Conidial Morphology Research Articles

First Report of Root Rot Disease in Green Gram (Vigna radiata) Caused by Ectophoma multirostrata in India.

A disease survey was conducted in the green gram (Vigna radiata) growing areas of Bharuch, Gujarat, India in February 2020. Root rot was observed on cultivar NVL-585 in 30 fields surveyed, with 15 to 20% of disease incidence. Infected plants showed foliar chlorosis, brown discoloration, dark black brittle roots with necrotic lesions, and dead plants. The root rot causal agent was isolated on potato dextrose agar (PDA) from 25 symptomatic samples collected from 10 diseased plants and pure cultures were obtained using single hyphal tip method. The cultures were grayish black with a smooth texture. Conidia were single celled, elliptical to oval in shape and were 5.2 to 6.5μm (n=25) in length and 2.7 to 3.4µm in width. Plastic pots (15 cm in diameter) were filled with autoclaved soil (1.5 kg/pot) inoculated with a 15-day-old culture of the fungus at 3 g/ kg (108 cfu/g). After 7 days, the local green gram cultivar HUM 16 was sown into the pots. Seedlings were thinned to 10 for each pot. Chlorosis of leaves, rotting roots, and even plant death were observed at 20, 30 and 40 days after seeding. The experiment was conducted twice to fulfill the Koch postulates. The fungus was reisolated from the root rot samples, and identical colony morphology was observed as seen previously. Based on the cultural characteristics and conidial morphology, the fungus belonged to Ectophoma sp. (Boerema et al. 2004). ITS, D1 and D2 regions of LSU and β-tubulin were used for molecular identification. The BLASTn analysis of ITS, D1 and D2 region of LSU gene and β-tubulin gene sequences of Isolate G80 showed a 100% similarity with accession number MH858623 (Vu et al. 2019), 99.82% similarity with MH870612 (Vu et al. 2019) and 100% similarity with MN983939 (Hou et al. 2020). Gene sequence has been submitted to NCBI GenBank, and accession numbers for ITS, D1 and D2 region of LSU and β-tubulin are MW165415, MW813868 and LC656357, respectively. The pathogen was previously reported from other hosts like Fuchsia× hybrida, Origanum vulgare, Lavandula angustifolia, Cicer arietinum and Coriandrum sativum. It can cause substantial economic damage to a wide range of commercially important crops. To the best of our knowledge, this is the first report of E. multirostrata causing root rot disease of green gram in India.

Isolation and identification of two Beauveria bassiana strains from silkworm, Bombyx mori.

Silkworm diseases caused by fungi infection occur frequently in sericulture and brought huge economic loss to sericulture. However, on the other hand, some fungi such as Beauveria bassiana, as an important entomological fungus, play an important role in biological control of insect pests. Here, two fungal pathogens causing yellow muscardine were isolated from the silkworm and named as SZY1 and SZY2. These two strains showed almost the same conidial morphology which were smooth, near-spherical, spherical, or ovoid and 2.7 ± 0.6µm × 2.5 ± 0.9µm in size, and the hyphal growth rate was also similar. However, the conidia production of SZY2 was almost twice as many as that of SZY1. The complete ribosomal RNA gene was sequenced and analyzed. As a result, the gene sequences of internal transcript space 1 (ITS1)-5.8S rRNA-internal transcript space 2 (ITS2) of SZY1 and SZY2 were identical in sequence and size, and for 18S rRNA, 28S rRNA, and intergenic spacer (IGS), the gene identity of SZY1 to SZY2 was 99%, 99%, and 98%, respectively. Results of phylogenetic analysis based on either ITS1-5.8S rRNA-ITS2 or 18S rRNA showed that both SZY1 and SZY2 were closely related to Beauveria bassiana. These results revealed that the pathogens of yellow muscardine SZY1 and SZY2 were identified as two different strains of Beauveria bassiana, which could provide diagnostic evidence for silkworm muscardine and was helpful for the research and development of novel Bombyx batryticatus and fungal biological insecticide.

First Report of Curvularia Leaf Spot of Field Corn, Caused by Curvularia lunata, in Mississippi.

