Types Of Conidia Research Articles

Characterization of Fusarium falciforme Inciting Wilt in Peace Lily (Spathiphyllum wallisii)

Severe wilt incidence with characteristic yellowing of older leaves was observed in Spathiphyllum plants of different commercial nurseries of Andhra Pradesh, India during 2021-23. Upon isolation on potato dextrose agar medium, the fungus displayed aerial greyish white mycelium on upper surface and with yellow pink pigmentation on the lower surface. Microscopic observation of the three types of conidia namely microconidia, macroconidia and chlamydospores confirmed the morphological identity of the pathogen as genus Fusarium. Multi-gene sequencing analysis, utilizing ITS, Tef-1α, RPB1, and RPB2 gene sequences, further confirmed the pathogen's identity at the species level as Fusarium falciforme. Pathogenicity assays in 30 days old Spathiphyllum plants demonstrated that yellowing of leaves was observed seven days after pathogen inoculation using a non-invasive method, followed by complete wilting of the plants within 15 days. In addition, host range analysis revealed that the isolated pathogen isolate from wilted Spathiphyllum plants could cause complete wilting within 12-15 days post-inoculation in other economically important solanaceous crops such as tomato, brinjal, chili and tobacco, further highlighting its economic significance. Studies evaluating different fungicides revealed that root zone application of carbendazim 50%WP at 1g/l and tebuconazole 25.9% EC at 1ml/l were effective in controlling wilt incidence, achieving 77.78% reduction as a prophylactic treatment and 66.67% reduction as a curative treatment. To the best of our knowledge, this is the first comprehensive study on Spathiphyllum wilt incidence and its management worldwide.

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

The co‐occurrence of two powdery mildew species on Cannabis sativa in the Czech Republic

AbstractIn late summer 2022 and 2023, powdery mildew symptoms were found on female plants of hemp (Cannabis sativa) growing in a private garden in Hradec nad Moravicí (Silesia, Czech Republic). On the upper sides of the leaves, white powdery colonies were observed which coalesced and finally covered the whole leaf surface. The stems and flowers were not affected. Microscopic examination revealed the formation of asexual (anamorphic) states characterized by conidiophores forming catenescent conidia (Euoidium type), but with two types of conidia, viz., ellipsoid to ovoid or doliiform conidia with fibrosin bodies (resembling conidia of Podosphaera spp.), and ellipsoid to doliiform conidia without fibrosin bodies (resembling conidia of Golovinomyces spp.). Molecular genetic analyses confirmed co‐infections in the studied hemp samples by two powdery mildew species, viz., Podosphaera macularis and Golovinomyces ambrosiae, which represent first records from the Czech Republic, as well as Europe and in the world. The authors discuss the latest information and ideas regarding this pathosystem.

Occurrence of leaf spot disease caused by Fusarium graminearum on white clover(Trifolium repens) in China

White clover (Trifolium repens L.) is a herbaceous, perennial plant that has become one of the most widely distributed legumes in the world. It is cultivated in a large area in China, mainly for animal husbandry feed, artificial grassland, garden greening and ecological construction. In 2022, leaf spot disease was observed on white clover in China's Sichuan province, and the strain was isolated. The pathogenicity of the isolates was confirmed using Koch's postulates. The fungus was/were red on PDA, and two types of conidia were observed after 6 days of cultivation on straw juice medium. The pathogen was identified as Fusarium graminearum by the amplification of part of the ITS and TEF-α genes and by phylogenetic analysis. Currently, this is the first report to indicate that the causal agent of leaf spot disease on white clover is F. graminearum.

First report of Diaporthe eres causing leaf spot on Rhododendron latoucheae Franch. in China.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

First Report of Fruit Rot Caused by Fusarium graminearum on Ponkan (Citrus reticulata Blanco cv. Ponkan) in China.

