressive segregation, hopeful monsters, and phenotypic selection drove rapid genetic gains and breakthroughs in predictive breeding for quantitative resistance to Macrophomina in strawberry.

Two decades have passed since the strawberry (Fragaria x ananassa) disease caused by Macrophomina phaseolina, a necrotrophic soilborne fungal pathogen, began surfacing in California, Florida, and elsewhere. This disease has since become one of the most common causes of plant death and yield losses in strawberry. The Macrophomina problem emerged and expanded in the wake of the global phase-out of soil fumigation with methyl bromide and appears to have been aggravated by an increase in climate change-associated abiotic stresses. Here we show that sources of resistance to this pathogen are rare in gene banks and that the favorable alleles they carry are phenotypically unobvious. The latter were exposed by transgressive segregation and selection in populations phenotyped for resistance to Macrophomina under heat and drought stress. The genetic gains were immediate and dramatic. The frequency of highly resistant individuals increased from 1% in selection cycle 0 to 74% in selection cycle 2. Using GWAS and survival analysis, we found that phenotypic selection had increased the frequencies of favorable alleles among 10 loci associated with resistance and that favorable alleles had to be accumulated among four or more of these loci for an individual to acquire resistance. An unexpectedly straightforward solution to the Macrophomina disease resistance breeding problem emerged from our studies, which showed that highly resistant cultivars can be developed by genomic selection per se or marker-assisted stacking of favorable alleles among a comparatively small number of large-effect loci.

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.

Exaggerated Plurivory of Macrophomina phaseolina: Accounting for the Large Host Range Claim and the Shifting of Scientific Language.

Macrophomina phaseolina is a plant pathogenic fungus that is frequently described as having a broad host range encompassing more than 500 species. We noticed that citations provided in support of this statement do not actually demonstrate such a broad host range. To elucidate the true documented host range of this fungus, we initiated a literature meta-analysis of 894 publications on M. phaseolina since 1913. We discovered that the first host range summaries did not require Koch's postulates or other experimental demonstrations of pathogenicity. Most of the available early host claims were based on tenuous associations between the fungus and symptoms, sometimes without reporting isolation or morphological examination in vitro. These statements apparently led to a pattern of increasingly exaggerated host range claims, without support from a primary reference, until the claim that M. phaseolina has 500 hosts became common in the early 2000s. At present, the scientific community typically requires Koch's postulates to characterize pathogenicity on a new host. Among all the available literature, we only found primary experimental evidence for M. phaseolina's pathogenicity on 97 hosts; 74 hosts confirmed by Koch's postulates and 23 hosts with all steps from Koch's postulates completed except for recovery of the pathogen from symptomatic tissues. This study demonstrates how scientific concepts can change over time and necessitate changes to historic axioms. We propose that the hyperbole surrounding the host range of M. phaseolina has obscured an accurate depiction of its biology.

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.

Pathotypes of Fusarium oxysporum f. sp. fragariae express discrete repertoires of accessory genes and induce distinct host transcriptional responses during root infection.

Convergent evolution of phytopathogenicity is poorly described, especially among multiple strains of a single microbial species. We investigated this phenomenon with genetically diverse isolates of Fusarium oxysporum f. sp. fragariae (Fof) that cause one of two syndromes: chlorosis and wilting (the 'yellows-fragariae' pathotype), or only wilting (the 'wilt-fragariae' pathotype). We challenged strawberry (Fragaria × ananassa) plants to root infection by five fungal isolates: three yellows-fragariae, one wilt-fragariae and one that is not pathogenic to strawberry. All Fof isolates had chromosome-level assemblies; three were newly generated. The two pathotypes triggered distinct host responses, especially among phytohormone-associated genes; yellows-fragariae isolates strongly induced jasmonic acid-associated genes, whereas the wilt-fragariae isolate primarily induced ethylene biosynthesis and signalling. The differentially expressed genes on fungal accessory chromosomes were almost entirely distinct between pathotypes. We identified an ~150 kbp 'pathogenicity island' that was horizontally transferred between wilt-fragariae strains. This predicted pathogenicity island was enriched with differentially expressed genes whose predicted functions were related to plant infection, and only one of these genes was also upregulated in planta by yellows-fragariae isolates. These results support the conclusion that wilt- and yellows-fragariae cause physiologically distinct syndromes by the expression of discrete repertoires of genes on accessory chromosomes.

