Conidial Length Research Articles

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates on Galleria mellonella

Background Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Characterization of Endophytic Entomopathogenic Fungal Isolates: Growth Dynamics, Viability, Density, and Morphological Attributes

Aims: This study aims to comprehensively characterize endophytic entomopathogenic fungal isolates, focusing on their growth dynamics, viability, density, and morphological attributes, with implications for biocontrol strategies against insect pests. Study Design: The research tracked radial growth rates of fungal isolates over 21 days, conducted conidial viability assessments at 24 and 48 hours, measured conidial density, and performed morphological analysis to identify distinct characteristics. Methodology: Endophytic entomopathogenic fungal isolates, including Beauveria bassiana and Lecanicillium lecanii, were subjected to radial growth rate tracking, viability assessments, density measurements, and morphological analysis. Statistical analysis was employed to identify significant variations among isolates and time intervals. Results: Significant variations in radial growth rates were observed among isolates, with Beauveria bassiana isolate ChBb exhibiting a rate of 3.66 mm/day, followed by B. bassiana isolate NaBb with 3.92 mm/day, and Lecanicillium lecanii isolate KmLl with 3.06 mm/day. Conidial viability assessments revealed temporal dynamics, with NaBb maintaining the highest viability at both 24 and 48 hours. Additionally, significant differences in conidial density were noted, with NaBb exhibiting the highest density followed by ChBb and KmLl. Morphological analysis unveiled distinct characteristics among isolates, including differences in conidia length, width, and length-to-width ratio. Conclusion: The findings underscore the potential of Beauveria bassiana and Lecanicillium lecanii isolates in biocontrol strategies against insect pests, highlighting their varying growth dynamics, viability, density, and morphological attributes. These insights contribute to the understanding of entomopathogenic fungi and their applications in sustainable pest management practices.

First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the β-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

EFFECT OF CULTURE MEDIA ON GROWTH AND SPORULATION OF Colletotrichum lindemuthianum (SACC AND MAGN) ISOLATES CAUSING ANTHRACNOSE OF FIELD BEAN

Anthracnose (Colletotrichum lindemuthianum) is the most important fungal disease affecting field bean. Growth and sporulation of thirty six isolates of C. lindemuthianum collected from five agro-climatic zones of Andhra Pradesh was evaluated in the year 2017-18 on four different solid and liquid culture media. Results revealed that potato dextrose agar was the best for both radial growth (66.74 mm) and dry mycelial weight (264.74 mg), whereas, host leaf extract agar significantly supported more sporulation 7.3 x104. The least mycelia growth of 39.32 mm, dry mycelial weight of 234.70 mg and sporulation 2.9 x104 were observed on Sabourband’s agar. Among the different isolates, Cl-33 grew fast with mycelial growth and sporulation (78.86 mm and 8.4x104) and Cl-32 produced the highest dry mycelial weight 266.75 mg). The least radial growth (31.35 mm), the lowest sporulation (4.1 x104) and lowest mycelial dry weight (227.75 mg) was recorded in CI-28, CI-1 and CI 17, respectively. Mean conidial length of 36 isolates ranged from 11.87 to 13.03 µm with the maximum length (13.03 µm) recorded in host leaf extract agar, while minimum was recorded in oat meal agar. Width of conidia varied from 3.59 to 4.11 µm on different media and 3.53 to 4.23 µm in different isolates.The growth phase study revealed that increase in the dry mycelial weight of fungus upto 15 days of incubation and growth declined from 17th day onwards.

