Genomic and transcriptomic insights into Raffaelea lauricola pathogenesis.

BACKGROUND: Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate. RESULTS: Relative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection. CONCLUSIONS: Through these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.

Pathogenicity, Symptom Development, and Colonization of Metrosideros polymorpha by Ceratocystis lukuohia.

Extensive mortality of Metrosideros polymorpha (`ohi`a) trees has been associated with Ceratocystis spp. on Hawai`i Island and was named rapid `ohi`a death (ROD). Both C. lukuohia and C. huliohia have been associated with ROD, although C. lukuohia appears to be the more important pathogen. Crown observations and dissections of forest trees either wound-inoculated with, or naturally infected by, C. lukuohia were conducted to confirm pathogenicity and document patterns of host colonization. In pathogenicity trials, one of three and two of three trees inoculated with the fungus in February and August, respectively, exhibited crown wilt symptoms at 92 and 69 days after inoculation. Extensive, radial, black staining of the sapwood was found in main stems, while no crown wilt or xylem staining was found in control trees. Xylem staining, necrotic phloem, and fungus presence was noted in six trees inoculated in May to June and harvested 37 to 42 days later, and these observations were compared with those in two naturally infected trees felled in early August. Contiguous xylem staining was found in the main stems and into crowns of all diseased trees, while discontinuous streaks of xylem staining extended into the main forks and side branches. Necrotic phloem associated with xylem staining occurred on the lower stems of inoculated trees. Aside from the necrotic phloem and radial staining of the sapwood, symptom development in `ohi`a infected with C. lukuohia is similar to other systemic wilt diseases on hardwood trees. We propose Ceratocystis wilt of `ohi`a as the official name of the disease.

The Progressive Spread of the Vascular Wilt Like Pathogen of Calophyllum Detected in Ranomafana National Park, Madagascar.

Pathogens are threatening crops worldwide, but little attention has been given to the threat to tree species in undisturbed rainforest. This communication reports the first case of a tree die off caused by a "wilt" in Madagascar. In 2016 while monitoring monthly tree phenology of Ranomafana National Park (RNP), the Centre ValBio research station observed that many Calophyllum adult trees had brown wilted leaves. There are three species of Calophyllum in this rainforest, C. paniculatum, C. drouhardii, and C. milvum, and all three have contracted this pathogen. Our goal was to document the spead of this suspected wilt in Calophyllum trees and determine if site, elevation and DBH had an influence on tree mortality. In 2019 we conducted an inventory of all Calophyllum trees in RNP and 42% of the observed trees were dead. The species with the highest mortality was C. paniculatum, with 53% of trees dead, followed by C. milvum with 18%, and C. drouhardii with only 2% of surveyed trees dead. Bark beetle traces were observed in all dead Calophyllum trees. Tree death caused by this suspected fungal pathogen has spread across a major river in the area and has been found at mid and high elevations. Our results show that C. paniculatum trees with a larger DBH have a higher mortality risk. Our report highlights the importance of fighting invasive pathogens that threaten protected ecosystems.

Putative Novel Effector Genes Revealed by the Genomic Analysis of the Phytopathogenic Fungus Fusarium oxysporum f. sp. physali (Foph) That Infects Cape Gooseberry Plants.

The vascular wilt disease caused by the fungus Fusarium oxysporum f. sp. physali (Foph) is one of the most limiting factors for the production and export of cape gooseberry (Physalis peruviana) in Colombia. A transcriptomic analysis of a highly virulent strain of F. oxysporum in cape gooseberry plants, revealed the presence of secreted in the xylem (SIX) effector genes, known to be involved in the pathogenicity of other formae speciales (ff. spp.) of F. oxysporum. This pathogenic strain was classified as a new f. sp. named Foph, due to its specificity for cape gooseberry hosts. Here, we sequenced and assembled the genome of five strains of F. oxysporum from a fungal collection associated to the cape gooseberry crop (including Foph), focusing on the validation of the presence of SIX homologous and on the identification of putative effectors unique to Foph. By comparative and phylogenomic analyses based on single-copy orthologous, we found that Foph is closely related to F. oxysporum ff. spp., associated with solanaceous hosts. We confirmed the presence of highly identical homologous genomic regions between Foph and Fol that contain effector genes and identified six new putative effector genes, specific to Foph pathogenic strains. We also conducted a molecular characterization using this set of putative novel effectors in a panel of 36 additional stains of F. oxysporum including two of the four sequenced strains, from the fungal collection mentioned above. These results suggest the polyphyletic origin of Foph and the putative independent acquisition of new candidate effectors in different clades of related strains. The novel effector candidates identified in this genomic analysis, represent new sources involved in the interaction between Foph and cape gooseberry, that could be implemented to develop appropriate management strategies of the wilt disease caused by Foph in the cape gooseberry crop.