In July 2021, foliar symptoms characterized by small, circular, light brown to tan lesions (0.5 to 3 mm diameter) with reddish-brown margins were observed on field corn (Zea mays L.) in two commercial fields in Hinds and Marion counties, Mississippi. Disease severity ranged from 2 to 15% on observed leaves. Symptomatic leaves were sealed in plastic bags, stored on ice, and transferred to the laboratory. Lesions were cut into small sections (≈4 mm2) and surface-sterilized with 70% ethanol for 30 s then rinsed with sterile water. Sterilized sections were transferred to potato dextrose agar (PDA) amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated at 25°C in the dark for 7 days. Gray to brown-black colonies with orange margins and melanized, curved conidia with three transverse septa were observed microscopically (Fig. 1; ×400). Conidia measurements ranged from 15 to 25 μm in length and 7.5 to 12.5 μm in width (x̄= 20 × 9.8 μm; n= 44). Colony and conidia morphology were consistent with previous descriptions of Curvularia lunata (Wakker) Boedijn (Mabadeje 1969; Ellis 1971). Pure cultures were obtained, and DNA was extracted from 9-day old cultures. Two isolates (TW003-21; TW008-21) were selected for sequencing of the internal transcribed spacer (ITS) region using ITS4 and ITS5 primers. The 530-bp consensus sequences were deposited in GenBank under the accession No. OK095277 and OK095278. BLASTn queries of NCBI GenBank showed that the sequences shared 100% identity with C. lunata isolate DMCC2087 from Louisiana (MG971304) and isolate CX-3 from China (KR633084). A pathogenicity test was performed on V4/V5 stage corn plants (Progeny 9114VT2P) grown in 10.2 cm pots in the greenhouse. Plants were transferred to a growth chamber one-week prior to inoculation. The two isolates were grown on amended PDA for 14 days at 25°C and an inoculum suspension was prepared for each isolate by rinsing culture plates with 2 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%) and re-suspended into 40 ml of RO water containing Tween 20. The final concentration was adjusted to 2.6×105 conidia/ml (TW003-21) and 2×105 conidia/ml (TW008-21). Ten corn plants were sprayed with 10 ml of inoculum suspension for each isolate using a Preval sprayer with a CO2 canister, and 10 plants were sprayed with water containing Tween 20 only. Plants were incubated in a growth chamber at ≈79% relative humidity and 25°C. Foliar symptoms including small, circular, and tan lesions, similar to those observed in the field, developed 3 days after inoculation. No symptoms were observed on control plants. Following incubation, symptomatic leaves were collected and C. lunata was re-isolated as described above. Colony, spore morphology and DNA sequences from inoculated plants were consistent with the original isolates as described above. The disease has been recently reported in Louisiana (Garcia-Aroca et al. 2018), Kentucky (Anderson et al. 2019), and Delaware (Henrickson et al. 2021). Although Curvularia leaf spot has been observed sporadically in MS corn fields since 2009 (Allen, personal communication), to our knowledge, this is the first official report of the disease in MS. While this disease has been more frequently encountered in MS, the economic impact associated with C. lunata is currently unknown. References Anderson, N. R., et al. 2019. Plant Dis. 103:2692. Chang, J., et al. 2020. J. Integr. Agr. 19:551-560. Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England, p. 452-458. Garcia-Aroca T., et al. 2018. Plant Health Prog. 19:140. Henrickson M., et al. 2021. Plant Dis. First Look. Mabadeje, S. A. 1969. Trans. Br. Mycol. Soc. 52:267-271. † Indicates the corresponding author. E-mail: twilkerson@drec.msstate.edu.

Isolation and Identification of Toxigenic Aspergillus species Associated with Sorghum Grains and Locally Brewed Beer (Burkutu) in Bukuru Metropolis of Jos South Local Government Area, Nigeria

Aim: This study is aimed at Isolating and Identifying the Toxigenic Aspergillus species Associated With Sorghum Grains and Locally Brewed Beer (Burkutu) in Bukuru Metropolis of Jos South Local Government Area, Nigeria.
 Methodology: Sorghum grains and locally brewed beer (Burkutu) were collected from three different locations; Gyel, Kugiya, and Doruwa Market of Bukuru Metropolis of Jos South Local Government Area, and Screened for their fungal and total aflatoxin level. The grains were blended for 30 seconds using a high-speed blender. 1 gram of the blended sorghum and 1ml of the Burkutu sample (local beer) were cultured into freshly prepared PDA supplemented with 0.5mg/ml of gentamycin and streptomycin to suppress bacterial growth. This was then incubated at a temperature of 37°C for 72 hours after which various fungi colonies were observed and counted with a colony counter. The fungi isolated were further sub-cultured to obtain pure cultures after which they were classified using conidial morphology as obtained from microscopy. Lactophenol cotton blue mount was also carried out to identify the exact fungi with reference to a mycological atlas. The colony-forming unit for each sample was determined, and the Aflatoxin test was carried out using a Mycotoxin kit (the Rida Quick Scan method) to determine the Total aflatoxin level.
 Results: The Aspergillus species found in both Sorghum grains and Burkuru (local beer) were: Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. For sorghum grains, Aspergillus niger had the highest frequency of occurrence (42.86%) while Aspergillus flavus & Aspergillus fumigatus occurred equally with (28.57%) respectively. Aspergillus flavus had the highest frequency of occurrence in Burkutu (50%), followed by Aspergillus niger (33.33%) and the least was Aspergillus fumigatus (16.67%). The highest aflatoxin level for grains was detected in Kugiya which had (3.6 parts per billion) followed by the grain sample from Gyel which had (3.0 Ppb) and the least was Doruwa (2.8 Ppb). Burkutu had the highest total level of aflatoxin (3.1Ppb) in the sample from Gyel, followed by Burkutu from Doruwa (2.6 Ppb) and the least was from Kugiya (2.4 Ppb).
 Conclusion: Both the sorghum grains and Locally Brewed Beer (Burkutu) collected from the Bukuru Metropolis of Jos South Local Government Area, Nigeria were significantly contaminated by the toxigenic Aspergillus species. This indicates possible health hazards for humans and animals ingesting these substances. However, effective screening of these grains for aflatoxins is advocated to ensure the safety of both grains and their products.

Transcription Factor Mavib-1 Negatively Regulates Conidiation by Affecting Utilization of Carbon and Nitrogen Source in Metarhizium acridum.

