Conidial Dimensions Research Articles

Identification and characterization of Botrytis cinerea causing gray mold on tomato in Malaysia

Botrytis cinerea, commonly known as gray mold, is a pervasive fungal pathogen that affects a wide range of plant species, leading to significant agricultural losses. The identification of Botrytis cinerea in Malaysia is crucial for protecting the agricultural sector, minimizing economic losses, ensuring food security, maintaining export quality, addressing environmental concerns, and advancing scientific research. In the present research, tomato fruits collected from Cameron Highlands, Pahang, Malaysia showed gray mold disease symptoms of B. cinerea. The fungal isolates were examined morphologically for colony colour, growth rate, conidiophores, conidia shape, and sclerotia on PDA and V8 agar. According to the results, conidiophores appeared in grape shape and length was range of 21.26-32.52 μm, ovoid conidial dimensions were in the range of 10.03-16.08 × 7.37-11.15 μm and sclerotia size was range 1.91-4.50 × 1.70-4.00 mm. All isolates were attributed to the morphospecies Botrytis cinerea on account of these characteristics. The resulting sequences deposited in GenBank were accessions MT012053 to MT012062, respectively. A BLAST analysis of the resulting 550-bp nucleotide sequences showed 99-100% identity closest matched to B. cinerea. The pathogenicity experiments showed P6 isolates of B. cinerea were highly pathogenic and caused gray mold development on tomato fruits that led to severe symptoms in five days. Meanwhile, the least pathogenic isolate was P9. In terms of temperature, B. cinerea grew faster on PDA at 20ºC, slower grew below 20ºC and did not grow at 25ºC. Identification and characterization of B. cinerea on tomato could potentially provide information to assist disease management strategies for B. cinerea.

First Report of Neofusicoccum parvum causing leaf spot on Camellia japonica in China.

Camellia japonica is an important garden landscape plant in southern China. In April 2022, leaf spot symptoms were observed at the camellia garden of Jiaying University (24°32'83″N, 17 116°12'31″E) in Meizhou city, Guangdong Province, China. The initial symptoms were grayish brown spots on the leaves, as the disease progressed, the lesions were enlarged and affected the whole leaf and eventually led to the loss of its ornamental value. The disease incidence was above 15%. Leaf pieces (5 × 5 mm) from 3 diseased Camellia leaves were sterilized in 75% ethanol for 1 min, then in 1% NaOCl for 1 min; and rinsed three times with sterile water. Leaf pieces were inoculated on potato dextrose agar (PDA) medium and incubated at 25 °C. Three days later, fungal colonies initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. Conidia were single-celled, hyaline, ellipsoidal and without septa. Dimensions of conidia (n≥50) were 14.27 to 20.65 × 4.28 to 6.56 μm. The morphological characteristics matched the genus Neofusicoccum (Pavlic et al. 2009). For molecular identification, the rDNA internal transcribed spacer (ITS1, 5.8S and ITS2) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) of a representative isolate SC6-2 were amplified using the primer pairs ITS1/ITS4, EF1/EF2 and BT2a/-BT2b, respectively (Golzar and Burgess，2011). The sequences obtained were deposited in GenBank (accession nos. PP064173, PP479650 and PP082457 for ITS, tef1-α and tub2, respectively). Nucleotide BLAST analysis showed a 99.81% homology with N. parvum (519/520 bp, OQ509869; 519/520 bp, KF294003; 518/519 bp KF293989) for ITS, 100% homology with N. parvum (398/398 bp, MN318108; 398/398 bp, MK294085; 398/398 bp, MH936021) for tub2, and >99% homology with N. parvum (259/259 bp, 100%, MW390561; 263/265 bp, 99.25%，MN175952; 263/265 bp, 99.25%, MK781982) for tef1-α. The combined phylogenetic analyses (ITS, tef1-α, and tub2) showed that the sequence of the tested isolate and the corresponding sequence of N. parvum (CMW9081, SHSJ1-2) in GenBank grouped in the same branch of the phylogenetic tree. Based on morphological characters, DNA sequencing, and the phylogenetic tree, it can be determined that the pathogen was Neofusicoccum parvum. Inoculation on Camellia leaves was performed to confirm pathogenicity. Nine healthy camellia leaves were pin-pricked with a sterile needle and inoculated with mycelial plugs of isolate SC6-2. Nine other healthy leaves were pin-pricked and inoculated with noncolonized PDA plugs as control leaves. The inoculated leaves were maintained on water agar solid medium at 25°C. To keep a high-humidity environment the inoculation sites were covered by moistened cotton for 2 days. The experiment was repeated three times. Five days after inoculation, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 6 days. The fungal isolates recovered from inoculated leaves were morphologically identical to the N. parvum isolates originally recovered from symptomatic leaves collected in the field, fulfilling Koch's postulates. Neofusicoccum parvum is an aggressive pathogen that causes severe disease on important tree and woody species (Liddle et al. 2019). It has been reported that N. parvum can infect the leaves and branches of grapes (Otoya-Martinez et al. 2023), dieback on Camellia japonica (Pintos, et al. 2012), Brazilian pepperwood (Bertetti et al. 2022), mango (Giancarlo et al. 2023) and other plants. To our knowledge, this is the first report of N. parvum causing leaf spot on Camellia japonica in China.

