Multilocus Phylogenetic Analysis Research Articles

Identification of Pseudopestalotiopsis ampullacea as a new pathogen causing tea gray blight in India and its management strategies

Gray blight is a serious threat to the tea [Camellia sinensis (L.) O. Kuntze] production in major tea cultivating countries including India. The disease is caused by Pestalotiopsis-like species. In this study, five isolates of Pseudopestalotiopsis species isolated from symptomatic tea leaf samples in North Bengal, India were investigated. Based on the multi-locus phylogenetic analysis using concatenated sequences of three (ITS, tef-1 alpha, and tub-2) loci, cultural and micromorphological characters, and host association, the fungal isolates were identified as Pseudopestalotiopsis ampullacea F. Liu & L. Cai. The morphological analysis also revealed that the fungal isolates were evidently differentiated from other Pseudopestalotiopsis species. To date, P. ampullacea has not been reported on tea plants in India. Among the five isolates studied, isolate NKT0P03 was randomly selected for pathogenicity tests and its sensitivity to fungicides and microbial antagonists. In pathogenicity test, the isolate showed weak to high virulence reactions on 25 different tea cultivars. The pathogen showed an avirulent reaction on the cultivar TV11. In order to identify an effective management strategy against this new pathogen, synthetic fungicides and microbial biocontrol agents were evaluated in the laboratory. Results revealed that carbendazim + mancozeb, hexaconazole, propiconazole, and valextra were effective fungicides with an 85.1% to 89.8% range of inhibitory activity against P. ampullacea NKT0P03. Among microbial agents, Trichoderma harzianum, T. reesei, T. hamatum, Bacillus subtilis, and Microbacterium barkeri were efficient bioagents against P. ampullacea NKT0P03 with antagonistic activity ranging between 66.6% and 84.2%. Thus, these fungicides and microbial bioagents can be recommended as effective agents for the management of P. ampullacea causing tea gray blight after their field evaluations.

Alternaria youyangensis sp. nov. (Ascomycota: Pleosporaceae) from leaf spot of Fagopyrum esculentum in China

Alternaria is a widely distributed fungal genus as saprophytes, endophytes, and pathogens. An Alternaria species was isolated from the leaf spot of Fagopyrum esculentum in Banxi Town, Youyang County, Chongqing municipality, China. The obtained strains were determined based on morphological characteristics and multi-locus phylogenetic analyses including the internal transcribed spacer (ITS) of the rDNA region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), RNA polymerase second largest subunit (RPB2), a major Alternaria allergen (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous region gene (OPA10-2) gene sequences. The results indicate that the present species is a novel species, which is described and illustrated as Alternaria youyangensis sp. nov. in this study.

First Report of Neocosmospora sp., Causing Stem Rot on Agave potatorum Zucc., in Mexico.