Conidium is the main infection unit and reproductive unit of pathogenic fungi. Exploring the mechanism of conidiation and its regulation contributes to understanding the pathogenicity of pathogenic fungi. Vib-1, a transcription factor, was reported to participate in the conidiation process. However, the regulation mechanism of Vib-1 in conidiation is still unclear. In this study, we analyzed the function of Vib-1 and its regulation mechanism in conidiation through knocking out and overexpression of Vib-1 in entomopathogenic fungus Metarhizium acridum. Results showed that the colonial growth of Mavib-1 disruption mutant (ΔMavib-1) was significantly decreased, and conidiation was earlier compared to wild type (WT), while overexpression of Mavib-1 led to a delayed conidiation especially when carbon or nitrogen sources were insufficient. Overexpression of Mavib-1 resulted in a conidiation pattern shift from microcycle conidiation to normal conidiation on nutrient-limited medium. These results indicated that Mavib-1 acted as a positive regulator in vegetative growth and a negative regulator in conidiation by affecting utilization of carbon and nitrogen sources in M. acridum. Transcription profile analysis demonstrated that many genes related to carbon and nitrogen source metabolisms were differentially expressed in ΔMavib-1 and OE strains compared to WT. Moreover, Mavib-1 affects the conidial germination, tolerance to UV-B and heat stresses, cell wall integrity, conidial surface morphology and conidial hydrophobicity in M. acridum. These findings unravel the regulatory mechanism of Mavib-1 in fungal growth and conidiation, and enrich the knowledge to conidiation pattern shift of filamentous fungi.

Molecular and Morphological Characteristics of a New Species Collected from an Insect (Cicindela transbaicalica) in Korea

To exploit insect-derived fungi, insects were collected from seven different regions in Korea, including Gyeongbuk, Goryeong, and several fungi were isolated from them. A fungal strain designated 21-64-D was isolated from riparian tiger beetle (Cicindela transbaicalica) and morphologically identified as a species belonging to the genus Oidiodendron. Phylogenetic analysis using the nucleotide sequences of internal transcribed spacer (ITS) regions and the partial sequence of the large subunit of the nuclear ribosomal RNA (LSU) gene revealed the distinct phylogenetic position of the isolate among recognized Oidiodendron species including its closest neighbors O. chlamydosporicum, O. citrinum, O. maius, and O. pilicola. The hyphal and conidial morphology of the strain, particularly club-shaped hyphae, clearly differentiated it from its close relatives. Results indicated that 21-64-D is a novel species in the genus Oidiodendron, for which the name Oidiodendron clavatum sp. nov. is proposed.

First report of halo blight of hop (Humulus lupulus) caused by Diaporthe humulicola in Quebec, Canada.

Halo blight of hop caused by Diaporthe humulicola has recently been reported in Michigan and Connecticut (Higgins et al. 2021, Allan-Perkins et al 2020). In August 2020 growers in Quebec, Canada reported necrotic foliar lesions and desiccation of the hop strobile (cone) on Chinook and Nugget cultivars. The foliar lesions were dry concentric circles with a chlorotic halo surrounding the lesions; no pycnidia were observed on leaves or cones. Up to 100% of the infected bract tissue was dry and easily shattered, the grower estimated that more than 90% of the plants in the hopyard exhibited symptoms. Twenty-six isolates were obtained from surface-sterilized leaf and cone tissue by plating the leading edge of lesions on potato dextrose agar. Fungal isolates were hyphal tipped and were incubated at 22°C with a 12 h photoperiod. After 21-days, all cultures were white to beige with pycnidia. DNA was extracted from cultures using the MagMAX Plant DNA Isolation Kit (Applied Biosystems, Foster City, CA). DNA amplification of a representative isolate (CD6C) was performed with primers ITS1/ITS4 (White et al. 1990) for the internal transcribed spacer (ITS), CYLH3F/H3-1b (Glass and Donaldson 1995) for histone 3 (HIS), and Ef1728f/EF1-986R (Carbone and Kohn 1999) for translation elongation factor 1-α (TEF). Amplification primers were used for bidirectional Sanger sequencing, reads were assembled using Geneious Prime (Biomatters, New Zealand), and identified using NCBI BLAST. BLAST results showed that the sequences for TEF, ITS, and HIS all had 100% pairwise identity to Diaporthe sp. 1-MI (MT909101, MT909099, MT909093, OK001342, MZ934713, OK001341). Futhermore, BLAST results showed that ITS and HIS have 100% pairwise identity D. humulicola (MN152929, MN180214). The TEF sequence also had 99.7% pairwise identity to D. humulicola (MN180209). Koch's postulates were conducted by inoculating six 3-mo-old 'Chinook' plants with conidia harvested from 28-day-old cultures and spraying 50 ml of inoculum (6 x 105 conidia/ml) or water to each plant. Plants were then stored in a greenhouse at 100% relative humidity at 22°C with a 14-h photo period. Lesions appeared on the adaxial side of the leaf after 21 days. D. humulicola was re-isolated from all infected leaf tissue, but not from any water inoculated plants and identified by conidial morphology using descriptions from Higgins et al. (2021). So far, Diaporthe sp. 1-MI appears to be synonymous with Diaporthe humulicola, but currently two names are being utilized (i.e. Diaporthe leaf spot and halo blight). In Higgins et al., (2021) it was proposed that the name halo blight might be more appropriate because disease symptoms are not confined to the leaves and cause significant blighting of cones. Halo blight caused by D. humulicola appears widespread in Michigan and Canada and may become an issue in other eastern North American growing regions with humid conditions.

