Searching for laurel wilt resistance in avocados of Mexican and Mexican-Guatemalan ancestry.

Laurel wilt (LW), a lethal vascular disease caused by the ambrosia fungus Harringtonia lauricola, has severely reduced avocado (Persea americana Mill.) production in Florida and decimated populations of native lauraceous trees across twelve U.S. states. All commercial avocado cultivars evaluated to date succumb to the disease, but the speed at which the tree declines varies. Cultivars with West Indian (WI) genetic background develop severe symptoms faster than those with Mexican (M) and Guatemalan (G) pedigree. Genetic resistance to LW is urgently needed, as management relies on costly cultural practices. We screened non-commercial open-pollinated progenies from 19 Mexican and 6 Mexican x Guatemalan accessions, and 2 Guatemalan x West Indian cultivars recognized as tolerant by growers. From the five disease response parameters evaluated, the final disease intensity index and disease severity on the last evaluation day were used to classify genotypes. A wide variability of responses was observed within and among families. Symptomatic plants were present in all families, while some individuals within 15 families remained asymptomatic. 'Colín V-33' (M×G) family was identified as tolerant, and Libres 3 (M), Bladimiro M-06 (M) 'Colinmex' (M×G), 'Collinson' (G×WI), Libres 5 (M) and Rag-13 (M) families were classified as moderately tolerant. This is the first-time tolerance to laurel wilt in avocado is formally reported, though surviving material needs to be propagated for response validation and field testing. Identifying tolerant accessions can help understand the underlying mechanisms and provide breeders with genetic resources for the future incorporation of resistance genes into commercial cultivars.

Essential Oils for Managing Anthracnose in Mango (Mangifera indica): Laboratory Results Do Not Translate into Field Efficacy.

Essential oil-based products with broad plant disease control claims are commercially available and may be a practical alternative to copper fungicides for crop protection in organic mango orchards. We evaluated the disease control efficacy and crop safety of thyme oil, savory oil, and tree tea oil through replicated in vitro, in vivo (detached leaf and potted trees), and field assays. Three Colletotrichum species associated with mango anthracnose were tested in vitro, whereas only C. siamense was used for in vivo assays. Within the range of concentrations tested in vitro (62.5 to 2,000 µl active ingredient [a.i.]/liter), thyme and savory oil displayed fungicidal activity, whereas no fungistatic or fungicidal activity was observed with tea tree oil. In the in vivo assays, none of the treatments based on a preventive application rate of thyme (1,150 µl a.i./liter), savory (2,000 µl a.i./liter), or tea tree oil (342 µl a.i./liter) were effective in preventing the development of anthracnose on wounded and artificially inoculated leaves. Although field applications of thyme or tea tree oil did not result in phytotoxicity or negative impacts on fruit yield, they were ineffective in reducing the incidence and severity of naturally occurring anthracnose. Applications of copper hydroxide approved for organic agriculture were effective in controlling anthracnose in the field, and no added benefits were found by premixing this compound with thyme oil. Results indicate that essential oil products based on thyme or tea tree oil are inefficient at controlling anthracnose in mangoes.

Susceptibility of centipede tongavine (Epipremnum pinnatum) commercially grown in nurseries in Florida to aroid leaf rust (Pseudocerradoa paullula).