Use of ginger extract and bacterial inoculants for the suppression of Alternaria solani causing early blight disease in Tomato

Early blight (EB), caused by Alternaria solani, is a serious problem in tomato production. Plant growth-promoting rhizobacteria promote plant growth and inhibit plant disease. The present study explored the bio-efficacy of synergistic effect of rhizobacterial isolates and ginger powder extract (GPE) against tomato EB disease, singly and in combination. Six fungal isolates from symptomatic tomato plants were identified as A. solani on the basis of morphological features i.e., horizontal septation (6.96 to 7.93 µm), vertical septation (1.50 to 2.22 µm), conidia length (174.2 to 187.6 µm), conidial width (14.09 to 16.52 µm), beak length (93.06 to 102.26 µm), and sporulation. Five of the twenty-three bacterial isolates recovered from tomato rhizosphere soil were nonpathogenic to tomato seedlings and were compatible with each other and with GPE. Out of five isolates tested individually, three isolates (St-149D, Hyd-13Z, and Gb-T23) showed maximum inhibition (56.3%, 48.3%, and 42.0% respectively) against mycelial growth of A. solani. Among combinations, St-149D + GPE had the highest mycelial growth inhibition (76.9%) over the untreated control. Bacterial strains molecularly characterized as Pseudomonas putida, Bacillus subtilis, and Bacillus cereus and were further tested in pot trials through seed bacterization for disease control. Seeds treated with bacterial consortia + GPE had the highest disease suppression percentage (78.1%), followed by St-149D + GPE (72.2%) and Hyd-13Z + GPE (67.5%). Maximum seed germination was obtained in the bacterial consortia + GPE (95.0 ± 2.04) followed by St-149D + GPE (92.5 ± 1.44) and Hyd-13Z + GPE (90.0 ± 2.04) over control (73.8 ± 2.39) and chemical control as standard treatment (90.0 ± 2). Ginger powder extracts also induce the activation of defence-related enzymes (TPC, PO, PPO, PAL, and CAT) activity in tomato plants. These were highly significant in the testing bacterial inoculants against A. solani infection in tomato crops.

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates in Galleria mellonella

Background Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Morph-Cultural Variability among Different Isolates of Alternaria alternata Causing Leaf Spot of Ber (Ziziphus mauritiana Lamk) in Eastern Uttar Pradesh

Alternaria alternata is one of the most important foliar fungi causing leaf spot of ber. Leaf spot diseased samples were collected from different districts of Uttar Pradesh. Six different isolates of A. alternata were obtained in pure culture and designated as Aa1, Aa2, Aa3, Aa4, Aa5 and Aa6. The results revealed that there is a greater variation among different isolates of A. alternata. The maximum mycelial growth after 10 DAI (days after incubation) was found in Aa2 (90.10 mm) followed Aa5 (88.36) and Aa6 (86.53). Colony characters like color varied as greenish (Aa1), dark olivaceous green (Aa2), brown blackish (Aa3 and Aa6), brownish green with black center (Aa5), Greenish brown with white center (Aa4) and growth was slow (Aa4 and Aa5), medium (Aa1 and Aa6) and fast (Aa2 and Aa3). Growth appearance varied from fluffy (Aa1, Aa4, Aa5 and Aa6) and compressed (Aa2 and Aa3) with margin regular (Aa2 and Aa4) or irregular margin (Aa1, Aa3, Aa5 and Aa6). Sporulation was recorded in all six isolates but very good sporulation was observed in Aa2, Aa4 and Aa5. Highest average conidial length was recorded in isolates Aa4 (38.88μm) followed by Aa3 (30.20μm). Highest average width was recorded in Aa5 (15.06μm) followed by Aa3 (14.04 μm). Maximum beak length was observed in Aa2 (17.28 µm). The number of septa also varied among isolates. Maximum number of transverse septa was found in Aa5 (4-5). Maximum number of longitudinal septa was found in Aa5 (2-4).

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

Variability of Alternaria brassicicola Causing Leaf Blight of Cabbage in Major Cabbage Growing Districts of Assam, India