First Report of Colletotrichum chrysophilum Causing Apple Bitter Rot in Spain.

Bitter rot of apple (Malus × domestica Borkh.) is a cosmopolitan disease affecting fruit and causes considerable losses worldwide. In September 2020, symptoms of bitter rot were observed on 'Pink Lady' apples in two orchards (~2.5 ha each) in Gualta, Catalonia, Spain (42.03803 N, 3.09831 E, and 42.03942 N, 3.10931 E). Early symptoms consisted of light brown and sunken circular lesions (1-4 mm) that enlarged over time, later becoming dark brown and water soaked, and extending cone-shaped toward the core. Sporulation was mostly noticed in larger lesions. Estimated incidence was 2% and 20% of 150 trees surveyed in each orchard, respectively. Twenty-one fungal isolates were obtained from diseased fruit by culturing small pieces of necrotic tissue on potato dextrose agar (PDA) amended with rifampicin at 50 mg/liter. Colonies on PDA looked identical. They were cottony, initially light-gray colored on top and darkening with age; colony reverse initially cream colored and darkening with age. Conidia were produced in orange acervular masses on Spezieller Nährstoffarmer Agar, and were aseptate, hyaline, cylindrical with obtuse ends, and measuring 10.1 to 14.7 × 4.5 to 7.1 μm (average 13.1 ± 1.04 × 5.3 ± 0.67 μm [mean ± SD], n = 50), with a mean length/width ratio 2.6 ± 0.39 (n = 16 isolates). Perithecia were not observed. Based on the conidial morphology, the isolates were tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from all isolates and six nuclear regions were amplified and partially sequenced: the internal transcribed spacer region of rDNA (ITS), the mating type protein 1-2-1 gene and the Mat1-2-1-Apn2 intergenic spacer region (ApMAT), actin (ACT), calmodulin (CAL), glyceraldehyde 3-P dehydrogenase (GAPDH), and tubulin (TUB2). The sequences for each region were 100% identical across all isolates. BLAST searches in GenBank showed 99-100% identity with sequences of various C. chrysophilum W.A.S. Vieira, W.G. Lima, M.P.S. Câmara & V.P. Doyle strains including the ex-type CMM4268 (Vieira et al. 2017). Sequences of the representative isolate CJL1080 were deposited in GenBank (ACT, MZ488944; ApMAT, MZ442299; CAL, MZ488945; GAPDH, MZ488946; ITS, MZ443972; TUB2, MZ442300). A multilocus phylogenetic analysis through Bayesian inference conducted with the obtained sequences and reference ones (Khodadadi et al. 2020) revealed that our isolates clustered well within C. chrysophilum, as suggested by BLAST results. To confirm Koch's postulates, isolates CJL1080 and CJL1095 were inoculated on 'Pink Lady' apples. Six surface-sterilized fruits per isolate were wound-inoculated four times each with either 20 μl of a conidial suspension (105 conidia/ml) or sterile distilled water (control). After 7 days of incubation in a moist chamber at 22°C, symptoms compatible with Colletotrichum infection were observed around the wounds, whereas control inoculations remained symptomless. The fungus was reisolated from all the lesions and identified through its morphological traits and DNA sequencing (ApMat, CAL, and GAPDH). No fungus was isolated from the controls. Taxa of the C. gloeosporioides species complex causing bitter rot have been recently reported in Europe (Grammen et al. 2019; Nodet et al. 2019). This is the first report of C. chrysophilum causing apple bitter rot in Spain, which expands the knowledge on the geographic distribution of this important pathogen of apple in Europe.

First Report of Molecular Detection of Leveillula taurica Associated with Powdery Mildew of Linseed (Linum usitatissimum) from India.