First Report of Anthracnose on Banana Peppers Caused By Colletotrichum scovillei in New York.

In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence was at least 60%. Affected developing and mature fruit had at least one tan, soft, sunken lesion with salmon-colored spore masses surrounded by brown, necrotic margins. Microscopic observation of the lesions identified acervuli and setae typical of Colletotrichum spp. Isolations were made from these lesions by spreading conidia from the acervuli on 2% water agar (WA) + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred onto clarified V8 juice agar (CV8) and incubated at 20°C. The isolation frequency was 100% and a total of six isolates were obtained: Coll23Pep001, Coll23Pep003, Coll23Pep005, Coll23Pep007, Coll23Pep008, and Coll23Pep010. After 10 days, colonies were subcultured to potato dextrose agar (PDA) and CV8. On PDA, colonies appeared off-white to dark gray with sparse aerial mycelia. On CV8, the colony was pale gray with acervuli and orange-colored spore masses in the center. Conidia were hyaline, smooth and fusiform to round, and tapered at both ends. Mean conidial dimensions (n = 20) were 20.2 (13.75 to 25) µm long × 4.7 (3 to 6.25) µm wide. To confirm the identity of the isolates, DNA was extracted, and PCR performed to amplify the internal transcriber spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), and actin (ACT) (primers ACT-512F/ACT-783R; Carbone and Kohn 1999) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (primers GDF1/GDR1; Guerber et al. 2003). Pairwise alignment of the sequences showed all isolates had 100% similarity to C. scovillei ex. holotype CBS 126529 (Damm et al. 2012). Sequences from all isolates were deposited in GenBank with accessions PP556967 to PP556972 (ITS), PP565766 to PP565771 (ACT), and PP565772 to PP565777 (GAPDH). For pathogenicity testing, all isolates were grown on CV8 at 20°C in the dark for 10 days. Conidia were harvested by flooding the plates of each isolate with sterile distilled water and filtering the suspension through a double layer of cheesecloth. The concentration of the conidial suspension was adjusted to 5 × 105 spores per ml. Pathogenicity of the six isolates was tested on banana pepper fruit by using a sterile toothpick to pierce the skin at the two opposite ends. A droplet (10 µl) of the conidial suspension was placed on each wound. The same number of fruit were inoculated without wounding, and non-inoculated control fruit received a droplet of sterile distilled water (either wounded or unwounded). The experiment was repeated twice. All fruit were placed in a humid box at room temperature for 7 days. All wounded and inoculated fruit developed sunken lesions filled with salmon-colored conidial masses. Disease did not occur on the unwounded, inoculated fruit nor the non-inoculated controls. C. scovillei was recovered from all inoculated fruit by reisolating onto CV8 media and isolates had similar morphology and conidial dimensions to the original isolates. To the best of our knowledge, this is the first report of C. scovillei causing anthracnose on pepper in NY. C. scovillei has been reported in South Carolina (Toporek and Keinath 2021), Brazil (Caires et al. 2014), eastern Asia (de Silva et al. 2019), and Kosovo (Xhemali et al. 2023). The pathogen is particularly aggressive on pepper and poses substantial threats to pepper production around the world.

Characterization of Alternaria blotch disease of apple in Himachal Pradesh, India: insights on morphology, pathogenicity, and molecular features.