The Emergence of Fusarium oxysporum f. sp. apii Race 4 and Fusarium oxysporum f. sp. coriandrii Highlights Major Obstacles Facing Agricultural Production in Coastal California in a Warming Climate: A Case Study.

Currently, Fusarium oxysporum f. sp. apii (Foa) race 4 in celery and F. oxysporum f. sp. coriandrii (Foci) in coriander have the characteristics of emerging infectious plant diseases in coastal southern California: the pathogens are spreading, yield losses can be severe, and there are currently no economical solutions for their control. Celery, and possibly coriander, production in these regions is are likely to have more severe disease from projected warmer conditions in the historically cool, coastal regions. Experimental evidence shows that Foa race 4 causes much higher disease severity when temperatures exceed 21°C. A phylogenomic analysis indicated that Foa race 4, an older, less virulent, and uncommon Foa race 3, and two Foci are closely related in their conserved genomes. These closely related genotypes are somatically compatible. Foa race 4 can also cause disease in coriander and the two organisms readily form "hetero" conidial anastomosis tubes (CAT), further increasing the likelihood of parasexual recombination and the generation of novel pathotypes. A horizontal chromosome transfer event likely accounts for the difference in host range between Foci versus Foa races 4 and 3 because they differ primarily in one or two accessory chromosomes. How Foa race 4 evolved its hyper-virulence is unknown. Although the accessory chromosomes of Foa races 3 and 4 are highly similar, there is no evidence that Foa race 4 evolved directly from race 3, and races 3 and 4 probably only have a common ancestor. Foa race 2, which is in a different clade within the Fusarium oxysporum species complex (FOSC) than the other Foa, did not contribute to the evolution of race 4, and does not form CATs with Foa race 4; consequently, while inter-isolate CAT formation is genetically less restrictive than somatic compatibility, it might be more restricted between FOSC clades than currently known. Other relatively new F. oxysporum in coastal California include F. oxysporum f. sp. fragariae on strawberry (Fof). Curiously, Fof "yellows-fragariae" isolates also have similar core genomes to Foa races 4 and 3 and Foci, perhaps suggesting that there may be core genome factors in this lineage that favor establishment in these soils.

Novel Fusarium wilt resistance genes uncovered in natural and cultivated strawberry populations are found on three non-homoeologous chromosomes.

KEY MESSAGE: Several Fusarium wilt resistance genes were discovered, genetically and physically mapped, and rapidly deployed via marker-assisted selection to develop cultivars resistant to Fusarium oxysporum f. sp. fragariae, a devastating soil-borne pathogen of strawberry. Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae, poses a significant threat to strawberry (Fragaria [Formula: see text] ananassa) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene (FW1) on chromosome 2B that confers resistance to race 1 of the pathogen, and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here, we show that resistance to both races is widespread in natural and domesticated populations and that resistance to race 1 is conferred by partially to completely dominant alleles among loci (FW1, FW2, FW3, FW4, and FW5) found on three non-homoeologous chromosomes (1A, 2B, and 6B). The underlying genes have not yet been cloned and functionally characterized; however, plausible candidates were identified that encode pattern recognition receptors or other proteins known to confer gene-for-gene resistance in plants. High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1-FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5, in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.

Spontaneous changes in somatic compatibility in Fusarium circinatum.

Filamentous fungi grow by the elaboration of hyphae, which may fuse to form a network as a colony develops. Fusion of hyphae can occur between genetically different individuals, provided they share a common allele at loci affecting somatic compatibility. Diversity in somatic compatibility phenotypes reduces the frequency of hyphal fusion in a population, thereby slowing the spread of deleterious genetic elements such as viruses and plasmids, which require direct cytoplasmic contact for transmission. Diverse somatic compatibility phenotypes can be generated by recombining alleles through sexual reproduction, but this mechanism may not fully account for the diversity found in nature. For example, multiple compatibility phenotypes of Fusarium circinatum were shown to be associated with the same clonal lineage, which implies they were derived by a mutation rather than recombination through sexual reproduction. Experimental tests of this hypothesis confirmed that spontaneous changes in somatic compatibility can occur at a frequency between 5 and 8 per million spores. Genomic analysis of F. circinatum strains with altered somatic compatibility revealed no consistent evidence of recombination and supported the hypothesis that a spontaneous mutation generated the observed phenotypic change. Genes known to be involved in somatic compatibility had no mutations, suggesting that mutation occurred in a gene with an as yet unexplored function in somatic compatibility.