Linseed (Linum usitatissimum L.) is an important oil yielding crop cultivated widely in India. During the field survey, 2018-19 (Rabi season; January-February), characteristic powdery mildew disease symptoms were observed in around 90 hectares of linseed in Northern Karnataka (Agricultural Research Station, Raichur). The incidence of the disease was estimated, and it was found in the range up to 96 per cent. Initially, the symptoms were appeared as tan lesions, which later turned into small, circular chlorotic lesions and yellowing of the abaxial leaf surface with white fungal mycelial growth on the abaxial leaf surface. As the disease progress, white fungal mycelial growth was also noticed on the adaxial leaf surface. In severe infection, drying and premature defoliation of infected leaves was observed (Figure S1 A-D). A total of 12 samples were collected and subjected to morphological and molecular identification. Microscopic examination revealed the presence of conidia borne on conidiophores (simple, sometimes branched). Conidiophores were measured 83.17 - 116.45 µm (n=90). Conidia were single-celled, hyaline, dimorphic (pyriform and cylindrical), and 70.2 - 82.7μm × 15.1 - 26.3 μm (n=100) (Figure S1 E&F). A reference specimen was deposited at Agharkar Research Institute's (ARI) Ajrekar Mycological Herbarium (AMH 2015-07/AKC 2231-03/SKS/DKM). Since the conidial morphology and characters were very similar to Leveillula taurica the fungus was identified as L. taurica (Lév.) G. Arnaud. (Braun and Cook, 2012). Further, to confirm the identity based on ITS-rDNA barcoding, genomic DNA was isolated from conidial suspension harvested from powdery mildew affected linseed samples. ITS was amplified using powdery mildew-specific ITS universal primer pair PN23/PN34 and sequenced directly (Chen et al., 2008). The ITS regions were amplified at ~700bp. nBLAST analysis revealed that the ITS sequence shared 100% similarity with reference sequence AB045148.1 respectively. The representative sequences were deposited in GenBank with accession numbers OK036584 (LuLtR1) and OK036585 (LuLtR2). Further amplification of L1/L2 primer pair from ITS region were PCR amplified (Reddy et al., 2013) and an expected amplicon of 374bp was analyzed. Though, the ITS-rDNA and L1/L2 specific region analyzed, they are not specific exclusively to the L. taurica. However, combination of morphology and molecular sequence was used to confirm the identity. Further to prove Koch's postulates, pathogenicity tests were conducted on healthy linseed plants grown under greenhouse condition (28±2° C; >80% Relative Humidity). Conidia were harvested from infected leaves and the suspension was sprayed on 30 (40 days old) linseed plants. The development of powdery mildew symptoms was recorded on 22 plants 10 days after inoculation. Control plants inoculated with sterile water remained healthy, and no powdery mildew symptoms were observed. The identity was confirmed upon microscopic observation of spores from artificially inoculated plants, thus confirming the association. Leveillula taurica is known to cause powdery mildew on tomato, chilli and other crops. Amano (1986) and Saharan and Saharan (1994) reported the occurrence of L. taurica on linseed, however, no further details are available on the association, as well as molecular confirmation of L. taurica associated with linseed from India (Farr and Rossman, 2021). To the best of our knowledge, this is the first report of L. taurica associated with powdery mildew of linseed in India. Since it is the first confirmed record through molecular sequence analysis, it is important to know its geographical distribution and impact on linseed production to chalk the management strategies.

Morphologically Diversity of Colletotrichum sp. Conidia Associated With Anthracnose on Chili

Abstract The research conducted to observe the diversity of Colletotrichum sp. associated with anthracnose on chili. The diversity was observed from morphology of conidia. Colletotrichum sp. was isolated from Capsicum frustescens, C. annum (big chili variety and red curly chili) with anthracnose symptoms. In the isolation process, two isolates were obtained from C. annum big chili variety (BA and BB), 5 isolates were isolated from C. annum red curly chili (DPTK1, DPTK2, KA, KC, and KD), and 7 isolates from C. frustescens (DPTR1, DPTR2, RA, RB, RC, RD, and UPN4). Conidia length, shape, and color were observed. Isolate BA from C. annum big chili variety has the longest conidia than other isolate in average (16.10). Isolate RA from C. frustescens has the shortest conidia than other isolates (10.05). All isolates have hyaline color. Isolate BA, DPT K-1, RC, RD, and UPN4 have Fusiform shape. Isolate with cylindrical with rounded apices is BB, DPT K-2, KA, KD, DPT R-1, DPT R-2, RA, and RB. Conidia of Colletotrichum sp. isolated from Capsicum frustescens and C. annum have hyaline colour and diversity on the length and shape.

Identification of Fungi in the Botryosphaeriaceae Family Associated with Stem Blight of Vaccinium spp. in the Southeastern United States

Stem blight is a major disease of blueberry caused by Botryosphaeriaceae fungi. Chemical and cultural management options are limited, putting emphasis on breeding efforts to identify sources of resistance. The efficacy and durability of host resistance could be impacted by the species composition of the pathogen population in a region and by the isolates employed in the screenings used to identify the resistance. Samples (365) were collected from southern highbush (SHB) and rabbiteye blueberry (REB) cultivars from 28 sites in the southeastern US (AL, FL, GA, NC, and SC). Colony morphology identified 86% of the isolates as Botryosphaeriaceae. Conidia morphology and Maximum Likelihood analysis of the Internal Transcribed Spacer rDNA regions (ITS), translation elongation factor one alpha (tef1-α), and β-tubulin were used to identify isolates at genera or species level. A PCR-restriction fragment length polymorphism (PCR-RFLP) test was used to identify isolates to genus. Neofusicoccum and Lasiodiplodia were the predominant genera. N. kwambonambiense, N. ribis, L. theobromae and L. pseudotheobromae were the most common species isolated. Phylogenies conducted with a limited number of isolates indicated non-clonal and potentially diverse populations occur on blueberry that warrant additional study. Botryosphaeria corticis, B. dothidea, and Diplodia seriata were isolated infrequently.

Functional Analysis of Two Affinity cAMP Phosphodiesterases in the Nematode-Trapping Fungus Arthrobotrys oligospora.