Alternaria blotch disease in Himachal Pradesh, India, caused by Alternaria spp., adversely affects apple cultivars, resulting in reduced fruit size and quality accompanied by premature leaf fall. Sixteen Alternaria isolates from apple growing regions underwent comprehensive analysis including morphology, pathogenicity, and molecular characterization. Variations in conidiophore and conidia dimensions, shapes, and divisions were observed among isolates. Pathogenicity assays revealed differences in incubation periods, latent phases, and disease responses. Molecular characterization via nuclear ITS rDNA and RAPD analysis indicated 99-100% homology with Alternaria alternata, Alternaria mali, and other Alternaria spp., with a close phylogenetic relationship to Chinese isolates. Differentiation of isolates based on origin, cultural characteristics, and morphology was achieved using RAPD markers. The study identifies diverse genotypes and morphotypes of Alternaria contributing to apple blotch disease in Himachal Pradesh. These findings highlight the complexity of the pathogenic environment and hold significant implications for disease management in apple orchards.

Discovery of Epichloë as novel endophytes of Psathyrostachys lanuginosa in China and their alkaloid profiling

The Epichloë genus represents a significant group of above-ground endophytes extensively researched for their potential applications in agriculture and ecology. Additionally, Epichloë species synthesize bioactive alkaloids, which generally cause health problems in livestock and have detrimental effects on the performance of insect herbivores. Psathyrostachys lanuginosa serves as a valuable forage grass for livestock owing to its high nutritional value and resilience in adverse environmental conditions. Nevertheless, to date, no reports have documented Epichloë as endophytes of P. lanuginosa. In this study, four strains (PF5, PF9, QG2, and QG4) were isolated and identified through morphological, molecular, and phylogenetic analyses as endophytes of P. lanuginosa. Morphological analysis indicated colony characteristics and conidia features consistent with symbiotic Epichloë, with no significant differences observed in growth rates or conidia dimensions among the four strains. Phylogenetic analysis confirmed all strains as E. bromicola. Additionally, alkaloid biosynthetic genes were detected, revealing differences in the potential synthesis of peramine and indole diterpenoid alkaloids among strains from different geographic origins. However, all four E. bromicola strains exhibited similar potential for synthesizing ergot alkaloids, but not loline alkaloids. Overall, this study identified P. lanuginosa as a novel host for E. bromicola and provided insights into the alkaloid profiles of these strains, laying a solid foundation for the scientific and rational utilization of Epichloë resources.

First Report of Alternaria alternata Causing Leaf Spot on Smooth Bromegrass (Bromus inermis Leyss.) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 μm by 8 to 9 μm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.

A new species of Fusichalara (Sclerococcaceae, Eurotiomycetes) from Taiwan.

Fusichalara pallida sp. nov. is described from decaying wood submerged in a freshwater stream in Taiwan. The phylogenetic relationship of Fusichalara species was sought among representative taxa from related fungal lineages, namely the Chaetosphaeriales and Glomerellalles in the Sordariomycetes, and various other ordinal groups in the Eurotiomycetes, by comparing the concatenated ITS and LSU sequences of their nuc rDNA. The novel Fusichalara species from Taiwan clustered with F. minuta within the Sclerococcales besides other ordinal groups in the Eurotiomycetes. Morphologically, F. pallida is comparable with F. dimorphospora and F. novae-zelandiae in having long-cylindrical first-formed conidia and fusiform subsequent conidia with paler end cells, however, they differ in conidial dimensions. With the addition of this novel taxon, Fusichala now comprises seven species. A synopsis of these species and a composite illustration of their conidial morphology are given to ease identification.

Fungal Bioagents and Botanicals Efficacy against Alternaria alternata Responsable for Leaf Blight Disease of Stevia rebaudiana