Ponkan (Citrus reticulata Blanco cv. Ponkan) is a Chinese citrus species with tasty fruit. In November 2021, an unknown postharvest disease of Ponkan fruit caused nearly 15% losses of 2000 fruits in Nanchang, Jiangxi Province (28.68° N, 115.85° E). The initial fruit's surface necrosis was brown (Xu et al. 2022) (Figure 1A). Disease spots spread to the entire fruit, white or grey hyphae appeared, and the fruit rotted. Twenty diseased fruits were surface-disinfested with 2% sodium hypochlorite and 75% ethanol, then rinsed with sterile distilled water to isolate the pathogen. Diseased tissue sections (5 × 3 mm) were incubated on potato dextrose agar (PDA) for 7 days at 25°C. Twelve of 15 monoconidial isolates have similar morphology. On PDA, the isolates produced copious white aerial mycelia. After 5-7 days on straw juice medium, two types of conidia appeared (Rice straw 60 g, Agar 20 g, distilled water 1000 mL) (Figure 1E-I). Macroconidia were abundant, falcate, slender, and slightly curved with 0-8 septa, mostly 4-5 septa (average 41.70 × 3.81 m, n=100) (Figure 1J). Microconidia were globose, oval, or piriform with 0-1 septa, 2.72 to 8.57 × 2.53 to 7.47 m (average 5.49 × 4.52 m, n=50) (Figure 1L), and chlamydospores were not observed. Conidial and colony morphology identified 12 monoconidial isolates as Fusarium graminearum (Fisher et al., 1982; Yulfo-Soto et al., 2021). Genomic DNA was extracted from three isolates using a DNA Extraction Kit (Yeasen, Shanghai, China). The ITS1/4 region combined with partial gene fragments of translation elongation factor-1 alpha (TEF-1α, primer TEF1/2, O'Donnell et al. 1998), RNA polymerase second largest subunit (RPB2, primer fRPB2-5F/7cR, Liu et al. 1999) and β-tubulin (β-tub, primer Bt2a/2b, Li et al. 2013) from the isolates were amplified and sequenced. The three tested isolates showed identical gene sequences. Sequences amplified from one representative isolate (PG16) have been submitted to GenBank. BLAST searches revealed that ITS (OM019317), TEF-1α (OM048103), RPB2 (ON364348), and β-tub (OM048104) had 99 to 100% identity compared with F. graminearum (MH591453.1, KX087136.1, MF662636.1, and MZ078952.1, respectively) in GenBank. The phylogenetic analysis combined ITS - TEF-1α - RPB2 (O'Donnell et al. 2015) concatenated sequences using MEGA7.0 (Mao et al. 2021) showed the isolate was clustered with the F. graminearum clade with 100% bootstrap support (Figure 2). The isolate PG16 was used for pathogenicity tests. Ponkan fruits were surface-disinfested with 75% ethanol and rinsed with sterile distilled water three times. Then, 30 punctured wound fruits (2-mm-diameter, 2-mm-depth) with a sterile needle and 30 unwounded fruits were inoculated with conidial suspension (10 µL, 3.0 × 105 conidia/mL). while the control fruits were inoculated with 10 µL sterile distilled water. All fruits were incubated at 25°C and 90% relative humidity. Two days later, all wounded fruits inoculated with conidial suspension showed disease spots, similar symptoms to the original rotten fruits (Figure 1D). Control and conidial-inoculated unwounded fruits were healthy (Figure 1B-C). The Pathogenicity test was repeated twice, and similar symptoms were observed. Morphologically and molecularly, the re-isolated fungus matched the inoculated isolate. First report of F. graminearum causing Ponkan fruit rot in China. As Ponkan is an important citrus crop with high economic value in China, identification of the causing agent, F. graminearum, for fruit rot allows the development of control measures to manage this disease.

First Report of Fusarium acuminatum Causing Dianthus chinensis root rot and foliage blight in China.