The research was conducted at Assam Agricultural University, Jorhat, Assam, India during September, 2021–May, 2022 to study the variation of Alternaria brassiciola infecting cabbage. Fifteen isolates of Alternaria brassicicola which infected cabbage were collected from six different districts in Assam and their variability was investigated. Within the in vitro setting, it was observed that all isolates exhibited significant variation in terms of conidial length, breadth, and septal count. The isolates displayed a range of average conidial length and breadth spanning from 20.45µm to 29.74µm and 6.56µm to 9.89µm, respectively. Additionally, the isolates exhibited a variation in transverse septa with counts ranging from 4 to 6 and longitudinal septa, ranging from 0.13 to 0.41. Regarding the colony colour, four distinct variations, ranging from dark grey-green to dark brown-green and from mild olive green to greyish olive green were observed. These colonies featured edges in shades of white, greyish, brownish, or green edges and their shapes varied between round and irregular, accompanied by either a fluffy or compressed growth pattern. The diameter of the radial growth, observed on the fifteenth day after inoculation, varied from 57.3 mm to 84.2 mm. In RAPD analysis it was evident that the Jorhat isolates (AbJor_2) and the Barpeta isolate, (AbBa_1) exhibited the highest genetic similarity, as indicated by the lowest genetic distance with the coefficient of similarity 0.484. Conversely, the Jorhat isolates (AbJor_3) and Barpeta isolate (AbBa_1) shared the least genetic similarity, with the coefficient of similarity 0.021.

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates on Galleria mellonella.

Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 7 spores ml -1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day -1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 8 conidial ml -1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

Uncovering the Morphological and Genetical Heterogeneity of Pyricularia oryzae (cooke) sacc. in Southwestern Region of Bangladesh

Significant yield losses are reported due to blast disease caused by Pyricularia oryzae across all rice growing areas of the world. Though, management strategies reduce disease significantly but blast epidemics are still common. This research was aimed to determine the variability among the 19 Pyricularia oryzae isolates of South-Western regions of Bangladesh. Morphological variability was determined based on nine morphological components like front mycelial color, reverse mycelial color, growth behavior, conidial length (μm), conidial width (μm), conidial color, radial growth at 12 days (mm), 14 days (mm) and 16 days (mm) after incubation. Cluster analysis considering nine morphological components showed five distinct groups. Front and reverse mycelial color, growth behavior and conidial length played more than 80% role in generating variation among the nine tested components. Higher genetic variation was also detected among the 19 isolates of P. oryzae. Here, larger numbers of amplified DNA fragments was formed by Primers UCB-155 which showed distinct polymorphism. Between two clusters, Cluster II consists of 18 isolates that were originated from different origin but having maximum 94% genetical similarity. Cluster I contain only one isolates PO-16. From pair wise distance matrix, maximum distance (80%) was found between PO-16 and PO-03 isolates and minimum distance (6%) was obtained between the isolates PO-11 and PO-12. Results of this experiment revealed that, higher genetic diversity among isolates of P. oryzae existed in the south western region of Bangladesh but there has no direct relation with genetic diversity and geographical origin.

Variability Among the Isolates of Alternaria Species Causing Blight in Rape-Seed Mustard (Brassica Juncea L.)

Variation in the mycelial growth was observed within the isolates of Alternaria brassicae and A. brassicicola on PDA medium. The isolates were also found to differ from each other in respect to cultural and conidial characters. The colonies were cottony or fluffy in appearance with varied colour from olivaceous green, white, off white, light brown to brown. Colony margins were smooth, rough or wavy and zonation was either present or absent. In Alternaria brassicae isolates, conidial length varied from 92.5 to 116.2 μm, conidial width varied from 11.8 to 17.2 μm, horizontal septa varied from 3.4 to 5.3, vertical septa varied from 0.3 to 1.4 μm and beak length varied from 68.4 to 88.4 μm. However, in A. brassicicola isolates, conidial length varied from 46.8 to 52.8 μm, conidial width varied from 12.8 to 14.6 μm, horizontal septa varied from 3.2 to 3.7 and vertical septa varied from 0.2 to 0.8 μm. Beak was absent in the isolates of A. brassicicola. In the isolates of A. brassicae and A. brassicicola, maximum per cent disease intensity and spot size on all the genotypes was observed in Ab11 and Abl2, respectively. Bangladesh J. Bot. 52(2): 331-336, 2023 (June)

Investigation on the Influence of Production and Incubation Temperature on the Growth, Virulence, Germination, and Conidial Size of Metarhizium brunneum for Granule Development.