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts.

Chitin-binding lysin motif (LysM) effectors contribute to the virulence of various plant-pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta.

Seed Transmission of Cephalosporium gramineum in Winter Wheat.

Although isolation of Cephalosporium gramineum from wheat (Triticum aestivum) seed has been reported, development of Cephalosporium stripe in plants from infected seed has not been demonstrated experimentally. Winter wheat seed was collected from three experimental field plots where Cephalosporium stripe was present, and C. gramineum was isolated from the seed following surface-disinfection and incubation on a semi-selective medium. C. gramineum was isolated from 0.10 to 0.88% of seed from 11 of 12 cultivars in a field experiment at Pullman, WA, and from 0.10 to 0.30% of seed from 3 of 4 genotypes in a field experiment at Fort Hall, ID; differences among cultivars were not significant in either experiment. C. gramineum was isolated from 0.35 and 0.55% of cv. Stephens plants with no symptoms and severe symptoms, respectively, from a uniform seeding in Pullman. Seed of the four genotypes from Fort Hall and Stephens from Pullman were grown under controlled environment in a soilless potting mix with no added inoculum and in which C. gramineum was not detected. Symptoms of Cephalosporium stripe developed in 0.08 and 0.17% of Stephens and breeding line 87-00314A plants, respectively, from Fort Hall, and from 0.18 and 0.55% of Stephens plants with no symptoms and severe symptoms, respectively. Although development of Cephalosporium stripe in plants grown from seed lots harvested from diseased plants was low, infected seed can provide an important source of inoculum for introducing the pathogen and initiating epidemics in areas where the pathogen did not occur previously.

How Phytohormones Shape Interactions between Plants and the Soil-Borne Fungus Fusarium oxysporum.

Plants interact with a huge variety of soil microbes, ranging from pathogenic to mutualistic. The Fusarium oxysporum (Fo) species complex consists of ubiquitous soil inhabiting fungi that can infect and cause disease in over 120 different plant species including tomato, banana, cotton, and Arabidopsis. However, in many cases Fo colonization remains symptomless or even has beneficial effects on plant growth and/or stress tolerance. Also in pathogenic interactions a lengthy asymptomatic phase usually precedes disease development. All this indicates a sophisticated and fine-tuned interaction between Fo and its host. The molecular mechanisms underlying this balance are poorly understood. Plant hormone signaling networks emerge as key regulators of plant-microbe interactions in general. In this review we summarize the effects of the major phytohormones on the interaction between Fo and its diverse hosts. Generally, Salicylic Acid (SA) signaling reduces plant susceptibility, whereas Jasmonic Acid (JA), Ethylene (ET), Abscisic Acid (ABA), and auxin have complex effects, and are potentially hijacked by Fo for host manipulation. Finally, we discuss how plant hormones and Fo effectors balance the interaction from beneficial to pathogenic and vice versa.

Seasonal Dispersal of the Oak Wilt Fungus by Colopterus truncatus and Carpophilus sayi in Minnesota.

Sap beetles (Nitidulidae) are considered important overland vectors of the oak wilt pathogen, Ceratocystis fagacearum, in the north central United States. Colopterus truncatus and Carpophilus sayi are thought to be the principal sap beetle vectors in Minnesota. Field studies using windoriented funnel traps baited with aggregation pheromones of the insects were conducted during 2 years in east central Minnesota. The studies compared temporal flight dynamics of Colopterus truncatus and Carpophilus sayi from April through October, measured the proportion of dispersing adults of each species carrying viable pathogen propagules, and estimated the populations of contaminated dispersing beetles in oak (Quercus spp.) stands with and without oak wilt. Abundance of Colopterus truncatus peaked in either April or May, but the pathogen was most frequently isolated (20 to 45%) from beetles obtained from oak wilt sites from July through September. However, the highest contaminated insect population (CIP) generally occurred during April and May. Abundance of Carpophilus sayi peaked in October of both years, but the pathogen was most commonly isolated from beetles collected during May and June. The CIP was highest in June. The infective insect potential, as indexed by CIP, was greatest for both species in the spring and was greater for Colopterus truncatus than for Carpophilus sayi.