Epipremnum pinnatum (L.) Engl., (Araceae, Monocots) known as dragon-tail plant or centipede tongavine, is the most cultivated aroid species worldwide (Boyce 2004). In 2022, symptomatic dragon-tail plants, collected from plant nurseries in south Florida (e-Xtra Fig.1). Symptoms included round leaf spots often with a yellow halo and erupting pustules mainly distributed in the underside of the leaves. Visits to the nurseries revealed a 60% incidence of approximability 50 mature plants, with some leaves showing up to 30% of tissue damage. The putative pathogen was identified morphologically as Pseudocerradoa paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese (Pucciniaceae, Basidiomycota) (Ebinghaus et al. 2022), characterized by the production of pseudosuprastomatal uredinia with globose to subglobose urediniospores, light-brown, echinulate (1 µm height), 24-31 µm diam with thick walls, 1.5-2.5 µm in height (n=30). Identical morphological features reported by Urbina et al. (2023) (e-Xtra Fig. 1). PCR amplification followed by Sanger sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal RNA genes (Aime 2006) together with LSU internal species specific primer (Urbina et al. 2023) were used to confirm the identification of the pathogen (GenBank ON887194-ON887196). MegaBlast (Chen et al. 2015) searches resulted in a >99% sequence similarity to a P. paullula specimen collected in Florida (2019-101665, GenBank ON887197). Host identification was made by using the Ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL. GenBank ON887186, ON887187) and Maturase K (matK) loci (GenBank ON887190, ON887191) (Fazekas et al. 2012). Both barcodes resulted in >99.13% sequence similarity to voucher J.R. Abbott 24912 FLAS (GenBank GU135198 and GU135036, respectively). Symptomatic dried specimens were deposited in the Plant Industry Herbarium (PIHG 16229 - 16232). Koch's postulates were fulfilled using urediniospores collected from an infected E. pinnatum sample that was kept in darkness at 4°C for seven days until inoculation. Eight potted dragon-tail plants were inoculated by hand rubbing urediniospores against upper and lower leaf surfaces and three plants were used as controls. All plants were misted with sterile water and covered with plastic bags (23 °C, >90% RH, 12/12 h daylight). Bags were removed 48 h after inoculation, plants were set in a climate-controlled greenhouse (~30 °C, ~65% RH, 12/12 h light cycle) and monitored daily for symptoms. Chlorotic spots appeared after 10 days, and pustules after 25 days while the non-inoculated controls remained symptomless. Aroid leaf rust is known to infect several aroid species, including dragon-tail (Shaw 1995), which some varieties capable to outdoors in USDA 9a hardiness zones (Wunderlin et al. 2023), but the rust fungus has not been observed on any species of Epipremnum in the landscape yet, suggesting that its susceptibility could be driven by plant growth conditions that favor pathogen infection (e.g., excess of humidity and nutrients, dense planting, overhead irrigation, etc.). Here we encourage dragon-tail plant growers to be aware of its susceptibility to P. paullula and to stay vigilant of the culture conditions to avoid plants from getting infected with this airborne pathogen.

Genome Sequence Resource of the Avocado Scab Pathogen Elsinoe perseae.

We present a draft genome sequence of Elsinoe perseae, an economically important plant pathogen of commercially grown avocados. The 23.5-Mb assembled genome consists of 169 contigs. This report represents an important genomic resource to guide future research aimed at understanding the genetic interactions of E. perseae with its host.

Genome Resources for the Colletotrichum gloeosporioides Species Complex: 13 Tree Endophytes from the Neotropics and Paleotropics.

Thirteen draft genome assemblies are presented for four Colletotrichum gloeosporioides complex species, namely, Colletotrichum aeschynomenes, Colletotrichum asianum, Colletotrichum fructicola, and Colletotrichum siamense, which were isolated from tropical tree hosts as endophytes.

Detection of aroid leaf rust Pseudocerradoa paullula on Swiss cheese plant Monstera deliciosa in the continental USA.