Phosphodiesterases are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Two phosphodiesterases, PdeH and PdeL, have been identified from yeast and filamentous fungi. Here, the orthologs of PdeH and PdeL were characterized in a typical nematode-trapping fungus Arthrobotrys oligospora by gene disruption and phenotypic comparison. Deletion of AopdeH caused serious defects in mycelial growth, conidiation, stress response, trap formation, and nematicidal efficiency compared to the wild-type strain. In contrast, these phenotypes have no significant difference in the absence of AopdeL. In addition, deletion of AopdeH and AopdeL resulted in a remarkable increase in cAMP level during vegetative growth and trap formation, and the number of autophagosomes was decreased in ΔAopdeH and ΔAopdeL mutants, whereas their volumes considerably increased. Moreover, metabolomic analyses revealed that many metabolites were downregulated in ΔAopdeH mutant compared to their expression in the wild-type strain. Our results indicate that AoPdeH plays a crucial role in mycelial growth, conidiation, stress response, secondary metabolism, and trap formation. In contrast, AoPdeL only plays a minor role in hyphal and conidial morphology, autophagy, and trap formation in A. oligospora. This work expands the roles of phosphodiesterases and deepens the understanding of the regulation of trap formation in nematode-trapping fungi.

First Report of Neopestalotiopsis mesopotamica Causing Root and Crown Rot on Strawberry in Ecuador.

In Ecuador, strawberry production is located in the Andean region with an area of 1000 ha. Albion is the most popular cultivar due to its conical fruit shape, fruit size, bright red color, and sweetness. Since June 2014, farmers reported a reduction in the production cycle from 24 months to 6-8 months and a decreased yield of around 50% due to an unknown soil pathogen. Plant symptoms presented a reddish discoloration on new leaves, coming through the leaf apex to the petiole until turning wholly brown in old leaves leading to plant death. Additionaly, a brown-reddish spot inside the strawberry crown and root rot were reported (Fig. S1). In 2020, in Ecuador's most extensive production area, called Yaruqui (Pichincha province), 25 diseased plants were collected for pathogen isolation. The pathogen was isolated on water agar medium from the crowns internal tissue using 0.5 cm diseased plant fragments, previously disinfected with 2% sodium hypochlorite, and rinsed with sterile water. After two days, single hyphal tip was reisolated on potato dextro agar (PDA). A total of 18 pure isolates were grown at 25°C for 12 days, then 3-7 days of blacklight treatment was applied to induce sporulation. All the isolates presented a cottony beige mycelium with undulate edges. The conidia were ellipsoidal (range between 20.73 to 29 µm in length and 6.2 to 8.77 µm in width; n=60), multiseptated (4 segments) showing hyaline apical (3.8 to 5 µm) and basal (4.87 to 8 µm) cells, and three brown median cells, the second and third were darker than the fourth one, with one basal and 2 to 4 apical appendages (26.09 to 38.7 µm; Fig. S1). According to colony and conidia morphology, the isolates were identified as Neopestalotiopsis sp. (Dung et al. 2016; Essa et al. 2018; Maharachchikumbura et al. 2011). Five isolates were select randomly for DNA extraction and sequencing of the internal transcribed spacer (ITS) region (ITS4/ITS5), β-tubulin (Bt2b/ T1), and translation elongation factor 1-alpha (TEF-1a) region (EF1-728/ EF1-986) (Maharachchikumbura et al. 2014). DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al. 2018). The consensus sequences were deposited in GenBank with the following accession numbers: ITS, MZ047602; β-tubulin, MZ054301; and TEF-1a, MZ054302. A multilocus Bayesian inference phylogenetic tree was constructed using the concatenated sequences in the Beast software (version 1.8.4)(Drummond et al. 2012; Maharachchikumbura et al. 2014). The isolate in our study clustered with isolates of Neopestalotiopsis mesopotamica with a posterior probability of 1, confirming its identity (Fig. S2). For Koch's postulates, healthy plants were grown in sterile soil for four months. Conidia of the pathogen were suspended in potato dextro broth (PDB) (1 x 104 conidia/ml), and it was sprayed on 15 healthy plants that previously had their crowns wounded with a sterile needle (0.6 cm deep) at the four cardinal points. The control treatment (15 plants) was wounded and sprayed with PDB alone. The plants were maintained at 25°C and more than 85% relative humidity (Sigillo et al. 2020). Twelve days after inoculation, plants showed reddish discoloration on new leaves, and old leaves presented low-level wilt, rusty color, and necrotic petioles. Forty-one days later, 75% of the treated plants had severe wilt or were dead, showing root and crown rot. Control plants presented no symptoms of the disease. Reisolation of the pathogen from the disease crown tissues was done on water agar and PDA as previously described. The isolates presented the exact morphology of pure cultures obtained from field diseased strawberry crowns. The pathogenicity test was performed twice. To our knowledge, this is the first report of Neopestalotiopsis mesopotamica being the causal agent of root and crown rot on strawberries in Ecuador. N. iranensis and N. mesopotamica have been reported as causal agents of strawberries fruit rot and leaf lesions in Iran (Ayoubi and Soleimani, 2016), and N. clavispora was reported to be causing root and crown rot on strawberry plants in Argentina (Obregon et al. 2018). Disease diagnosis contributes to providing strategies against this new disease. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in strawberries in Ecuador.