Important for the infection of an insect with an entomopathogenic fungus and its use as a plant protection agent are its growth, conidiation, germination, and virulence, which all depend on the environmental temperature. We investigated not only the effect of environmental temperature but also that of production temperature of the fungus. For this purpose, Metarhizium brunneum JKI-BI-1450 was produced and incubated at different temperatures, and the factors mentioned as well as conidial size were determined. The temperature at which the fungus was produced affects its subsequent growth and conidiation on granule formulation, the speed of germination, and the conidial width, but not its final germination or virulence. The growth and conidiation was at its highest when the fungus was produced at 25 °C, whereas when the germination was faster, the warmer the fungus was produced. The incubation temperature optimum of JKI-BI-1450 in relation to growth, speed of germination, and survival time was 25-30 °C and for conidiation 20-25 °C. Conidial length decreased with increasing incubation temperature. Although the fungus could not be adapted to unfavorable conditions by the production temperature, it was found that the quality of a biological control agent based on entomopathogenic fungi can be positively influenced by its production temperature.

The chalara-like anamorphs of Leotiomycetes

The chalara-like anamorphs of Leotiomycetes are phialidic hyphomycetes with cylindrical collarettes and deeply seated sporulating loci, and hyaline, aseptate or septate, cylindrical conidia. They are commonly found on plant litters in both terrestrial and submerged environments, and with broad geographical distribution. This paper reports our research result of diversity, taxonomy and phylogeny of these fungi in China, which is based on a systematic study by using an integrated approach of literature study, morphological observation and phylogenetic analyses of 153 chalara-like fungal species with diversified morphology in conidiomata, setae, conidiophores, phialides and conidia. The phylogenetic analyses employing different datasets of SSU, LSU and ITS sequences of 116 species showed that these chalara-like fungi were paraphyletic and scattered in 20 accepted genera belonging to five families of Leotiomycetes: Arachnopezizaceae, Hamatocanthoscyphaceae, Helotiaceae, Neolauriomycetaceae and Pezizellaceae. Additional six genera, Ascoconidium, Bioscypha, Chalarodendron, Didonia, Phaeoscypha and Tapesina, all reported with chalara-like anamorphs in literatures, are also accepted as members of Pezizellaceae or Leotiomycetes genera incertae sedis. Among of these 26 accepted genera of chalara-like fungi in Leotiomycetes, 17 genera are asexually typified genera (Ascoconidium, Bloxamia, Chalara, Chalarodendron, Constrictochalara, Cylindrochalara, Cylindrocephalum, Leochalara, Lareunionomyces, Minichalara, Neochalara, Neolauriomyces, Nagrajchalara, Parachalara, Stipitochalara, Xenochalara and Zymochalara), and 9 are sexually typified genera (Bioscypha, Bloxamiella, Calycellina, Calycina, Didonia, Hymenoscyphus, Mollisina, Phaeoscypha and Tapesina). The phylogenetic significance of conidial septation in generic delimitation was further confirmed; while other morphologies such as conidiomata, setae, conidiophores, phialides, conidial length, and conidial ornamentation have little phylogenetic significance, but could be used for species delimitation. The polyphyletic genus Chalara s. lat. is revised with monophyletic generic concepts by redelimitation of Chalara s. str. in a narrow concept, adaption of the emended Calycina to also include asexually typified chalara-like fungi, reinstatement of Cylindrocephalum, and introduction of six new genera: Constrictochalara W.P. Wu & Y.Z. Diao, Leochalara W.P. Wu & Y.Z. Diao, Minichalara W.P. Wu & Y.Z. Diao, Nagrajchalara W.P. Wu & Y.Z. Diao, Parachalara W.P. Wu & Y.Z. Diao and Stipitochalara W.P. Wu & Y.Z. Diao. Chaetochalara becomes a synonym of Chalara s. str., and the known species are disassembled into Chalara s. str. and Nagrajchalara. The polyphyletic genus Bloxamia is also redefined by introducing the new genus Bloxamiella W.P. Wu & Y.Z. Diao for B. cyatheicola. Five existing species of Chalara s. lat. were excluded from Leotiomycetes and reclassified: Chalara breviclavata as Chalarosphaeria breviclavata W.P. Wu & Y.Z. Diao gen. et sp. nov. in Chaetosphaeriaceae, C. vaccinii as Sordariochalara vaccinii W.P. Wu & Y.Z. Diao gen. et sp. nov. in Lasiosphaeriaceae, and three other Chalara species with hyaline phialides, C. hyalina, C. schoenoplecti and C. siamense as combinations of Pyxidiophora in Pyxidiophoraceae. For biodiversity of these fungi in China, a total of 80 species in 12 genera, including 60 new species, 17 new records and 1 new name, were discovered and documented in this paper. In addition, five species including three new species are reported from Japan. In connection to this revision, a total of 44 new combinations are made. The identification keys are provided for most of these genera. Future research area of these fungi should be the phylogenetic relationship of several sexually typified genera such as Bioscypha, Calycellina, Calycina, Didonia, Phaeoscypha, Rodwayella and Tapesina, and systematic revision of existing names under the genera Bloxamia, Chaetochalara and Chalara.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