Monstera deliciosa Liebm. (Araceae, Monocots), sometimes referred to as Swiss cheese plant, is one of the most common aroids used as an indoor and landscape ornamental plant (Cedeño et al. 2020). Production of M. deliciosa and other closely related Araceae species represents an important sector of the ornamental nursery business worldwide. Swiss cheese plant is believed to have originated in the tropical forests of southern Mexico, where its fruit is considered a delicacy due to its sweet, exotic flavor (Cedeño et al. 2020). Since 2019, symptomatic Monstera plants from two plant nurseries and residential properties in South Florida were submitted for disease diagnosis to the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS-DPI) in Gainesville, Florida, and to the University of Florida, Tropical Research and Education Center Plant Clinic in Homestead, Florida. Symptoms included small chlorotic spots on the leaf surface, which expanded and became brown to reddish-brown often with a yellow halo and produced uredinia with abundant urediniospores. The pathogen was identified morphologically as the rust fungus Pseudocerradoa (=Puccinia) paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese (Pucciniaceae, Basidiomycota) (Ebinghaus et al. 2022), characterized by the production of pseudosuprastomatal uredinia. Uredinospores light-brown and globose, echinulate (1 µm height), reddish to light brown, 24 - 31 µm diameter, with thick walls, 1.5 - 2.5 µm height (n=15). Teliospores 2-celled, light-yellow and ellipsoidal, 23 - 28 × 19 - 24 µm (n =15) were observed in sori appearing as dark-brown leaf spots on the adaxial side of the leaves (e-Xtra Fig. 1). Molecular characterization of the fungal pathogen was based on the small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) of the ribosomal RNA genes (Aime 2006) with the addition of a LSU internal primer specific for the rust species Ppaullula_int-forward 5'ATAGTTATTGGCTTTGATTTACA-3' designed in this study to increase the quality and the sequence read length due to a 3'- ~21-Ts-homopolymer (e-Xtra Fig. 2) (GenBank accession number ON887196, ON887197, OQ275200, OQ275201). In addition to morphological identification, the host plant was identified using the Ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and Maturase K (matK) genes (Fazekas et al. 2012) (GenBank accession numbers ON887189, ON887193, respectively). MegaBlast searches confirmed the morphological identification with 100% identity to M. deliciosa vouchers GQ436772 and MK206496, respectively (Chen et al. 2015). Dried specimens were deposited in the Plant Industry Herbarium Gainesville (PIHG 16226, 16227, 17154, 17155). Molecular identification of the rust pathogen P. paullula was carried out through megaBlast (Chen et al. 2015) searches together with a phylogenetic analysis performed in RAxML v8 (Stamatakis 2014) (e-Xtra Fig. 3). Koch's postulates were performed by using urediniospores, collected from an infected sample and were kept for 7 days at 4 C, as an inoculum source. Healthy rooted M. deliciosa plants were inoculated by rubbing the inoculum on both leaf surfaces at >90% RH, room temperature, 12/12 light cycle. After the incubation period (48 h), plants were placed in a climate-controlled greenhouse and watered twice a week, ~30 C, ~65 RH, 12/12 light cycle. After three weeks, all inoculated plants developed symptoms resembling those observed on the samples submitted for disease diagnosis. Controls did not show symptoms. Spores from the pustules of inoculated plants were identified as P. paullula by both morphology and molecular means. The genus Pseudocerradoa comprises P. paullula and its sister species P. rhaphidophorae (Syd.) M. Ebinghaus & Dianese. Both species can be distinguished by size and coloration of urediniospores and their host range within the Araceae. Pseudocerradoa rhaphidophorae produces smaller urediniospores and only occurs on Rhaphidophora species (Shaw 1995). Pseudocerradoa paullula is not considered fully established in Florida, since the host distribution is mainly restricted to indoors and M. deliciosa is rarely used as an outdoor ornamental (Wunderlin et al. 2023). Here we name the disease caused by P. paullula as "aroid leaf rust", due to its ability to infect several species in this plant family. Other closely related hosts reported as susceptible to this pathogen are Monstera standleyana G.S.Bunting (as M.s. cv. variegata), Monstera adansonii var. laniata (Schott) Mayo & I.M. Andrade, Monstera subpinnata (Schott) Engl., Typhonodorum lindleyanum Schott, and Stenospermation sp. (Shaw 1991, 1992, 1995). To date, the aroid leaf rust was only known from Australia, China, Japan, Malaysia, and Philippines (Lee et al. 2012; Shaw 1991). Based on our review, P. paullulla was intercepted once from Malaysia in 2014 at the port of Los Angeles, USA (BPI voucher 893085). This present study reports the establishment of P. paullula in Florida, USA infecting M. deliciosa.

Native ectomycorrhizal fungi from the endangered pine rocklands are superior symbionts to commercial inoculum for slash pine seedlings.

The south Florida pine rocklands is a critically endangered, fire-dependent ecosystem dominated by the overstory tree Pinus densa (South Florida slash pine). Because pine recruitment in this ecosystem has proven problematic, restoration efforts need to include replanting slash pine trees. Even though ectomycorrhizal fungi are known to be critical symbionts of young pines and are necessary for the development of healthy pines, virtually nothing is known about these mutualists and their role in pine establishment and survival in the pine rocklands. One approach to improve pine establishment is to grow seedlings in a nursery before outplanting, facilitating early associations with ectomycorrhizae, and therefore improving seedling health. In this study, we compared health metrics (height, stem diameter, final needle length, root length, root colonization, needle greenness, root volume, and root:shoot ratio) of seedlings grown in soil amended with five commercially available mycorrhizal inocula versus field soil collected from three pine rockland fragments. Seedlings grown with native field soil from the pine rocklands generally performed better than those grown with commercial inoculum in all metrics except root length. According to their labels, each commercial inoculum contained between 4 and 10 ectomycorrhizal fungi species. However, no ectomycorrhizal fungi were recovered from two of the inoculum products and only three ectomycorrhizal fungi in total were recovered from the other three products. In contrast, seedlings grown with field soil are associated with ten ectomycorrhizal species. Our results highlight the importance of incorporating native ectomycorrhizal fungi into pine seedling replanting as part of restoration efforts in the pine rocklands.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Lasiodiplodia iraniensis, a New Causal Agent of Tuber Rot on Yam (Dioscorea Species) Imported into the United States and Implications for Quarantine Decisions.