Stevia rebaudiana, a herbaceous perennial prized for its natural sweetness, has gained global acclaim and found in various parts of India including Karnataka. An exploration into leaf spot disease (caused by Alternaria alternata (FR.) Keissler) in Stevia was conducted under the conditions of southern Karnataka to identify effective management strategies. The symptoms initially manifested as petite circular spots of a light brown colour, subsequently evolving into irregular shapes ranging from dark brown to grey. Some spots maintained their circular form, exhibiting concentric rings or zones. Severely affected leaves exhibited the merging of numerous spots, forming expansive necrotic areas. On older leaves, concentric spots were predominantly found at the tips. The diameter of the leaf spots ranged from 2 to 18 mm. The conidial dimensions varied from 10 to 40 × 6-12 mm, displaying a mid to dark brown or olive-brown color. They were short-beaked, arranged in long chains, and had an oval and bean-shaped structure with 3–5 transverse septa. Considering the adverse effects of chemical fungicides, the exploration for a safer alternative to control the pathogen became a preferable option. This led to experiments involving the use of bioagents for pathogen control. The six known bioagents were evaluated by dual culture, pathogen at periphery and pathogen at the center technique to monitor antagonistic effect. The results revealed that out of all the six bioagents used, two bioagents namely Trichoderma viride (74.77%, 69.04% and 79.45%) and T. harzianum (71.25%, 59.96% and 74.78%) showed maximum growth inhibition in dual culture, pathogen at periphery and pathogen at the center methods, respectively. among the botanicals used neem (36.63%) and ginger (36.42%) found to be effective in inhibiting mycelial growth. Unraveled the strong antagonistic effect to inhibit the mycelia growth of the pathogen significantly.

Phytochemicals reduced growth, sporulation and conidial dimensions of Fusarium verticillioides, cause of fumonisin contamination in maize grains

The objective of the study was to evaluate the effectiveness of methanolic extracts of plants against radial growth and spore dimensions of Fusarium verticillioides. Leaf extracts of 25 plants were tested against the fungus. Of which, thirteen extracts were potent against the fungus and evaluated using food poising technique. Growth was evaluated on PDA medium amended with the extracts at 5 mg ml–1. Control treatments included plates without (negative) extracts and with synthetic (positive) fungicide. Spore dimension was determined using PDB. The results showed T. vulgaris extract completely inhibited mycelial growth of the fungus as equivalent as the fungicide. Similarly, G. parviflora, C. citratus, R. officinalis, R. chalepensis, and Agave sp. also recorded growth reductions ranging from 71.04 to 81.35 % at day seven. In addition, extracts of Agave sp., C. citratus and T. vulgaris did not support sporulation. Overall, the results indicate that T. vulgaris extracts could be safe source of bioactive chemicals to control F. verticillioides.

Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India

Apple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.

Effects of Blue-Light Laser Irradiation on the Enzymatic Activities and Sporulation of Trichoderma atroviride Grown on Rice Husks

Light is known to impact various aspects of Trichoderma, with possible implications for industrial and agricultural applications. In this study, we investigated the irradiation of Trichoderma atroviride with blue light using a laser source system. We determined the cellulase and protease activities and the effects of UV–Vis absorption of the filtrated culture on conidia formation, dimension, and behavior, and on the apparent chlamydospore abundance, as a function of irradiation duration, dose of irradiation, and moment of irradiation. We show that the effects on the enzymatic activities range from positive to neutral to negative. Compared with previous studies, our stimulation with light does not show a drastic negative effect on the cellulase (CMC-ase) activity, but it seems to delay the maximum activity over time. The effects on the proteases are partially in agreement with the only previous study reported for the light effects on protease activity. The number of conidia is increased upon irradiation, representing an expected behavior, and we show for the first time to the best of our knowledge that the conidia dimensions and chlamydospore number also increase. Our data also suggest that the highest dose resulted in the conidia clustering around the liquid–air interface, suggesting an increased hydrophobic character.

Peronosclerospora neglecta sp. nov.—a widespread and overlooked threat to corn (maize) production in the tropics

Downy mildew is a serious threat to corn (maize) production in the tropics and subtropics. Corn is native to Central America, and was introduced into South-East Asia by the Spanish colonisers in the 1700s. Corn is evolutionarily naïve to downy mildews of the genus Peronosclerospora. Consequently, corn monocultures are particularly susceptible to a variety of Peronosclerospora species, which spread to the crop from local grasses. Globally, corn is one of the most important crops for both humans and livestock. Several downy mildews of corn have been identified as potential threats to global food security, and trade with corn seeds is strictly regulated to avoid spreading the pathogens. Despite their importance, little is known about the biodiversity of graminicolous downy mildews, because their identification has often relied on variable morphological features, such as conidial dimensions. DNA barcodes for most species have become available only recently. During surveys for downy mildews on corn in Indonesia, a previously unrecognised species of Peronosclerospora was found and investigated using a combination of morphological characters and molecular phylogenetic analyses. The new species, introduced here as Peronosclerospora neglecta, is widely distributed in South-East Asia from Thailand to eastern Indonesia. The impact of this downy mildew can be severe, with complete crop losses in heavily affected fields. Given the aggressiveness of the species, close surveillance is warranted to restrict its further spread.