Cytospora paraplurivora sp. nov. isolated from orchards with fruit tree decline syndrome in Ontario, Canada.

A new species of Cytospora was isolated from cankered wood of Prunus spp. during a survey of orchards exhibiting symptoms of fruit tree decline syndrome in southern Ontario, Canada. We found isolates that are morphologically similar to species in the Cytosporaceae family, which is characterized by single or labyrinthine locules, filamentous conidiophores or clavate to elongate obovoid asci and allantoid, hyaline conidia. Multi-gene phylogenetic analysis of ITS, LSU, act and tef1- α showed that the isolates form a distinct clade, sister to Cytospora plurivora. Morphologically, our isolates showed differences in the length of conidia and culture characteristics compared to C. plurivora, suggesting the establishment of a new species. The species is described as Cytospora paraplurivora sp. nov. and placed in the family Cytosporaceae of Diaporthales. Additionally, we sequenced, assembled and characterized the genome of the representative isolate for this new species. The phylogenomic analysis confirms the species order and family level classification. C. paraplurivora sp. nov. has the potential to severely affect stone fruits production, causing cankers and dieback in stressed trees, and eventually leads to tree decline. Pathogenicity tests show that the species is pathogenic to Prunus persica var. persica.

Variability in Alternaria lini causing leaf blight disease of linseed (Linum usitatissimum L.)

Alternaria blight is an important disease of linseed that hampers its productivity and oil content. The pathogen is geneticallydiverse showing variability in respect of cultural, morphological and pathogenic characters. Variability in seventeen isolates of Alternaria lini was carried out. Studies on cultural variability showed considerable variation among the isolates. The colony diameter varied from 46.67- 71.67 mm after 96 hrs. of incubation and most of the isolates recorded medium growth rate (50-64mm). The isolates Alt-6,Alt-7 Alt-12 showed fast growth habit (>65mm). The colony colour varied from grayish white, grey, dark olive green to grayish black. Sporulation in most of the isolates were late and concentric ring was present only in four isolates (Alt 3, Alt 8, Alt 13, Alt 14) while zonation was completely absent in other isolates. Morphologically different isolates of A. lini revealed variation with respect to mycelium width, size, shape, colour and septation of conidia. Mycelial width of most of the isolates having moderate to long width. Conidial length ranged from 24.87-33.68 mm similarly breadth of conidia ranged from7.54-10.67 mm. Transverse septation were more than longitudinal septation. Variation in beak size ranged from 3.5-6.4 mm while colour of conidia varied from brown to dark brown colour and most of the isolates exhibited obclavate, oval to obclavate shaped conidia while one isolate appeared as oval shaped. Studies on pathogenic variations revealed that the variation observed in accordance with the latent period, lesion size, lesion shape and plant disease index. The latent period of A. lini isolates varied from4.75 to 6.25 days with shortest (4.75 days) latent period recorded by isolates Alt-3 and Alt-11 and longest latent period of 6.25 days was observed in isolates Alt-6, Alt-14, Alt-16. The size of lesion varied from 2.25 to 4.75 mm and maximum lesions size (4.75 mm) was observed in isolate Alt-4 whereas minimum lesion size (2.25 mm) was observed in isolate Alt-5. The lesion produced after inoculation were elongated, oval and irregular in shape. The average number of lesions ranged from 2.75 to 4.00 with maximum number (4.00) of lesion recorded in isolate Alt-13. Plant disease index revealed that Meera and Nagarkot were more severe/virulent compare to Divya and Priyam in all the isolates.