One negative consequence of international trade of agricultural commodities is the inadvertent global spread of crop diseases. Yam (Dioscorea spp.) is a staple food crop in many countries and is traded globally. Most of the commercially traded yams in the United States are imported. In late 2020, samples of yam tubers from a commercial facility were submitted to the plant diagnostic clinic at the UF/IFAS Tropical Research and Education Center in Homestead, Florida. Samples showed rotten symptoms and were drawn from lots that were marked to be destroyed because the source of the rotting symptoms was unknown. Preliminary isolation showed that a fungus was consistently associated with the symptoms and was confirmed in the subsequent pathogenicity test as the causal agent. The fungus grew profusely on potato dextrose agar (PDA) with highly melanized hyphae. Matured conidia showed longitudinal striations. Based on its growth pattern and morphology, it was suspected that this fungus may be in the genus Lasiodiplodia. DNA-based identification using partial sequences of the internal transcribed spacer (ITS), ß-tubulin (TUB2), 28S rDNA (LSU), and elongation factor alpha (EF1-α) genes confirmed the identity of the isolates as Lasiodiplodia iraniensis Abdollahz., Zare & A.J.L. Phillips (synonym: L. iranensis). This is the first report of L. iraniensis affecting yam and has implications for international trade. This finding will provide an important foundation for making quarantine decisions to prevent spread of this disease.

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Prevalence and Epidemics of Neoscytalidium Stem and Fruit Canker on Pitahaya (Hylocereus spp.) in South Florida.

Since the 2000s, production of pitahaya (Hylocereus spp.) has increased significantly in South Florida. However, very limited information is available on the main diseases affecting this crop, particularly in regard to disease epidemiology and economic impact on the commodity. In this study, we surveyed five local pitahaya orchards and documented the most prevalent diseases and their causal agents. Three genera of fungal pathogens (Neoscytalidium, Alternaria, and Colletotrichum) were the major groups associated with symptoms on pitahaya cladodes (stems) during the early growing season. Among these, N. dimidiatum was identified as the most prevalent pathogen, with an overall isolation frequency of 29.8% (range, 13.9 to 47.2%). Hence, the temporal progress of N. dimidiatum stem canker infection was monitored and the relationship between stem canker intensity (incidence and severity) and fruit canker incidence was investigated. A significant positive correlation was found between fruit canker incidence and the standardized area under the disease incidence or severity curve on cladodes, suggesting that high stem canker intensity in the early season may contribute to high fruit canker incidence and thereby impact the aesthetic and market value of fruits. In vitro assays showed that both conidial germination and mycelial growth of N. dimidiatum are positively correlated with increasing temperature, with a maximum growth area at 32°C. This finding suggests a higher risk of infection, under an environment with high temperatures, which is common in South Florida. Data obtained in this study represent baseline knowledge for the future development of integrative management programs for controlling major diseases of pitahaya in South Florida.

Fungal functional ecology: bringing a trait-based approach to plant-associated fungi.

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro-organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function. Trait-based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and -omics-based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (FunFun ). FunFun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait-based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.

Exploration of stem endophytic communities revealed developmental stage as one of the drivers of fungal endophytic community assemblages in two Amazonian hardwood genera.

Many aspects of the dynamics of tropical fungal endophyte communities are poorly known, including the influence of host taxonomy, host life stage, host defence, and host geographical distance on community assembly and composition. Recent fungal endophyte research has focused on Hevea brasiliensis due to its global importance as the main source of natural rubber. However, almost no data exist on the fungal community harboured within other Hevea species or its sister genus Micrandra. In this study, we expanded sampling to include four additional Hevea spp. and two Micrandra spp., as well as two host developmental stages. Through culture-dependent and -independent (metagenomic) approaches, a total of 381 seedlings and 144 adults distributed across three remote areas within the Peruvian Amazon were sampled. Results from both sampling methodologies indicate that host developmental stage had a greater influence in community assemblage than host taxonomy or locality. Based on FunGuild ecological guild assignments, saprotrophic and mycotrophic endophytes were more frequent in adults, while plant pathogens were dominant in seedlings. Trichoderma was the most abundant genus recovered from adult trees while Diaporthe prevailed in seedlings. Potential explanations for that disparity of abundance are discussed in relation to plant physiological traits and community ecology hypotheses.