First Report of Halo Blight on Hop Caused by Diaporthe humulicola in New York.

In late July and August 2015, foliar disease was observed in three hop (Humulus lupulus; unknown cultivars) yards in Ontario, Otsego, and Putnam counties, New York (NY). Disease incidence ranged between 70 and 90% of plants, and up to 25% of the leaves per plant were affected. Leaf symptoms were large, necrotic patches with a chlorotic halo (2 to 10 cm diam.). Leaves and dry, easily shattered cones were placed at high humidity for 10 days. Pycnidia were abundant in leaf lesions which extruded conidia. Pycnidia were also observed on cone bracts and bracteoles. Fifteen isolations were made from each yard by placing a pycnidium onto 2% water agar + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred to potato dextrose agar (PDA) before incubation at 20°C with a 12-h photoperiod. Colonies on PDA had flat mycelia and were white to cream in color. The isolation frequency was 100%. To induce sporulation, five isolates were grown on PDA with autoclaved alfalfa stems for 7 to 10 days. Alpha conidia were hyaline, and oval with obtuse ends. Mean alpha conidial dimensions were (n = 20): 9.1 m × 3.4 µm (BE1; Ontario Co.); 11.8 × 3.8 µm (BE34; Ontario Co.); 9.6 × 4.1 µm (BE10; Ontario Co.); 10.2 × 3.7 µm (BE52; Otsego Co.); and 10.3 × 3.6 µm (BE69; Putnam Co.). Beta conidia were not observed. DNA was extracted and PCR performed to amplify the internal transcribed spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), translation elongation factor 1-α (TEF; EF1-728F/EF1-986R; Carbone and Kohn 1999), a partial region of β-tubulin (TUB; Bt2a/Bt2b; Glass and Donaldson 1995), a partial region of histone 3 (H3) (H3; CYLH3F/H3-1b Crous et al. 2004), and calmodulin (CAL; CAL-228F/CAL2Rd; Groenewald et al. 2013) genes. For all NY isolates, sequence similarity was >99% to D. humulicola CT2018-3 for the ITS region, and TEF, HIS, and CAL genes. Sequence similarity to CT2018-3 for the TUB region ranged from 86.96% (BE-1) to 96.15% (BE-10). . Analyses with the ITS, TEF, CAL, and HIS sequences supported our identification of the NY isolates as D. humulicola. Sequences were deposited in GenBank (OM370960 to OM370984). For pathogenicity testing, BE-34 and BE-69 were grown on PDA + autoclaved alfalfa stems at room temperature and a 12-h photoperiod for 10 days. Conidia were harvested by flooding the plate with sterile water. Conidial concentration was quantified, and the inoculum suspension diluted to ~5  105 (+ 0.01% polysorbate-20)/ml. Five cv. Cascade plants were sprayed with inoculum until run-off and covered with a plastic bag for 72 h. Non-inoculated control plants were sprayed with 0.01% polysorbate-20 and bagged. Plants were placed in a misting chamber and exposed to alternating 25°C light/18°C dark with a 16 h photoperiod. Mist was applied for 1 h daily. Necrotic lesions like the field specimens were observed on all inoculated plants after 28 days with no symptoms on control plants. Diseased leaves were detached and placed in a humid chamber for 2 days, and pycnidia observed in lesions. The reisolation frequency of D. humulicola was 100%. Conidia from the isolates had similar morphology to the original isolates. This is the first report of halo blight caused by D. humulicola on hop in NY. Halo blight has been reported on hop and associated with significant yield loss through cone shattering in MI (Higgins et al. 2021), CT (Allan-Perkins et al. 2020), and Quebec, Canada (Hatlen et al. 2021). Research is needed to determine if management is warranted.

First Report of Stemphylium lycopersici Causing Leaf Spot on Hevea brasiliensis in China.