First Report of Colletotrichum boninense Causing Anthracnose on Aucuba japonica in Guizhou Province of China.

Aucuba japonica is a plant with colorful leaves that is widely used in landscaping. Due to its shade tolerance and cold resistance (Li et al. 2013), A. japonica has become a dominant plant species in Guiyang city, China. From 2018 to 2021, an anthracnose disease was observed on A. japonica in Huaxi District (26°23'03'' N, 106°37'58'' E), Guiyang, Guizhou Province. The incidence of anthracnose in newly planted A. japonica reached 90%, resulting in a mortality rate of 30%. This has become a major disease for A. japonica in Guiyang. Typical symptoms include faded spots that initially appear at the infection center, followed by black-brown lesions with irregular edges. At the latter stage, slightly raised black spots are seen arranged in a wheel pattern. In severe cases, diseased leaves fall off. To identify the pathogen, leaf pieces (5 mm × 3 mm) containing symptomatic tissue with healthy margins were excised. The pieces were sterilized with 75% ethanol for 1 min, rinsed three times with sterile water, and cultured on potato dextrose agar (PDA) in Petri dishes at 26°C. White to cream colonies were developed after growth on PDA for 2 days. Mycelial growth ranged from 4.8 to 5.0 mm/day. Conidia were cylindrical, obtuse apex, and protruding base, with dimensions of 18.5 to 20.0 × 7.0 to 9.0 μm (n = 20). Conidial length:breadth ratio was 2-3 (n = 20). Acervuli and sclerotia had not been seen on PDA. To confirm the species of the isolate, PCR was performed on extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the calmodulin (CAL) gene, and the large subunit (LSU) region by using the primer pairs ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), CAL228F/CAL737R (Carbone & Kohn 1999), and NL1/NL4 (Ziemiecki et al. 1990), respectively. The obtained 553 bp ITS sequence (Genbank accession no. MZ424503) showed 99.82% similarity with several C. boninense sequences (MN429163, MN542218, KM520024, and KM520014). The obtained 233 bp GAPDH sequence (OM643394) showed 99.07% similarity with C. boninense (MT602277, GQ221769, MK544890, and MK544888). The obtained 763 bp CAL sequence (OM313386) showed 99.32% similarity with C. boninense (MK569127). The obtained 584 bp LSU sequence (OK513272) showed 99.83% similarity with C. boninense (MH876452, MH877053, and MH876450). Phylogenic trees based on the sequences of ITS, GAPDH, and CAL (Damm et al. 2012), the species of isolate was confirmed as C. boninense. To confirm pathogenicity and to fulfill Koch's postulates, leaves of five A. japonica plants were wounded with a sterilized hypodermic needle and inoculated with 1 μL of a conidial suspension (106 conidia/mL). As control, 5 plants were inoculated with 1 μL of sterile water. After 7 days in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, symptoms appeared on all inoculated leaves, while the control leaves remained healthy. The pathogen was reisolated from the inoculated leaves, and was confirmed to be C. boninense using the same morphological and molecular methods as before. C. boninense has been reported to infect many plant species, including Capsicum annuum, Rosa chinensis, and Eucalyptus robusta (Ding et al. 2021; Tozze et al. 2009; Zhang et al. 2018). Another Colletotrichum pathogen, C. fructicola, was reported to cause anthracnose on A. japonica in China (Gong et al. 2016). To our knowledge, this is the first report of A. japonica anthracnose disease caused by C. boninense in Guizhou province, China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.