First report of Pythium schmitthenneri on olive trees and in Morocco

In a grove in north-eastern Morocco in 2020, severe root rot and crown rot symptoms were noticed on olive trees (Olea europaea). Pythium schmitthenneri was identified as the pathogen based on conidia morphology and cytochrome oxidase subunit II (COXII) gene sequence analysis. Koch's postulates were verified by re-isolation of the fungus P. schmitthenneri from artificially inoculated olive seedlings after a pathogenicity test. This is the first time P. schmitthenneri has been identified as the cause of olive tree root and crown rot in Morocco.

Morphological and pathogenic characterization of Corynespora cassiicola isolates reveals specific genotypic interactions in soybean

AbstractSoybean target spot, caused by the fungus Corynespora cassiicola, is an important disease in northwestern Argentina (NWA). A cultural, morphological, and virulence characterization of 24 C. cassiicola isolates from different geographic localities in NWA was performed. The isolates were classified into five different cultural phenotypes, one of which has not been previously described. The ribosomal DNA internal transcribed spacer region sequence from five representative isolates (587 bp) exhibited 100% identity with C. cassiicola. A high variability of conidial morphology was observed among isolates, but no correlation with cultural phenotype was observed. When the soybean cultivar A 8000 RG was challenged with the 24 isolates, different degrees of virulence were observed, ranging from highly virulent to nonvirulent. No association of virulence with cultural or morphological characters was observed, but a relationship with geographical origin was demonstrated. Histopathological studies were performed on a nonvirulent and a highly virulent isolate. Microscopic observations of infected tissues of the former showed low mycelium development restricted to the upper epidermis, a thickening of the cuticle and primary wall of subepidermal cells, and accumulation of callose in phloematic vessels. In tissue infected with the latter, there was abundant mycelium development accompanied by cellular disorganization in mesophyll cells. Pathological challenges of isolates on nine different cultivars indicated that the degree of virulence of isolates depends on the plant genotype, demonstrating that the C. cassiicola–soybean interaction is highly specific. Understanding the genetic basis of this interaction will provide new information for better disease management and breeding strategies.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

The Small GTPase FgRab1 Plays Indispensable Roles in the Vegetative Growth, Vesicle Fusion, Autophagy and Pathogenicity of Fusarium graminearum.

Rab GTPases are key regulators of membrane and intracellular vesicle transports. However, the biological functions of FgRab1 are still unclear in the devastating wheat pathogen Fusarium graminearum. In this study, we generated constitutively active (CA) and dominant-negative (DN) forms of FgRAB1 from the wild-type PH-1 background for functional analyses. Phenotypic analyses of these mutants showed that FgRab1 is important for vegetative growth, cell wall integrity and hyphal branching. Compared to the PH-1 strain, the number of spores produced by the Fgrab1DN strain was significantly reduced, with obviously abnormal conidial morphology. The number of septa in the conidia of the Fgrab1DN mutant was fewer than that observed in the PH-1 conidia. Fgrab1DN was dramatically reduced in its ability to cause Fusarium head blight symptoms on wheat heads. GFP-FgRab1 was observed to partly localize to the Golgi apparatus, endoplasmic reticulum and Spitzenkörper. Furthermore, we found that FgRab1 inactivation blocks not only the transport of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane but also the fusion of endocytic vesicles with their target membranes and general autophagy. In summary, our results indicate that FgRab1 plays vital roles in vegetative growth, conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking in F. graminearum.

Diagnostic Guide for Alternaria Leaf Spot on Sugar Beet, Red Beet, and Chard

Alternaria leaf spot, caused by Alternaria spp., is a common foliar disease of sugar beet and other Beta vulgaris crop types. Known to be present in the United States since at least the 1950s, this disease has been found in all major sugar beet growing regions of the United States as well being reported as present everywhere beets are grown. Alternaria leaf spot symptoms are similar to several other common foliar diseases of beets including Cercospora leaf spot and the early stages of Phoma leaf spot. Symptoms and signs of this disease in the field do not provide enough information to help distinguish between diseases unless the viewer is readily familiar with the pathogen, especially when viewed with the unaided eye. Alternaria cultures can be identified to species using colony and conidial morphology on select culture media or using molecular markers. While not as common as Cercospora leaf spot, Alternaria leaf spot can result in significant reductions in yield and quality when not managed.