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. In May 2020, a disease was found at several landscape sites in Xuanwu District, Nanjing, China, causing symptoms of foliage blight and root discoloration onapproximately 52% of one-year old D. chinensis plants. To recover the causal pathogen, samples of infected roots and leaves were cut into 5×5 mm2 pieces, surface-disinfected in 75% ethanol for 30 sec, followed by 1% NaClO for 90 sec, rinsed with sterile water three times and placed on potato dextrose agar (PDA) with 0.1 mg/mL of ampicillin at 25 ⁰C. Hyphae growing on PDA were visible from both root and leaf tissues after three days. Individual hyphal tips were transferred to new PDA plates to obtain pure isolates. Three representative isolates were deposited in the China Forestry Culture Collection Center (CFCC 57545,57546, 57547). The hyphae grew radially, densely, and the aerial hyphae were velvety, white, yellow-white, or pink-white. Representative isolate Facu-DCY5 produced three types of conidia (microconidia, macroconidia, and chlamydospores). Macroconidia were sickle-shaped, measuring 25.7-55.4 µm × 3.2-4.6 µm (n=50). Microconidia were numerous, oval or kidney-shaped, measuring 6.8-11.9 µm × 3.5-4.8 µm (n=50). Conidia produced in the aerial mycelium were 16-34 × 2.2-5.3 µm (n=50). The ITS region, TEF1, calmodulin (CMDA), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4, EF1/EF2, CL1/CL2A and 5F2/7CR , respectively and sequenced at Sangon Biotech (Nanjing, China). The ITS sequence of isolate Facu-DCY5 (GenBank No. ON307073.1) was identical to HQ165938.1, ON306850.1, OM964482.1. TEF1 (ON331997.1) was identical to LC546967.1, HQ165866.1, MZ158155.1. CMDA (ON331996.1) was identical to HQ412345.1, MZ921595.1 and MZ921597.1. RPB2 (ON331995.1) was identical to MZ997370.1. Maximum parsimony and maximum likelihood phylogenies of the Facu-DCY5 multilocus sequence data and those of several species within the F. tricinctum species complex identified the isolate from D. chinensis as F. acuminatum . Pathogenicity tests were performed using a conidial suspension (104conidia/mL). Each plant (approx. 0.3 m in -height) was inoculated with 1 mL of the conidial suspension by mixing it into the potting soil (500 g). Control plants were treated with sterile distilled water. All inoculated plants (n=9) in three repeats of the assay exhibited foliage blight and root rot after 15 days, whereas all control plants (n=9) remained asymptomatic. Fusarium isolates with identical morphological features and molecular marker sequences to those of Facu-DCY5 were recovered from foliage blight and root tissues of all the inoculated plants. In China, F. acuminatum has been reported as a pathogen of Cucurbita maxima, Actinidia arguta, Polygonatum odoratumand Schisandra chinensis. This is the first report of F. acuminatum on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nurseries and landscaping industries, we recommend that diseased plants be removed to prevent the spread of F. acuminatum, and that identification of the infecting isolates from D. chinensis at other sites and landscape locations be performed.

Rapid Identification of Diaporthe citri by Gene Sequence Analysis

Citrus melanoses caused by <i>Diaporthe citri</i>, has been one of the serious diseases in many citrus orchards of Jeju Island. To protect melanose in citrus farms, a fast and exact diagnosis method is necessary. In this study, diseased leaves and dieback twigs were collected from a total of 49 farms within March to April in 2022. A total of 465 fungal isolates were obtained from a total of 358 isolated plant samples. Among these fungal isolates, 40 representatives of <i>D. citrii</i> isolates which were isolated from 22 twigs and 18 leaves on 23 farms were found based on cultural characteristics on potato dextrose agar and conidial morphology. Additionally, the molecular assay was carried out and compared with those by morphological diagnosis. All isolates were identified as <i>D. citrii</i> by analyzing the sequences at the internal transcribed spacer (ITS) rDNA region using primers of ITS1/ITS4 or at β-tubulin using primer Btdcitri-F/R. Therefore, based on the present study, where the results of morphological identification of conidial type were consistent with DNA sequence analysis of certain gene, choosing a suitable method for a fast diagnosis of citrus melanose was suggested.

A new species of Papiliomyces (Clavicipiteae, Hypocreales) from China.