Hevea brasiliensis is widely planted in tropical and subtropical regions and is the main source of natural rubber production. The growth of rubber trees is plagued by various leaf diseases, resulting in decreased rubber production. From January to March in 2020, a severe leaf spots disease on Hevea brasiliensis found in Agricultural Science Base in Haidian campus of Hainan University (20° 03' 31″ N, 110° 19' 07″ E), Haikou, Hainan province, China. Spots were only observed on the mature green rather than young and bronze-colored leaves. This symptom has never been reported on the leaves of Hevea brasiliensis. During the early stages of the disease, gray leaf spots were concentrated to the leaf margins, but later expanded forming irregular gray lesions with chlorotic edges (Figure 1A). Eventually, lesions became necrotic shot holed, and leaves curled, wilted, and dropped. Five small pieces were cut from the margin of spots from different infected leaves, and were surface disinfected with 75% alcohol three times for five seconds each time and 1% sodium hypochlorite solution (NaClO) for 60 s. After washing twice with sterile water, leaf pieces were placed in the center of plates with Potato Dextrose Agar (PDA) medium and incubated for one week at 28 °C. After 7 days, mycelium developed and colonies were single-spore cultured for further study. One of the strains labeled HN01 developed a yellowish-brown to reddish-brown pigment on PDA, and the colonies were gray and cottony. The colony and pigment feature very consistent with Stemphylium sp. (Figure 2) (Li et al. 2017). Conidiophore were solitary, transparent to pale, mostly 102.1-228.8 µm × 4.0-5.8 µm, with 2-3 septa and apical vesicular swellings 6.5-7.9 µm. The dimensions of conidia were 28.3-45.1 × 11.5-17.5 µm and one septum (Figure 3). Conidia of S. lycopersici were solitary, oblong with a conical end at the apex, with 1-2 septa, and constricted at the transverse septum. The internal transcribed spacer region of rDNA was amplified with primers ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3'/5'-TCCTCCGCTTATTGATATGC-3'), glyceraldehyde-3-phosphate dehydrogenase (gpd) was amplified with primers GPD-F/R (5'-GCACCGACCACAAAAATC-3'/ 5'-GGGCCGTCAACGACCTTC-3'), calmodulin region (cmdA) was amplified with the primers CALDF1/CALDR2 (5'-AGCAAGTCTCCGAGTTCAAGG-3'/5'-CTTCTGCATCATCAYCTGGACG3') from genomic DNA of strain HN01 (Xie et al. 2018), and PCR products were sequenced. The ITS sequence of strain HN01 (GenBank Accession No. MZ496930) had 99.64% identity with isolates sl001, sl110, sl111, and sl112 of Stemphylium lycopersici (GenBank Accession No. KX858848.1, MF480547.1, MF480548.1, MF480549.1). Similarly GPD sequences (GenBank Accession No. MZ505106) had 100% identity with strain xiqing, HZ2114 and HZ2115 of Stemphylium lycopersici (GenBank Accession No. KR911809.1, KR911810.1, KT957742.1 and KT957743.1), and CMDA sequences (GenBank Accession No. MZ505105) had 99.85% identity with Stemphylium lycopersici strain LJ1609270201 (GenBank Accession No. MG742412.1). A phylogenetic analysis constructed by MEGA6.0 based on concatenated sequences of the HN01 and another 17 strains from GenBank by using the maximum-likelihood (ML) method showed that the HN01 was clustered and matched with Stemphylium lycopersici LJ1609270201 (Figure 4). To satisfy Koch's postulates, we inoculated mature green leaves of Hevea brasiliensis with mycelial plugs (diameter = 5 mm) of pure cultured strain HN01. All leaves of Hevea brasiliensis were wrapped in a freezer bag to maintain relative humidity >85%, and the temperature of greenhouse is 28ºC. The disease developed on the inoculated leaves after 2-3 days, but not on control leaves (Figure 1B). We used the same method as before to re-isolate the pathogen, which had the same morphology and genotypes as the original isolate. S. lycopersici has been reported to infect the leaves of a variety of plants, including pepper, tomato, eggplant, watermelon, Physalis alkekengi. (Yang et al.2017; Ben et al. 2017; Yang et al. 2020). To our knowledge, this is the first record of S. lycopersici causing leaf spot of Hevea brasiliensis in China, and Hevea brasiliensis is the global new host of S. lycopersici. Hevea brasiliensis is the main source of natural rubber and is widely planted in southern China. Therefore, it is imperative to implement disease management measures to prevent potential threats.

First Report of Leaf Spot Caused by Penicillium sclerotiorum on Aegiceras corniculatum in China.

First Report of Leaf Spot Caused by Penicillium sclerotiorum on Aegiceras corniculatum in China.