Endophytes from Wild Rubber Trees as Antagonists of the Pathogen Corynespora cassiicola.

The Corynespora leaf fall disease of rubber trees, caused by the necrotrophic fungus Corynespora cassiicola, is responsible for important yield losses in Asian and African plantations, whereas its impact is negligible in South America. The objective of this study was to identify potential antagonists of C. cassiicola among fungal endophytes (i.e., Pestalotiopsis, Colletotrichum, and Trichoderma spp.) isolated from wild and cultivated rubber trees distributed in the Peruvian Amazon. We first tested the endophytes in dual in vitro confrontation assays against a virulent C. cassiicola isolate (CCP) obtained from diseased rubber trees in the Philippines. All Trichoderma isolates overran the CCP colony, suggesting some antagonistic mechanism, while species from the other genera behaved as mutual antagonists. Trichoderma isolates were then tested through antibiosis assays for their capacity to produce growth-inhibiting molecules. One isolate (LA279), recovered as an endophyte from a wild Hevea guianensis specimen and identified as Trichoderma koningiopsis, showed significant antibiosis capacity. We demonstrated that LA279 was also able to endophytically colonize the cultivated rubber tree species (H. brasiliensis). Under controlled laboratory conditions, rubber plants were inoculated with three Trichoderma strains, including LA279, in combination with the pathogenic CCP. Results showed that 1 week preinoculation with the endophytes differentially reduced CCP mycelial development and symptoms. In conclusion, this study suggests that T. koningiopsis isolate LA279-and derivate compounds-could be a promising candidate for the biological control of the important rubber tree pathogen C. cassiicola.

Assessment of Alternative Candidate Subcortical Insect Vectors From Walnut Crowns in Habitats Quarantined for Thousand Cankers Disease.

Thousand cankers disease (TCD) results from the combined activity of the fungal pathogen, Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat and its principle vector, Pityophthorus juglandis (Blackman) (Coleoptera: Curculionidae: Scolytinae) in Juglans L. spp. and Pterocarya Kunth spp. host plants. TCD has been reported from the eastern and western United States. To evaluate potential for other beetle species to vector the fungus in east Tennessee, specimens were collected using ethanol-baited traps that were suspended beneath crowns of TCD-symptomatic trees. Associations of G. morbida with insect species collected in traps were assessed in an unsuccessful, preliminary culture-based fungal assay, and then with a molecular-based detection method. For culture-based assays, rinsate from washed, individual insects was plated on nutrient media and growing colonies were subcultured to obtain axenic G. morbida cultures for identification. For the molecular-based method, G. morbida presence was detected by amplifying the previously developed, species-specific microsatellite locus GS004. Capillary electrophoresis was used to detect the amplified amplicons and representative reactions were validated using Sanger sequencing. Eleven beetle species were found to carry G. morbida, including Cnestus mutilatus (Blandford), Dryoxylon onoharaensum (Murayama), Hylocurus rudis (LeConte), Monarthrum fasciatum (Say), Monarthrum mali (Fitch), Xyleborinus saxesenii (Ratzeburg), Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford) (all Coleoptera: Curculionidae: Scolytinae), Stenomimus pallidus (Boheman) (Coleoptera: Curculionidae: Cossoninae), Oxoplatypus quadridentatus (Olivier) (Coleoptera: Curculionidae: Platypodinae), and Xylops basilaris (Say) (Coleoptera: Bostrichidae). These findings raise concerns that alternative subcortical insect species that already occur within quarantined habitats can sustain incidence of introduced G. morbida and contribute to spread within the native range of black walnut, Juglans nigra L., in the eastern United States.

Mycobiota associated with insect galleries in walnut with thousand cankers disease reveals a potential natural enemy against Geosmithia morbida.

Thousand Cankers Disease (TCD) affects Juglans and Pterocarya species. This disease poses not only a major threat to the nut and timber industries but also to native stands of walnut trees. Galleries created by Pityophthorus juglandis (vector) are colonized by the fungus Geosmithia morbida (causal agent of necrosis). It is unknown if other fungi colonizing these galleries might act antagonistically towards G. morbida. The objectives of this study were to: (1) characterize the fungal community associated with TCD-infected trees and (2) develop a pilot study addressing their potential antagonism towards G. morbida. We collected non-Geosmithia fungi from ten TCD-infected walnut trees from California and Tennessee. Four hundred and fifty-seven isolates, representing sixty-five Operational Taxonomic Units (99 % ITS similarity) were obtained. Fungal communities were found to be highly diverse. Ophiostoma dominated the communities associated with TCD-compromised trees from California, whereas Trichoderma dominated TCD-compromised trees in Tennessee. Six Trichoderma isolates showed varying levels of antagonism against three isolates of G. morbida, suggesting potential applications for the biological control of TCD. Furthermore, results from this study contribute to the growing knowledge about the observed differential disease development between the western and eastern USA and could overall impact our understanding of TCD etiology.