Agave potatorum Zucc., also known as "agave tobalá", is an endemic species in the Mexican states of Oaxaca and Puebla and is one of the most important wild species for producing the Mexican alcoholic distillate called mezcal. In September 2021, symptoms of dry stem rot and subsequent death of plants were observed, in a commercial field of 10 acres located in the municipality of Villa Sola de Vega, Oaxaca, Mexico. The disease incidence was 45%. Twenty symptomatic agave stem plants were sampled to isolate the pathogen. Pieces (5 × 5 mm) of the symptomatic tissues from diseased plants were surface disinfested with 1% NaOCl (60 s) and 70% ethanol (1 min), rinsed with sterile water (2 × 60 s), placed on potato dextrose agar (PDA Bioxon®, Mexico), and incubated at 25°C for 6 days with natural light. Next, we prepared single-spore derived cultures on PDA and carnation leaf-piece agar (CLA). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 14 monoconidial isolates were obtained. On PDA, aerial mycelium was white to beige with diffuse pink pigment in the center of the culture and colonies were appressed with a regular shape after 7 days of incubation at 25℃. Macroconidia (n=100) were falcate, almost straight, 1 to 4-septate, 18.5 to 47.9 × 3.9-5.7 μm. Microconidia (n=100) were ellipsoidal to subcylindrical, slightly curved, hyaline, 8 to 16 × 3.5 to 4.5 μm, 0 to 1-septate, and grouped in false heads. Chlamydospores were globose to subglobose, terminal or intercalary in hyphae, and were solitary or in chains. Morphological features were consistent with the description of Neocosmospora spp. (Crous et al. 2021; Sandoval-Denis et al. 2019). Three isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited as IPN-AP1, IPN-AP2, and IPN-AP3 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. For molecular identification, genomic DNA was extracted, and partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (rpb2) genes were amplified and sequenced with the primer sets EF1-728F/EF1-986R (Carbone and Kohn 1999) and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree using concatenated EF1-α and rpb2 sequences was constructed for Neocosmospora spp. based on Maximum Likelihood. The phylogenetic analysis suggested that the three isolates belong to a new species, closely related to N. noneumartii, as they formed a sister clade with this species. The sequences were deposited in GenBank with accession nos. OP902594 to OP902596 for EF1-α, and OP902591 to OP902593 for rpb2. A pathogenicity test for each isolate was performed by inoculating five healthy A. potatorum plants (1-year-old) grown in pots. Conidia were cultured on PDA for 7 days and the conidial solution was prepared with sterile water and adjusted to 1×105 spores/ml. A total of 1 ml of the conidial suspension was placed on the basal stem of each plant. Five plants mock-inoculated with sterilize water served as the controls. All plants were kept in a greenhouse for 45 days at temperatures ranging from 20 to 30°C. At 35 days after inoculation, dry stem rot was visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected plants and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Neocosmospora spp., has been previously associated with dry root rot in Citrus sinensis in South Africa (Guarnaccia et al. 2021) as well as rot of Cactaceae and other succulent plants in Iran (Kamali-Sarvestani et al. 2022). To our knowledge, this is the first report of Neocosmospora sp. causing stem rot of A. potatorum in Mexico and worldwide. Further studies should focus on determining the distribution, impact, and management of this disease in the production areas of agave tobalá in Mexico.

Identification and Characterization of Colletotrichum Species Associated with Maize in Sichuan, China.

Colletotrichum species are the most common cause of maize anthracnose, which often manifests as leaf spots. However, field observations often reveal symptoms similar to those caused by other leaf spot pathogens, such as Curvularia and Bipolaris. In this study, 99 isolates were identified using tissue separation and single-spore isolation techniques. As preliminary measures of species diversity, all 99 isolates were identified morphologically, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were phylogenetically analyzed. Furthermore, 48 representative strains were selected for molecular identification using multi-locus phylogenetic analyses based on five gene loci (ITS, TUB, ACT, GAPDH, and CAL). Finally, 10 species of Colletotrichum isolated from maize leaf spots were identified. Colletotrichum cliviicola was the most dominant species (24.2%), followed by C. fructicola (18.2%), C. karstii (16.1%), C. siamense (13.1%), C. boninense (7.1%), C. kahawae (7.1%), C. brevisporum (6.1%), C. truncatum (5.1%), C. gigasporum (2.0%), and C. gloeosporioides (1.0%). For the first time, pathogenicity tests revealed that C. cliviicola, C. fructicola, C. siamense, C. karstii, and C. truncatum are the causative agents of maize anthracnose. Additionally, C. boninense was identified as an endophytic fungus on healthy maize. In conclusion, this study is the first to identify the pathogen of maize anthracnose in Sichuan Province. It provides valuable insights for accurately diagnosing and managing maize anthracnose.

Identification, Pathogenicity, and Fungicide Sensitivity of Colletotrichum Species Associated with Anthracnose on Italian Ryegrass in Southwestern China.