Horizontal chromosome transfer and independent evolution drive diversification in Fusarium oxysporum f. sp. fragariae.

The genes required for host-specific pathogenicity in Fusarium oxysporum can be acquired through horizontal chromosome transfer (HCT). However, it is unknown if HCT commonly contributes to the diversification of pathotypes. Using comparative genomics and pathogenicity phenotyping, we explored the role of HCT in the evolution of F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry, with isolates from four continents. We observed two distinct syndromes: one included chlorosis ('yellows-fragariae') and the other did not ('wilt-fragariae'). All yellows-fragariae isolates carried a predicted pathogenicity chromosome, 'chrY-frag ', that was horizontally transferred at least four times. chrY-frag was associated with virulence on specific cultivars and encoded predicted effectors that were highly upregulated during infection. chrY-frag was not present in wilt-fragariae; isolates causing this syndrome evolved pathogenicity independently. All origins of F. oxysporum f. sp. fragariae occurred outside of the host's native range. Our data support the conclusion that HCT is widespread in F. oxysporum, but pathogenicity can also evolve independently. The absence of chrY-frag in wilt-fragariae suggests that multiple, distinct pathogenicity chromosomes can confer the same host specificity. The wild progenitors of cultivated strawberry (Fragaria × ananassa) did not co-evolve with this pathogen, yet we discovered several sources of genetic resistance.

Accuracy of genomic selection and long-term genetic gain for resistance to Verticillium wilt in strawberry.

Verticillium wilt, a soil-borne disease caused by the fungal pathogen Verticillium dahliae, threatens strawberry (Fragaria × ananassa) production worldwide. The development of resistant cultivars has been a persistent challenge, in part because the genetics of resistance is complex. The heritability of resistance and genetic gains in breeding for resistance to this pathogen have not been well documented. To elucidate the genetics, assess long-term genetic gains, and estimate the accuracy of genomic selection for resistance to Verticillium wilt, we analyzed a genetically diverse population of elite and exotic germplasm accessions (n = 984), including 245 cultivars developed since 1854. We observed a full range of phenotypes, from highly susceptible to highly resistant: < 3% were classified as highly resistant, whereas > 50% were classified as moderately to highly susceptible. Broad-sense heritability estimates ranged from 0.70-0.76, whereas narrow-sense genomic heritability estimates ranged from 0.33-0.45. We found that genetic gains in breeding for resistance to Verticillium wilt have been negative over the last 165 years (mean resistance has decreased over time). We identified several highly resistant accessions that might harbor favorable alleles that are either rare or non-existent in modern populations. We did not observe the segregation of large-effect loci. The accuracy of genomic predictions ranged from 0.38-0.53 among years and whole-genome regression methods. We show that genomic selection has promise for increasing genetic gains and accelerating the development of resistant cultivars in strawberry by shortening selection cycles and enabling selection in early developmental stages without phenotyping.

Detection of Fusarium oxysporum f. sp. fragariae from Infected Strawberry Plants.

Isolates of the Fusarium oxysporum species complex have been characterized as plant pathogens that commonly cause vascular wilt, stunting, and yellowing of the leaves in a variety of hosts. F. oxysporum species complex isolates have been grouped into formae speciales based on their ability to cause disease on a specific host. F. oxysporum f. sp. fragariae is the causal agent of Fusarium wilt of strawberry and has become a threat to production as fumigation practices have changed in California. F. oxysporum f. sp. fragariae is polyphyletic and limited genetic markers are available for its detection. In this study, next-generation sequencing and comparative genomics were used to identify a unique genetic locus that can detect all of the somatic compatibility groups of F. oxysporum f. sp. fragariae identified in California. This locus was used to develop a TaqMan quantitative polymerase chain reaction assay and an isothermal recombinase polymerase amplification (RPA) assay that have very high sensitivity and specificity for more than 180 different isolates of the pathogen tested. RPA assay results from multiple field samples were validated with pathogenicity tests of recovered isolates.

