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.

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.

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.

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.

First Report of Watermelon Mosaic Virus Naturally Infecting Coriander (Coriandrum sativum) and causing a leaf mottling disease in California.

Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) is a species of considerable economic importance to cucurbit crops worldwide (Keinath et al. 2017). This virus has a wide host range that includes more than 170 plant species from 27 families (Dong et al. 2017; Lecoq et al. 2011). In 2018, leaves of coriander (Coriandrum sativum) plants in a student garden (C-SG) at UC Davis, and in a home garden in Davis, CA (C-Pet) (~1.1 miles apart) showed symptoms of light green mottling and crumpling. Symptomatic leaves from each location were weakly positive with the general potyvirus immunostrip test (Agdia, Elkhart, IN). In RT-PCR tests with total RNA extracts (RNeasy Plant Mini Kit Qiagen, Germantown, MD) of these leaves and the potyvirus degenerate primer pair CIFor/CIRev (Ha et al. 2008), the expected-size ~0.7 kb fragment was amplified. These fragments were gel-purified and sequenced, and a BLASTn search revealed highest identities of 91.6% (C-SG) and 97.9% (C-Pet) with the sequence of an isolate of WMV from watermelon in the U.S. (TX29, KU246036). Thus, these isolates are designated WMV-C-SG-18 and WMV-C-Pet-18. Mechanical inoculation experiments were next performed with sap prepared with symptomatic coriander leaf tissue in ice-cold 0.01 M phosphate buffer (pH 7.0) in a 1:4 wt/vol ratio. First, to obtain pure isolates, sap was inoculated onto celite-dusted leaves of Chenopodium quinoa plants (3-4 leaf stage). As expected for WMV, leaves inoculated with sap of each isolate developed chlorotic local lesions ~9 d post-inoculation (dpi) (Moreno et al. 2004). One lesion for each isolate was excised, ground in phosphate buffer, and the sap was mechanically inoculated onto leaves of Nicotiana benthamiana plants. By ~14 dpi, newly emerged leaves showed mild mottling and crumpling, and were weakly positive with the potyvirus immunostrip test. To confirm that these plants were only infected with WMV, total RNA was extracted from symptomatic leaves and used for high throughput sequencing (HTS) (Soltani et al. 2021) at the Foundation Plant Services at UC Davis. The HTS analyses revealed infection with only WMV, i.e., no other viral contigs were identified, and allowed for determination of the complete sequences (~10,000 nt) of WMV [US-CA-C-SG-18] and WMV [US-CA-C-Pet-18] with GenBank accession numbers: OM746964 and OM746965, respectively. Whole genome sequence comparisons revealed that the sequences are 99.0% identical, and 97.3% identical to the sequence of WMV TX29. Sap from symptomatic N. benthamiana leaves infected with each isolate was mechanical inoculated onto leaves of coriander plants (30-35 d old). Newly emerged leaves developed epinasty, crumpling and light green mottling by 14 dpi, and WMV infection was confirmed by RT-PCR with the WMV-specific primer pair WMV-UNI-1F and WMV-UNI-1R (Kim et al. 2019). Thus, Koch's postulates were fulfilled for this leaf mottling disease of coriander. Furthermore, the isolates from coriander induced stunting and distortion and mosaic in leaves of melon, pumpkin and squash plants by 7 dpi, whereas watermelon plants developed stunting and small leaves with mild mottling by 20 dpi. Similar results were obtained with sap prepared from infected coriander leaves. Thus, infected coriander plants are a potential inoculum source for cucurbits via several aphid vectors (Keinath et al. 2017). This is the first report of a mottle disease of coriander caused by WMV, and adds to the wide host range of the virus.

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.

Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.

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.

Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii.

BACKGROUND: Members of the F. oxysporium species complex (FOSC) in the f. sp. apii (Foa) are pathogenic on celery and those in f. sp. coriandrii (Foci) are pathogenic on coriander (=cilantro). Foci was first reported in California in 2005; a new and highly aggressive race 4 of Foa was observed in 2013 in California. Preliminary evidence indicated that Foa can also cause disease on coriander, albeit are less virulent than Foci. Comparative genomics was used to investigate the evolutionary relationships between Foa race 4, Foa race 3, and the Foci, which are all in FOSC Clade 2, and Foa race 2, which is in FOSC Clade 3. RESULTS: A phylogenetic analysis of 2718 single-copy conserved genes and mitochondrial DNA sequence indicated that Foa races 3 and 4 and the Foci are monophyletic within FOSC Clade 2; these strains also are in a single somatic compatibility group. However, in the accessory genomes, the Foci versus Foa races 3 and 4 differ in multiple contigs. Based on significantly increased expression of Foa race 4 genes in planta vs. in vitro, we identified 23 putative effectors and 13 possible pathogenicity factors. PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified. Finally, mixtures of conidia that were pre-stained with different fluorochromes indicated that Foa race 4 formed conidial anastomosis tubes (CATs) with Foci. Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other. CONCLUSIONS: There was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4; Foa race 2 and 4 are CAT-incompatible. Although Foa races 3 and 4 and the Foci are closely related, there is no evidence that either Foci contributed to the evolution of Foa race 4, or that Foa race 4 was the recent recipient of a multi-gene chromosomal segment from another strain. However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range.

Pharmacological ablation of the airway smooth muscle layer-Mathematical predictions of functional improvement in asthma.

Airway smooth muscle (ASM) plays a major role in acute airway narrowing and reducing ASM thickness is expected to attenuate airway hyper-responsiveness and disease burden. There are two therapeutic approaches to reduce ASM thickness: (a) a direct approach, targeting specific airways, best exemplified by bronchial thermoplasty (BT), which delivers radiofrequency energy to the airway via bronchoscope; and (b) a pharmacological approach, targeting airways more broadly. An example of the less well-established pharmacological approach is the calcium-channel blocker gallopamil which in a clinical trial effectively reduced ASM thickness; other agents may act similarly. In view of established anti-proliferative properties of the macrolide antibiotic azithromycin, we examined its effects in naive mice and report a reduction in ASM thickness of 29% (p < .01). We further considered the potential functional implications of this finding, if it were to extend to humans, by way of a mathematical model of lung function in asthmatic patients which has previously been used to understand the mechanistic action of BT. Predictions show that pharmacological reduction of ASM in all airways of this magnitude would reduce ventilation heterogeneity in asthma, and produce a therapeutic benefit similar to BT. Moreover there are differences in the expected response depending on disease severity, with the pharmacological approach exceeding the benefits provided by BT in more severe disease. Findings provide further proof of concept that pharmacological targeting of ASM thickness will be beneficial and may be facilitated by azithromycin, revealing a new mode of action of an existing agent in respiratory medicine.

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.

Accurate real time localization tracking in a clinical environment using Bluetooth Low Energy and deep learning.

Deep learning has started to revolutionize several different industries, and the applications of these methods in medicine are now becoming more commonplace. This study focuses on investigating the feasibility of tracking patients and clinical staff wearing Bluetooth Low Energy (BLE) tags in a radiation oncology clinic using artificial neural networks (ANNs) and convolutional neural networks (CNNs). The performance of these networks was compared to relative received signal strength indicator (RSSI) thresholding and triangulation. By utilizing temporal information, a combined CNN+ANN network was capable of correctly identifying the location of the BLE tag with an accuracy of 99.9%. It outperformed a CNN model (accuracy = 94%), a thresholding model employing majority voting (accuracy = 95%), and a triangulation classifier utilizing majority voting (accuracy = 95%). Future studies will seek to deploy this affordable real time location system in hospitals to improve clinical workflow, efficiency, and patient safety.