Italian ryegrass (Lolium multiflorum L.) is widely cultivated as an important forage grass worldwide because of its high nutritional value and good palatability. Anthracnose caused by Colletotrichum species was a common and new emerging disease of Italian ryegrass. In this study, 88 Colletotrichum isolates were collected from diseased leaves of Italian ryegrass planting regions in Sichuan, Chongqing, and Guizhou provinces of southwestern China between 2019 and 2022. By the pure culture technique, 15 representative single-spore isolates were obtained for further study. Multilocus phylogenetic analysis coupled with morphological features showed that these isolates were finally identified as six new record species: C. cereale of the C. graminicola species complex, C. fioriniae and C. nymphaeae of the C. acutatum species complex, C. boninense and C. citricola of the C. boninense species complex, and C. nageiae. Pathogenicity tests indicated that all species could induce anthracnose symptoms; of these, C. cereale was more invasive than other species, followed by C. fioriniae, C. nageiae, C. citricola, and C. boninense; C. nymphaeae was the weakest pathogenic species to Italian ryegrass plants (P ≤ 0.05). Fungicide sensitivity assays showed that iprodione, propineb, and oxime·tebuconazole had strong inhibitory effects on the mycelial growth of six Colletotrichum species; in addition, azoxystrobin and fludioxonil also significantly inhibited the mycelial growth of C. nymphaeae and C. fioriniae, respectively. These results provide the basis for the diagnosis and detection in the field, pathogen identification, and management of anthracnose on Italian ryegrass.

Diversity of Colletotrichum species causing cherry postharvest anthracnose in China

Cherry postharvest anthracnose (CPA) is an important fungal disease that endangers cherry production. To characterize the pathogen diversity, 43 Colletotrichum isolates were isolated from 175 cherry fruits. The multi-locus phylogenetic analysis (ACT, CAL, CHS-1, GAPDH, HIS3, ITS, TUB2) revealed that strains of the C. gloeosporioides species complex (CGSC) associated with CPA included C. aenigma, C. fructicola, C. gloeosporioides, C. siamense, and strains of the C. acutatum species complex (CASC) included C. fioriniae, C. godetiae, C. salicis. There were significant differences in biological characteristics between CGSC and CASC. The mycelium growth rate, sporulation and conidial germination ability of CGSC was significantly higher than that of CASC. However, CASC was more adaptable to low temperatures. CGSC was more pathogenic than CASC toward cherry fruits. On other tested hosts, all species showed pathogenicity only on wounded condition and the pathogenicity of CGSC and CASC to other host fruits was significantly different too. C. aenigma and C. siamense exhibited significantly higher pathogenicity towards grapes and blueberries compared to C. gloeosporioides and C. fioriniae, while the pathogenicity to cherry tomatoes was reversed. In general, CGSC was more pathogenic than CASC to testes hosts. Our results showed that there were seven species of two species complex of Colletotrichum responsible for CPA in China, and the biological characteristics and pathogenicity of different species were significantly different.

Fusarium and Neocosmospora Species Associated with the Decline of Metasequoia glyptostroboides in China.

During surveys conducted in 2020, severe symptoms associated with death and decline were observed on >30-year-old Metasequoia glyptostroboides (Chinese redwood) trees in the shelter-forests along Yangtze River in Jingzhou city, Hubei province, China. A previous study showed that Phytophthora acerina was one of the causal agents of the decline of the Chinese redwood. In this study, a total of 147 fungal isolates were obtained from the diseased roots and xylem of trunks of declining M. glyptostroboides trees. Through morphology and multi-locus phylogenetic analysis, these isolates were identified as eight species belonging to the genera Fusarium and Neocosmospora including F. fujikuroi, F. irregulare, F. odoratissimum, F. reticulatum， N. falciformis, N. keratoplastica, N. solani, and N. tonkinensis. Single inoculation and co-inoculation with P. acerina assays of these Fusarium and Neocosmospora species were then performed to test pathogenicity on three-year-old seedlings of M. glyptostroboides. Lesions (i.e., on seedling stems) caused by species of the genera Neocosmopora and Fusarium were smaller than those caused by P. acerina. Co-inoculation of F. fujikuroi and P. acerina, as well as the co-inoculation of F. reticulatum and P. acerina caused larger lesions than inoculations with P. acerina alone. All these species of Fusarium and Neocosmospora were shown to have the potential to be pathogenic to M. glyptostroboides. This study provided evidence that the decline of M. glyptostroboides in Jingzhou is a disease complex.