Characterization, pathogenicity, and fungicide sensitivity of Alternaria isolates associated with preharvest fruit drop in California citrus

Alternaria rot has been recently described as an emerging fungal disease of citrus causing significant damage in California groves. A survey was conducted to determine latent infections on fruits, twigs, and leaves and investigate their seasonal patterns during 2019 and 2020. On fruits, latent infections were more associated with the stem end than with the stylar end, except during spring when a significantly high percentage of flowers (86%) had latent infections. Latent infections on twigs varied markedly between years (28% in 2019 and 9.5% in 2020), while Alternaria spp. were also recovered from citrus leaves. Alternaria isolates collected during the survey were identified based on multigene sequence analysis, confirming that Alternaria alternata and Alternaria arborescens are the two species associated with infections of citrus fruits. Of the 23 isolates, 19 were identified as A. alternata and demonstrated the dominance of this species over A. arborescens. Isolates representing populations of these two species were selected as representative isolates for physiological and morphological studies. A. alternata and A. arborescens showed similar conidial dimensions but differed in the number of conidia produced. Growth rates demonstrated that A. alternata grows faster than A. arborescens at all the temperatures evaluated, except at 25 and 35 °C. The growth patterns were similar for both species. The sporulation rate of the Alternaria isolates was influenced differently by temperature. This parameter also influenced conidial germination and appressorium formation, and no significant differences were observed between Alternaria species. Pathogenicity and aggressiveness tests on detached fruit demonstrated the ability of A. alternata and A. arborescens to cause internal lesions and produce fruit drop in the orchards with no quantitative differences between them (disease severity indexes of 58 and 68%, respectively). The fungicide sensitivity tests showed that DMI fungicides are the most effective fungicides in reducing mycelial growth. The SDHI fungicides had intermediate activity against the mycelial growth but also suppressed spore germination. The spore germination assay suggested that some of the isolates included in this study might have some level of resistance to QoI and SDHI fungicides. The findings of this study provide new information about the pathogens associated with the excessive fruit drop recently observed in some California citrus groves.

Diplodia parva sp. nov., a novel species of the family Botryosphaeriaceae isolated from soil in Korea

A fungal strain designated KNU16-007, belonging to the family Botryosphaeriaceae, was isolated from soil in Daegu, Korea. Phylogenetic analyses based on the concatenated nucleotide sequences of the ITS and TEF-1α regions, showed that the isolate resides in a clade together with Diplodia species but occupies a distinct phylogenetic position. Conidial dimensions of strain KNU16-007 (22.7–29.3 μm × 8.9–10.9 μm) or its conidial length:width ratio were significantly differed from those of the closely related D. sapinea, D. intermedia, D. scrobiculata, D. seriata, D. crataegicola, D. rosacearum, and D. citricarpa clearly indicating morphological differences from these species. Detailed descriptions, illustrations, and discussions regarding the morphological and phylogenetic analyses of the closely related species are provided to support the novelty of the isolated species. The results of phylogenetic analysis and morphological observations indicate that strain KNU16-007 represents a novel species in the genus Diplodia, for which the name Diplodia parva sp. nov. is proposed.

Novel species of Alternaria section Nimbya from Iran as revealed by morphological and molecular data

ABSTRACT In this study, the diversity of Alternaria species in section Nimbya associated with symptomatic plants in the Cyperaceae and Juncaceae families was assessed. Multilocus sequence analyses of the rDNA internal transcribed spacer (ITS) region and parts of Alternaria major allergen (Alt a 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), second largest subunit of RNA polymerase II (RPB2), and translation elongation factor 1-alpha (TEF1) genes revealed the presence of two previously known species, A. scirpivora and A. caricicola, and three new species, which are described here as A. cypericola, sp. nov., A. heyranica, sp. nov., and A. junci-acuti, sp. nov. These new species were characterized morphologically with respect to the dimensions of conidia, the number of pseudosepta in mature conidia, and the type of conidium apical beak. According to the results of phylogenetic analyses, the presence of long, filiform true beak is not a reliable morphological indicator for grouping species in sections Alternantherae and Nimbya and phylogenetic species recognition should be used. All identified species were described, illustrated, and their morphology and phylogenetic relationships with other species in Alternaria section Nimbya were discussed.