A novel molecular toolkit for rapid detection of the pathogen and primary vector of thousand cankers disease.

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.

Improving taxonomic accuracy for fungi in public sequence databases: applying 'one name one species' in well-defined genera with Trichoderma/Hypocrea as a test case.

The ITS (nuclear ribosomal internal transcribed spacer) RefSeq database at the National Center for Biotechnology Information (NCBI) is dedicated to the clear association between name, specimen and sequence data. This database is focused on sequences obtained from type material stored in public collections. While the initial ITS sequence curation effort together with numerous fungal taxonomy experts attempted to cover as many orders as possible, we extended our latest focus to the family and genus ranks. We focused on Trichoderma for several reasons, mainly because the asexual and sexual synonyms were well documented, and a list of proposed names and type material were recently proposed and published. In this case study the recent taxonomic information was applied to do a complete taxonomic audit for the genus Trichoderma in the NCBI Taxonomy database. A name status report is available here: https://www.ncbi.nlm.nih.gov/Taxonomy/TaxIdentifier/tax_identifier.cgi. As a result, the ITS RefSeq Targeted Loci database at NCBI has been augmented with more sequences from type and verified material from Trichoderma species. Additionally, to aid in the cross referencing of data from single loci and genomes we have collected a list of quality records of the RPB2 gene obtained from type material in GenBank that could help validate future submissions. During the process of curation misidentified genomes were discovered, and sequence records from type material were found hidden under previous classifications. Source metadata curation, although more cumbersome, proved to be useful as confirmation of the type material designation. Database URL:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353

The genome of Xylona heveae provides a window into fungal endophytism.

Xylona heveae has only been isolated as an endophyte of rubber trees. In an effort to understand the genetic basis of endophytism, we compared the genome contents of X. heveae and 36 other Ascomycota with diverse lifestyles and nutritional modes. We focused on genes that are known to be important in the host-fungus interaction interface and that presumably have a role in determining the lifestyle of a fungus. We used phylogenomic data to infer the higher-level phylogenetic position of the Xylonomycetes, and mined ITS sequences to explore its taxonomic and ecological diversity. The X. heveae genome contains a low number of enzymes needed for plant cell wall degradation, suggesting that Xylona is a highly adapted specialist and likely dependent on its host for survival. The reduced repertoire of carbohydrate active enzymes could reflect an adaptation to intercellulary growth and to the avoidance of the host's immune system, suggesting that Xylona has a strictly endophytic lifestyle. Phylogenomic data resolved the position of Xylonomycetes as sister to Lecanoromycetes and Eurotiomycetes and placed the beetle-endosymbiont Symbiotaphrina as a member of this class. ITS data revealed that Trinosporium is also part of the Xylonomycetes, extending the taxonomic and ecological diversity of this group.

Synthesis of phylogeny and taxonomy into a comprehensive tree of life.

Reconstructing the phylogenetic relationships that unite all lineages (the tree of life) is a grand challenge. The paucity of homologous character data across disparately related lineages currently renders direct phylogenetic inference untenable. To reconstruct a comprehensive tree of life, we therefore synthesized published phylogenies, together with taxonomic classifications for taxa never incorporated into a phylogeny. We present a draft tree containing 2.3 million tips-the Open Tree of Life. Realization of this tree required the assembly of two additional community resources: (i) a comprehensive global reference taxonomy and (ii) a database of published phylogenetic trees mapped to this taxonomy. Our open source framework facilitates community comment and contribution, enabling the tree to be continuously updated when new phylogenetic and taxonomic data become digitally available. Although data coverage and phylogenetic conflict across the Open Tree of Life illuminate gaps in both the underlying data available for phylogenetic reconstruction and the publication of trees as digital objects, the tree provides a compelling starting point for community contribution. This comprehensive tree will fuel fundamental research on the nature of biological diversity, ultimately providing up-to-date phylogenies for downstream applications in comparative biology, ecology, conservation biology, climate change, agriculture, and genomics.