Cytospora azadirachtae sp. nov.—A new species associated with dieback disease in Azadirachta indica from southern India

Stem cankers associated with dieback disease of neem (Azadirachta indica) shoots were collected from Manasagangotri and Doddamaragowdanahalli, Mysore, Karnataka (India) and based on microscopic, cultural and initial blast results of ITS-rDNA identified the isolated fungus to the genus Cytospora. The phylogenetic analysis based on nrITS-rDNA-tef1-α-tub2 sequence data showed it represents a new species and is described as Cytospora azadirachtae. The concatenated phylogeny revealed that Cytospora azadirachtae is phylogenetically distinct and closely related to Cytospora shoreae and C. rhizophorae. Cytospora rhizophorae differs from C. azadirachtae in that it has shorter conidia and conidiogenous cells. Further, C. rhizophorae is halotolerant and saprobic and shows host-specificity usually associated with Rhizophora sp. (R. mangle and R. mucronata). Similarly, C. shoreae differs from C. azadirachtae in having larger flask-shaped conidiomata and larger conidia. Based on multi-locus sequence and phylogenetic analysis of the canker and dieback disease symptoms, the present study introduces C. azadirachtae as a new species.

Two new species of Cytospora (Diaporthales, Cytosporaceae) causing canker disease of Malusdomestica and M.sieversii in Xinjiang, China.

Apple tree canker is a serious disease caused by species of Cytospora. Xinjiang Uygur Autonomous Region is one of the most important apple-producing areas in China. However, losses due to apple Cytospora canker have seriously damaged the apple industry and affected the economic development of the apple growers in this region. In this study, we used morphological characteristics combined with multilocus phylogenetic analyses of the ITS, act, rpb2, tef1 and tub2 loci to identify isolates from apple (Malusdomestica) and wild apple (M.sieversii). As a result, C.hippophaopsis sp. nov. from M.sieversii and C.shawanensis sp. nov. from M.domestica were discovered and proposed herein. Pathogenicity tests were further conducted on 13 varieties of apple and wild apple, which confirmed C.hippophaopsis and C.shawanensis as canker pathogens. Meanwhile, C.hippophaopsis is generally more aggressive than C.shawanensis on the tested varieties of apple and wild apple.

Diversity, divergence time, and biogeography of the genus Albatrellus (Agaricomycetes, Russulales)

ABSTRACT The genus Albatrellus is an important group of stipitate terrestrial fungi in the order Russulales. Some species in the genus form ectomycorrhizae, mostly with trees of Pinaceae; some are well-known edible mushrooms. However, its diversity and biogeography are unclear. Taxonomic and phylogenetic studies on Albatrellus were carried out by morphological examination, which included detailed observations of the fruiting body, spore shape and size, and other key features, together with potential hosts. These observations were then compared and analysed using multi-locus molecular phylogenetic analyses, including the internal transcribed spacer regions (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), the translation elongation factor 1-α gene (tef1), the largest subunit of RNA polymerase II (rpb1), the second largest subunit of RNA polymerase II (rpb2), the small subunit mitochondrial rRNA gene (mtSSU), and the small subunit of the nuclear ribosomal RNA gene (nucSSU). The results demonstrated that the species of Albatrellus formed eight clades. Nine new species are described and illustrated, and two new combinations are proposed. A total of 38 species are accepted in Albatrellus worldwide. Of those species, 26, 7, and 8 species are distributed in Asia, Europe, and North America, respectively. The divergence time indicated that the maximum crown age of Albatrellus was approximately 70.5 million years ago, and East Asia and North America are the likely ancestral areas. Dispersal and differentiation to other continents occurred during the late Paleocene and Miocene. Three kinds of dispersal routes are proposed: East Asia and Europe, East Asia and North America, and Europe and North America.

First report of Sclerotinia minor causing soft rot of Scabiosa atropurpurea in California.