Morpho-phylogenetic evidence reveals Lasiodiplodia chiangraiensis sp. nov. (Botryosphaeriaceae) associated with woody hosts in northern Thailand

Lasiodiplodia species are commonly as endophytes, saprobes and pathogens in tropics and subtropics. During an investigation of Botryosphaeriaceae in Thailand, two Lasiodiplodia taxa were isolated. Morphological characteristics and phylogenetic analyses based on combined ITS, tef and tub2 sequence data support the establishment of a novel species, Lasiodiplodia chiangraiensis, isolated from woody hosts. Lasiodiplodia chiangraiensis is phylogenetically close to L. iraniensis and L. thailandica, but represents a distinct lineage. The new species could be distinguished from extant Lasiodiplodia species by its mature conidial dimensions. A detailed description and illustration are provided, as well as an updated phylogenetic tree (ITS, tef and tub2) including all species (with available molecular data) of Lasiodiplodia. In addition, the accepted genera in Botryosphaeriaceae based on recent studies are given.

First Report of Didymosphaeria rubi-ulmifolii Brown Spot infection of Chinese quince fruit in South Korea.

Chinese quince (Pseudocydonia sinensis (Thouin) CK Schneid.), a deciduous tree in the family Rosaceae, is native to China, Japan, and South Korea; the fruit is known as mogwa in South Korea. The ripened yellow fruit has been used as a traditional therapeutic for respiratory ailments and as an additive in health products such as syrups, tea, and candies (Sawai et al. 2008). From May to August 2020, Chinese quince trees showing symptoms of brown spots were observed on the Kyungpook National University premises, Daegu, South Korea, with an incidence of 30%-40%. The disease first appeared as small, round, yellow specks on the fruits, which necrotized over time and gradually enlarged to 0.7-2.7 cm in diameter. To isolate the pathogen, symptomatic tissues obtained from disease fruit were surface sterilized for 1 min with 70% ethanol, rinsed in sterile distilled water, and plated onto potato dextrose agar (PDA). The inoculated plates were incubated at 25°C for 7 days. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained from 25 fruit. Colonies on PDA reached a diameter of 30-40 mm. After incubation for 7 days at 25°C, spreading with an even, colorless-to-buff glabrous margin, a submarginal ring of conidiomata developed from day 5 to 12 and was visible as scattered dots on either side of the plate. Conidiogenous cells were discrete (3.5-6 × 3.5-5 μm); conidia were ellipsoid to short-cylindrical [3-5 × 2.1-3.5 μm (n = 60)] and olivaceous in color. These conidial dimensions corresponded to those of Didymosphaeria rubi-ulmifolii Ariyaw., Camporesi & K.D. Hyde (basionym: Paraconiothyrium brasiliense), which has been found on Rubus ulmifolius in Italy (Ariyawansa et al. 2014). Sequence data for the rDNA internal transcribed spacer (ITS), large subunit ribosomal RNA (LSU), and partial translation elongation factor 1-α (TEF) (White et al. 1990, Rehner and Buckley 2005) were obtained for one of the pure culture isolate (BT1) with GenBank accession numbers MW020087, MW020060 and MW027220, respectively. The sequences of BT1 isolate using a BLASTn analysis showed 100% identity with the ex-type MFLUCC 14-0023 of D. rubi-ulmifolii in ITS, and LSU portions (accession nos. MT310602, and MT214555, respectively) and 99% identity in TEF portion (accession no. MT394734). The procedure for Koch's postulates was followed to confirm fungal pathogenicity using 3-day-old mycelial discs. Fifteen healthy fruit were divided into three groups of five fruit each, with each group receiving a different treatment. One group of fruit was wounded by puncturing with a sterile pin and inoculated using 5-mm agar discs with mycelium on the wounds. Mycelium covered agar discs were also placed on the surfaces of five unwounded fruits. The remaining five fruit were maintained as a control and inoculated with sterile PDA plugs. The pathogenicity test was replicated thrice. The wounded fruits showed symptoms similar to those observed in the field. The control group remained asymptomatic and the morphology of the fungus re-isolated from the inoculated fruit was the same as that of D. rubi-ulmifolii. The phylogeny, together with the morphological identification and inoculation results, confirmed the identity of the fungus as D. rubi-ulmifolii (Ariyawansa et al. 2014). To the best of our knowledge, this is the first report of D. rubi-ulmifolii causing brown spot in Chinese quince.