Nigrospora sphaerica Causing Leaf Blight Disease on Sesame in Pakistan

Sesame (Sesamum indicum L.) commonly known as 'til' is the most ancient and widely grown oilseed crop of Pakistan. During 2020, field survey conducted in various research fields of Faisalabad (31°26'00.2″N, 73°04'25.4″E) revealed the occurrence of characteristic leaf blight disease with an incidence of 10 to 13%. The symptoms were characterized by yellow-brown and irregular lesions. At later stages, the lesions expanded and the affected leaves turned grayish to dark-brown and finally became wilted. Symptomatic leaves (both the diseased and healthy tissue) were cut into approximately 2 × 2 mm pieces, surface sterilized with 1% sodium hypochlorite for 30 s, 70% ethanol for 30 s, and finally, three times in sterile distilled water prior to culturing on Potato Dextrose Agar (PDA) and incubated at 25 °C under a 12-h photoperiod for 7 days. To obtain pure cultures, hyphal tips of growing mycelia from leaf tissues were carefully isolated and transferred onto fresh PDA plates. Fungal colonies of 11 isolates were initially white, becoming light to dark-gray. The conidia were black, spherical to subspherical, and single-celled (12 to 14 × 18 to 20 μm) in diameter, which were borne on a hyaline vesicle at the tip of the conidiophore. Further, to identify the pathogen to the species level, genomic DNA was extracted using a modified CTAB protocol described by (Guo et al. 2000). The internal transcribed spacer (ITS) region of the ribosomal DNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. The sequences were submitted to GenBank (accession no. MW287214 for ITS and MW325222 for TEF1. The sequences comparison revealed 99% and 100% similarity to multiple sequences of N. sphaerica (GenBank accessions KX834822 and MN995332). On the basis of cultural features, conidial morphology and molecular data, the fungus was identified as Nigrospora sphaerica (Sacc.) Mason (Wang et al. 2017; Chen et al. 2018). To test the pathogenicity, fresh and healthy leaves of ten 6-week-old growth stage sesame plants were spray inoculated with a conidial suspension (105 conidia/ml), collected from a 7-day-old culture on PDA. In addition, 10 plants sprayed with sterile distilled water served as controls. Inoculated plants were covered with polyethylene bags to maintain high humidity and kept at 28°C, and observations were made at regular intervals. After 8-10 days of inoculation, leaves developed blight symptoms similar to those observed on naturally infected leaves, whereas control leaves remained asymptomatic. The pathogen was re-isolated from the inoculated leaves, and its identity was confirmed by morphological and molecular (ITS and TEF1) means, thus fulfilling Koch's postulates. N. sphaerica is distributed on a wide range of hosts and has been reported from different host genera including monocotyledonous and dicotyledonous (Wang et al. 2017). Previously, N. sphaerica has been reported to cause leaf blight of Cunninghamia lanceolata in China (Xu and Liu, 2016). To the best of our knowledge, this is the first report of Nigrospora sphaerica as the causal agent of leaf blight of sesame in Pakistan. Because sesame is an important oilseed crop of Pakistan, appropriate disease management practices should be developed and implemented.

First report of Colletotrichum fioriniae and C. nymphaeae as Causal Agents of anthracnose on walnut in Italy.

English walnut (Juglans regia L.) is species grown either for high quality wood or fruit production. In Italy walnut cultivation occupies an area of about 4600 ha (FAOSTAT, http://www.fao.org/faostat, 2020). In 2019-2020, walnut fruits (cv Lara) with anthracnose symptoms were collected from walnut orchards in Province of Venice (Northern Italy). Affected fruits showed necrotic and circular lesions with acervuli in the center causing the complete mummification of the fruit as described by Da Lio et al., 2018. Orange conidial masses appeared under wet conditions. The fungus was isolated from necrotic tissues and conidial masses were put on potato dextrose agar (PDA) medium. Plates were incubated at 25°C for 5 to 7 days. The colonies were white to pink on the upper side and pink with black spots on the reverse. Acervuli formed and produced conidial masses on PDA after 6 days. Culture and conidial morphology were in concordance with published descriptions of C. acutatum sensu lato (Damm et al., 2012). To confirm the identity, internal transcribed spacers (ITS), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White at al. 1990), GDF1/GDR1 (Guerber et al., 2003), ACT512F/ACT783R and BT2Fd/BT4R primers (Da Lio et al., 2018). The isolates belonged to two different species of Colletotrichum acutatum complex: C. fioriniae (Marcelino & Gouli) and C. nymphaeae (Pass). Sequences of two representative isolates C. fioriniae CREADC-F2317 and C. nymphaeae CREADC-F2372 were deposited in GenBank with accession numbers MZ153170 and MZ191794 (ITS), MZ203522 and MZ224013 (GAPDH), MZ203521 and MZ224012 (ACT), and MZ203523 and MZ224014 (TUB2). For all the genes, isolates had a 100% similarity to multiple C. fioriniae and C. nymphaeae accessions, respectively. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA 6.0 (Tamura et al., 2013). The phylogenetic analysis grouped the isolates in the C. fioriniae and nymphaeae clusters respectively. The two isolates CREADC-F2317 and CREADC-F2372 were used to confirm pathogenicity on walnut fruits. Fruits of cv Lara were surface disinfected by dipping in 3% NaOCl for 1 min, rinsed in sterile distilled water, and arranged in sterile humid chambers. Fruits were wounded with a sterile needle then inoculated with 20 μl of 106 conidia/ml suspensions of each isolate (one wound per fruit). Fruit treated with sterile distilled water served as a control. Inoculations were conducted on three fruits per replicate and three replicates per treatment arranged in a complete block randomized design. After 7 days incubation at 25 ± 1°C, all the inoculated fruits showed typical anthracnose symptoms and lesions with cream to salmon pink acervuli, whereas the controls remaied healthy. The species C. nymphaeae and C. fioriniae were reisolated from the rotted fruit. Pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates. The species C. fioriniae and C. nymphaeae have been described affecting numerous species worldwide (Damm et al., 2012). C. fioriniae and C. nymphaeae have been previously reported to cause severe anthracnose on walnut, C. fioriniae in France (Da Lio et al., 2018) and Hungary (Varjas et al., 2019) and C. nymphaeae in France (Da Lio et al., 2018) and Brazil (Savian et al., 2019). To our knowledge, this is the first report of C. fioriniae and C. nymphaeae as causal agents of walnut anthracnose in Italy.