In May of 2019, Scabiosa atropurpurea samples with brown discoloration, soft rot of the crown and lower stem, with presence of white mycelium and black sclerotia (Supp. Fig. 1A, B) were collected from a 0.10 ha open field diversified cut flower production in San Luis Obispo County, CA. Approximately 30 to 40% of the scabiosa crop planted in a quarter of the field, exhibited symptoms. Symptomatic crowns and lower stems from five plants were surface disinfested by rinsing in 0.1% Tween 20, soaking in 70% ethanol for 30 s, 1% sodium hypochlorite for 2 min and sterile water. Disinfested tissue was placed in 1/10 potato dextrose agar (PDA) and incubated at 20°C (12 h photoperiod). Resulting colonies (n = 5) formed abundant white mycelia, with black sclerotia formed on the outer edge of the plates after two weeks (Suppl. Fig. 1C). Sclerotia (n = 50) had an average size of 1.6 (± 0.19) mm in diameter. Morphological identification resulted in Sclerotinia sp. (Hao et al., 2003). The pathogen was further identified by DNA extraction of two hyphal tipped isolates, followed by amplification and sequencing of the rDNA internal transcribed spacer (ITS) region, ITS1/ITS4 (White et al. 1990), calmodulin (CaM), CAL-228F/CAL-737R (Carbone and Kohn, 1999), and DNA replication licensing factor Mcm7 Mcm7-709for/Mcm7-1348rev (Schmitt et al., 2009). NCBI BLAST searches with consensus sequences for each maker revealed 99 to 100% identity with S. minor ex-types for all loci (Supp. Table 1). A maximum parsimony multilocus phylogenetic analysis clustered Californian isolates with reference strains of S. minor (Supp. Fig. 1F). Sequences were deposited in GenBank (Supp. Table 1). Pathogenicity tests were conducted with isolate CS435, which was transferred onto PDA plates and incubated at 20°C for one week. Inoculum consisted of CS435 infested PDA plugs (1 cm3). In the greenhouse, the experiment was set as a complete randomized design and observed for six weeks. Fourteen-week-old scabiosa 'Merlot Red' grown in 3.78 L pots (n = 6), were inoculated by wounding plants at 0.5 cm above the crowns with a 1 mm probe. Inoculum was placed directly on top of the wound and was secured with parafilm. Negative control plants (n = 6) were wounded as above and inoculated with PDA plugs. In experiment 1(19.4 (± 3) °C, RH 46.9), 83% of plants exhibited yellowing of the lower leaves and wilting at one week post inoculation (wpi). Symptoms progressed over time until wilting, major leaf and stem necrosis, was observed in all inoculated plants (Supp. Fig 1E, D). Plant mortality incidence at five wpi was 83%. Pathogen signs including white mycelia and black sclerotia were also observed. In experiment 2 (20.0 (± 10) °C, RH 39.6), 66% of the total plants were symptomatic at five wpi: 33% exhibited yellowing of the lower leaves and wilting, and 33% of plants died. Disease did not develop in non-inoculated plants in either experiment. S. minor was successfully reisolated from surface disinfested tissue of at least 50% (Exp. 1) and 100% (Exp. 2) of inoculated plants, yielding identical sequences to those of the inoculated isolate. S. minor is a soil-borne pathogen that infects tomato, lettuce, brassica, and sunflower crops in California. This study stablishes S. minor as a new soil-borne pathogen of Scabiosa in California. Soil-borne pathogens are a recurrent issue in the cut flower industry, characterizing new pathogens in these crops can inform crop advisors and disease diagnostician to improve disease management in the ornamental industry.

Morphological and phylogenetic analyses reveal eight novel species of Pestalotiopsis (Sporocadaceae, Amphisphaeriales) from southern China.

Plants play an important role in maintaining the ecological balance of the biosphere, but often suffer from pathogenic fungi during growth. During our continuing mycological surveys of plant pathogens from terrestrial plants in Jiangxi and Yunnan provinces, China, 24 strains of Pestalotiopsis isolated from diseased and healthy tissues of plant leaves represented eight new species, viz. P.alpinicola, P.camelliicola, P.cyclosora, P.eriobotryae, P.gardeniae, P.hederae, P.machiliana and P.mangifericola. Multi-locus (ITS, tef1-α and tub2) phylogenetic analyses were performed using maximum-likelihood and Bayesian inference to reveal their taxonomic placement within Pestalotiopsis. Both molecular phylogenetic analyses and morphological comparisons supported them as eight independent taxa within Pestalotiopsis. Illustrations and descriptions of these eight taxa were provided, in conjunction with comparisons with closely related taxa in the genus. This work highlights the large potential for new fungal species associated with diseased plant leaves.