Genome Sequence of a California Isolate of Fusarium oxysporum f. sp. lycopersici Race 3, a Fungus Causing Wilt Disease on Tomato.

Fusarium wilt of tomato, caused by the soilborne fungus Fusarium oxysporum f. sp. lycopersici, is an increasingly important disease of tomato. This paper reports the high-quality draft genome assembly of F. oxysporum f. sp. lycopersici isolate D11 (race 3), which consists of 39 scaffolds with 57,281,978 bp (GC content, 47.5%), an N 50 of 4,408,267 bp, a mean read coverage of 99.8×, and 17,682 predicted genes.

Persistence of Fusarium oxysporum f. sp. fragariae in Soil Through Asymptomatic Colonization of Rotation Crops.

Asymptomatic plant colonization is hypothesized to enhance persistence of pathogenic forms of Fusarium oxysporum. However, a correlation between pathogen populations on living, asymptomatic plant tissues and soilborne populations after tillage has not been demonstrated. Living and dead tissues of broccoli, lettuce, spinach, wheat, cilantro, raspberry, and strawberry plants grown in soil infested with F. oxysporum f. sp. fragariae (the cause of Fusarium wilt of strawberry) were assayed to quantify the incidence of infection and extent of colonization by this pathogen. All crops could be infected by F. oxysporum f. sp. fragariae but the extent of colonization varied between plant species. Pathogen population densities on nonliving crown tissues incorporated into the soil matrix were typically greater than those observed on living tissues. Crop-dependent differences in the inoculum density of F. oxysporum f. sp. fragariae in soil were only observed after decomposition of crop residue. Forty-four weeks after plants were incorporated into the soil, F. oxysporum f. sp. fragariae soil population densities were positively correlated with population densities on plant tissue fragments recovered at the same time point. Results indicate that asymptomatic colonization can have a significant, long-term impact on soilborne populations of Fusarium wilt pathogens. Cultural practices such as crop rotation should be leveraged to favor pathogen population decline by planting hosts that do not support extensive population growth on living or decomposing tissues.

Finished Genome Sequences of Xanthomonas fragariae, the Cause of Bacterial Angular Leaf Spot of Strawberry.

Xanthomonas fragariae is a foliar pathogen of strawberry that is of significant concern to nursery production of strawberry transplants and field production of strawberry fruit. Long-read sequencing was employed to generate finished genomes for two isolates (each with one chromosome and two plasmids) from symptomatic plants in northern California.

Inhibition of Xanthomonas fragariae, Causative Agent of Angular Leaf Spot of Strawberry, through Iron Deprivation.

In commercial production settings, few options exist to prevent or treat angular leaf spot (ALS) of strawberry, a disease of economic importance and caused by the bacterial pathogen Xanthomonas fragariae. In the process of isolating and identifying X. fragariae bacteria from symptomatic plants, we observed growth inhibition of X. fragariae by bacterial isolates from the same leaf macerates. Identified as species of Pseudomonas and Rhizobium, these isolates were confirmed to suppress growth of X. fragariae in agar overlay plates and in microtiter plate cultures, as did our reference strain Pseudomonas putida KT2440. Screening of a transposon mutant library of KT2440 revealed that disruption of the biosynthetic pathway for the siderophore pyoverdine resulted in complete loss of X. fragariae antagonism, suggesting iron competition as a mode of action. Antagonism could be replicated on plate and in culture by addition of purified pyoverdine or by addition of the chelating agents tannic acid and dipyridyl, while supplementing the medium with iron negated the inhibitory effects of pyoverdine, tannic acid and dipyridyl. When co-inoculated with tannic acid onto strawberry plants, X. fragariae's ability to cause foliar symptoms was greatly reduced, suggesting a possible opportunity for iron-based management of ALS. We discuss our findings in the context of 'nutritional immunity,' the idea that plant hosts restrict pathogen access to iron, either directly, or indirectly through their associated microbiota.