Calonectria eucalyptorum sp. nov., a new leaf blight pathogen of Eucalyptus from India.

In this study, Calonectria eucalyptorum sp. nov. is described from the blighted leaves of Eucalyptus in Indiausing morphological and multi-locus phylogenetic analyses. The new species belongs to the Calonectria cylindrospora species complex, and its unique microscopic features and DNA sequence information enable clear separation from the12 currently accepted species in this complex. Conidia of the new taxon are slightly longer than those of its phylogenetic neighbors. Additionally, this species produces central as well as lateral stipe extensions, which is a feature not known for the other members ofthe C. cylindrospora species complex. Analyses of the combined partial calmodulin, histone, translation elongation factor-1α, and β-tubulingene regions revealed a distinct phylogenetic position for C. eucalyptorum. Recombination analysis provided additional support for the new species hypothesis. Koch's postulates for the new taxon as a foliar pathogen of Eucalyptus were fulfilled. The discovery of novel and pathogenic Calonectria species is important because it sheds light on species diversity, potential threats, and disease control.

Genomic Characterization and Establishment of a Genetic Manipulation System for Trichoderma sp. (Harzianum Clade) LZ117.

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction.

First report of leaf spot caused by Paramyrothecium roridum on Coffea arabica in Hawai'i, USA.

During the 2022-2023 season, the harvested coffee crop in Hawai'i (Coffea arabica) was valued at $57.1 million (USDA NASS 2023). In September 2022, coffee leaf samples with foliar leaf spots affecting the Kona Typica variety were collected from Hōnaunau, Hawai'i, incidence <10%. The symptoms were circular, necrotic leaf spots with yellow margins, which merged, resulting in complete leaf blade coverage and subsequent leaf drop. Sporodochia were present on the abaxial leaf surface. Symptomatic leaf tissue was disinfected in 10% bleach solution for 60 seconds and chlorotic leaf tissue from the spot margins were excised and placed onto water agar and potato dextrose agar (PDA; Difco, USA). After a 7-day incubation period, pure cultures with white aerial mycelium having sporodochia arranged in concentric rings with olivaceous to black conidial masses were isolated. The conidia were aseptate, hyaline, smooth, cylindrical with rounded ends, measuring 5.1 to 6.8 μm long and 1.7 to 2.3 μm wide (n=50). Based on symptomology and cultural/morphological characteristics (Huaman-Pilco et al. 2023; Lombard et al. 2016; Pelayo-Sanchez et al. 2017), the isolates were initially identified as Paramyrothecium roridum (Tode) L. Lombard & Crous, comb. nov. (syn. Myrothecium roridum Tode). Fungal identification of isolate P22-81-2 was further confirmed using BLAST analysis of bulk sequenced PCR products of the ribosomal DNA internal transcribed spacer (ITS) region (White et al. 1990), β-tubulin (βtub), RNA polymerase II (RPB2), and calmodulin genes (Lombard et al., 2016; Huaman-Pilco et al., 2023). The gene sequences (GenBank accession nos. PP211198, PQ192517-19) were >98.4% identical to the P. roridum type specimen (CBS 357.89). A multilocus maximum likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al., 2016; Pinruan et al. 2022) confirmed species identification. To prove pathogenicity, four, 26-month-old Kona Typica variety seedlings were foliar inoculated with a 1 X 106 conidia/ml suspension using a perfume atomizer. An additional four plants were inoculated in a similar manner with sterile water which served as controls. All plants were sprayed to drip on both the upper and lower leaf surfaces and incubated in a clear plastic bag to keep the humidity levels between 90 to 100% for 48 hours at 24°C. After 48 hours, the plants were removed from the bags, placed on a greenhouse bench, and observed weekly for symptom development. Within seven days light brown sunken spots had developed on all inoculated plants. The spots continued to enlarge having a dark distinct margin, light tan center, chlorotic halo, and formed concentric rings, which were identical to the original diseased samples. Leaf spots were not present on any of the control plants. The test was conducted twice. A fungus was consistently reisolated from the leaf spot margins of inoculated plants and morphologically (PDA) and molecularly (ITS, βtub, RPB2, calmodulin) identified as P. roridum, thus fulfilling Koch's Postulates. To the best of our knowledge, this is the first report of P. roridum causing leafspots on C. arabica plants in Hawai'i. This pathogen has been reported on coffee in other parts of the world including Colombia, Costa Rica, Guatemala, Puerto Rico, and Mexico (USDA Fungus-Host Database). Under the right conditions, P. roridum has the potential to cause leafspots and defoliation resulting in economic losses for coffee growers in Hawai'i.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

Isolation, identification and multi-locus sequence typing of Phytophthora capsici from capsicum fields and its cross-infectivity in different crop species

Phytophthora capsici is a major soil-borne oomycete plant pathogen, which severely affects the productivity of peppers including Capsicum annuum L., by causing fruit rot, root rot, and foliar blight diseases. It has a broad host range beyond Capsicum species and evolves rapidly to find new potential hosts for survival under different climatic conditions. In the present study, P. capsici was isolated from soil using castor seed baiting, and characterized through growth patterns and spore morphology. The species identification and molecular characterization were achieved through multi-locus sequence and phylogenetic analysis of three genomic loci (ITS, tef1 and cox), and also attributed given P. capsici isolate Pc_UHF into India-specific ITS-based evolutionary lineage Pc-X. Interestingly, dN/dS estimates revealed reasonably high positive selection in the tef1 gene, which could have possible implications on the pathogen's adaptation and/or virulence. Pathogenicity of this isolate was tested in three commercially growing bell pepper varieties and also in other crop species using detached leaf and/or in planta disease screening. Differential disease symptoms were induced by Pc_UHF in three bell pepper varieties, such as both collar rot and foliar blight symptoms observed in "Yolo Wonder" and "California Wonder", whereas only foliar blight symptoms were recorded in "Solan Bharpur". Furthermore, cross-pathogenicity was also recorded in tomato, citrus, cucumber and eggplant. This is the first report of molecular characterization and cross-pathogenically assessment of P. capsici isolate from farm soils in the North Western Himalayan region of India, which has a dynamic virulence profile and distinct host range.

First Report of Fusarium kyushuense Causing Leaf Spot on Sweet Persimmon in China.

Sweet persimmon (Diospyros kaki L.) is a fruit of significant nutritional and commercial value in Asia. In summer 2023, leaf spots were observed affecting 20 to 30% of sweet persimmon trees in a commercial orchard located in Gongcheng City, Guangxi, China. Initially, the infected leaves exhibited sparse light brown spots on their upper surface, which subsequently evolved into brown circular to irregular lesions encircled by a yellow halo. Eventually, these lesions became densely distributed across the leaves leading to insufficient nutrient accumulation in the fruit. To isolate the pathogen, diseased leaves were cut into small pieces (5×5 mm), disinfected with 75% ethanol for 15 seconds, followed by 1% NaClO for 1minute, rinsed three times with sterile water, and then transferred onto potato dextrose agar (PDA) plates. The plates were then incubated in darkness for 3 days at 25°C. Pure cultures were obtained using the hyphal-tip method and single-spore isolation. On PDA, the colonies initially appeared fluffy and white after 24 hours, turning yellowish or red after 3 days. Macroconidia (average length of 26.1 μm in length × 4.3 μm in width, n = 50) exhibited dorsiventral curvature and were hyaline, with 3 to 5 septa. Microconidia (average length of 9.45 μm in length × 3.4 μm in width, n = 50) were hyaline, aseptate, and oval. Two representative isolates, Gxfky1 and Gxfky2, were selected for further molecular analyses. Their internal transcribed spacer (ITS) region rDNA gene were amplified via PCR and sanger sequenced (GenBank Accession Nos. PP506475, PP506593) using the primer pair ITS1/ITS4 (White et al. 1990), showing more than 99% sequence identity with Fusarium kyushuense type-material strain NRRL3509 (NR_152943) according to BLASTn analysis in NCBI. To further confirm the identity of the isolates, four gene sequences were amplified: RPB1 (PP532864, PP532865), RPB2 (PP532866, PP532867), TEF1 (PP580505, PP580506), and TUB2 (PP532862, PP532863), using the F5/G2R, 5f2/11ar, EF1/EF2, and T1/T2 primer sets, respectively (O'Donnell et al., 1997; O'Donnell et al., 2010). A multi-locus maximum likelihood phylogenetic analysis revealed that Gxfky1 and Gxfky2 clustered with strains F. kyushuense with 100% bootstrap support. Pathogenicity tests using Gxfky1 and Gxfky2 were conducted on leaves of two-year-old sweet persimmon plants using non-wound inoculation. Specifically, 5-mm mycelial plugs and sterile agar plugs were placed on six leaves and secured with cling film, with six plugs each for the inoculation treatment and negative control, respectively. They were then incubated in a greenhouse at room temperature (25 ± 2°C) with a relative humidity of 70 to 80%. After 5 days, the same symptoms on naturally infected plants were observed on leaves inoculated with mycelium, while no symptoms were observed on the controls. The same fungus were reisolated from the inoculated leaves and identified based on morphology and the TEF1 gene sequence, thus fulfilling Koch's postulates. Fusarium kyushuense has previously been reported to cause diseases in various plant species, including maize (Cao et al., 2021), rice (Wang et al., 2024), and tobacco (Wang et al., 2013). To our knowledge, this is the first report of F. kyushuense causing leaf spot on sweet persimmon in China, which expands the known host range of this pathogen.

Characterization, Phylogenetic Analyses, and Pathogenicity of Eutiarosporella dactylidis on Sorghum in China.

Sorghum, the fifth-largest cereal crop globally and a C4 crop, mainly grows in arid and semi-arid areas. In 2021-2023, a new foliar disease of sorghum occurred in China. The diseased leaves showed water-soaked symptoms in the leaf tip and margins, resulting in half- and full-leaf desiccation and necrosis, thus affecting plant photosynthesis. A total of 24 Eutiarosporella strains were isolated from symptomatic leaves. Based on morphological characteristics and multi-locus phylogenetic analysis involving ITS, LSU, and EF1-α sequences, and the pathogenicity test, the pathogen of sorghum causing leaf blight in China was identified as Eutiarosporella dactylidis. The virulence of all E. dactylidis strains was evaluated using the spray-mycelium method. Different strains showed significantly different pathogenicities toward a susceptible cultivar, Longza 10, with disease indexes ranging from 23.76 to 60.37. This study first reported leaf blight of sorghum caused by E. dactylidis and named it "Eutiarosporella leaf blight", which provides a theoretical basis for farmers in disease management.

Novel report of Bipolaris heliconiae causing frog-eye-like leaf spot on Dypsis lutescens in Indian Sub-Continent

Dypsis lutescens, commonly known as areca palm, is a highly valued ornamental species due to its aesthetic value. However, the foliage is vulnerable to various pathogens, particularly those responsible for fungal leaf spot diseases. In October 2023, a severe incidence (93 %) of destructive leaf spots was recorded on Dypsis lutescens at the University of Agricultural Sciences, GKVK, Bangalore, and surrounding areas. The leaf spot symptoms manifested as frog-eye-like lesions, leading to complete leaf desiccation and significantly reducing the palms ornamental value. The pathogen exhibited the highest radial growth (90.00 mm) and prominent sporulation on oat meal agar, whereas Richard's synthetic agar resulted in the lowest radial growth (38.00 mm) with no sporulation. Morphological and multilocus phylogenetic analyses confirmed the pathogen as Bipolaris heliconiae. Pathogenicity tests fulfilled Koch's postulates, confirming that Bipolaris heliconiae is the causative agent of leaf spot disease in Dypsis lutescens in India. This novel finding underscores the emergence of a new disease and highlights the urgent need for effective management strategies.