Papiliomyces (Hypocreales, Sordariomycetes) was introduced to accommodate two species: Papiliomycesliangshanensis and Papiliomycesshibinensis. Later, Papiliomycesliangshanensis was renamed Ophiocordycepsliangshanensis. However, the Papiliomycesliangshanensis molecular data (Nepalese) used to establish the Papiliomyces genus was different from Ophiocordycepsliangshanensis (China) strains. This paper describes a new species, Papiliomyceslongiclavatus, found in Yangchang District, Guiyang City, Guizhou Province, China. It is proposed, based on morphology and multilocus phylogeny (ITS, SSU, LSU, TEF1, RPB1 and RPB2). The new species is phylogenetically most closely related to Papiliomycesliangshanensis (Nepalese collections). However, Papiliomycesliangshanensis (Nepalese collections) requires morphological details and additional detection. The new species differs from other Papiliomyces species in having robust stroma with completely immersed perithecia, multi-septate ascospores, cylindrical secondary ascospores, two types of phialides and two types of conidia:longer α-conidia and longer β-conidia.

Root Rot Disease of Torreya grandis Caused by Fusarium fujikuroi in China.

Torreya grandis is an evergreen plant endemic of China and widely grown in Southern China. Its fruit is a precious nut in China, rich in vitamins and minerals, can be directly eaten, can also be used as medicinal plants with functions of lowering blood lipids and softening blood vessels (Wang 2022). From 2018 to 2020, typical root rot symptoms of Torreya grandis was found in plantations in Huangshan and surrounding areas of Huangshan, Anhui province, China. About 15 to 32% of root rot disease incidence was recorded at the plantation. Diseased plants were observed with symptoms such as yellow to brownish leaves without lesions and later drying, and rotten roots looked dark brown while the roots of heathy plants showed white, and eventually leading to the death of the diseased plant. The root rot symptomatic plants were collected in June of 2020. Tissues were cut to the length of 0.3 to 0.5 cm, then surface sterilized by 2% sodium hypochlorite for 2 min and 75% alcohol for 1 min, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 9 days. Eight isolates with similar morphology were isolated from single spores. On PDA, the isolates produced abundant aerial white mycelia with septation and turned violet to dark pink on the reverse side of the culture. Morphological characteristic was determined using a pure culture grown on synthetic low nutrient agar (SNA). Two types of conidia, microconidia and macroconidia, were observed on SNA. Macroconidia were long and slender, usually 3 to 5 septate, measuring 2.7 to 4.3 × 22.3 to 49.6 μm (n=30), and narrowed at the both ends. Microconidia were abundant, oval, clavate or ovate, zero to one septate and measured 1.6 to 3.9 × 4.4 to 13.0 μm (n=50). According to the culture and conidial characteristics, the isolates were tentatively identified as Fusarium species (Leslie and Summerell 2006). Four isolates were random selected for molecular identification. The general primers ITS1/ITS4 for internal transcribed spacer (ITS) (White et al. 1990), EF1/EF2 for translation elongation factor (TEF1) (O'Donnell et al. 1998), 5F2/7cR for the second largest subunit of RNA polymerase Ⅱ(RPB2) (O'Donnell et al., 2007), H3-1a/H3-1b for Histone H3 (Jacobs et al., 2010), F5/R8 for subunits 1 of DNA-directed RNA polymerase Ⅱ (RPB1) (O'Donnell et al. 2010) and MS3F/MS3R for mitochondrial small subunit (mtSSU) (Stenglein et al. 2010) were amplified, respectively. The products were sequenced and deposited in GenBank with accession numbers of MW350689, MW029444, ON077156, ON077158, ON077157, ON054432, respectively. Blast analysis showed 99.40 to 100% sequence homology with known F. fujikuroi isolates. A phylogenetic analysis based on the concatenated sequences clustered from the combined datasets (TEF1, RPB2, Histone H3, RPB1 and mtSSU) revealed the isolate most closely related to the F. fujikuroi (100% bootstrap). Fifteen 2-year-old healthy plants of Torreya grandis were selected for the pathogenicity test. A conidial suspension (1×106 conidia/ml) was prepared by collecting spores from 10-day-old cultures on PDA. The root of each plants inoculated with 200 ml of a 106 conidia/ml suspension, and the five control plants inoculated with sterilized water. The plants were incubated in green house with 25℃ (14 h light)/22℃ (10 h dark) at 85% humidity. Two weeks later, 100% of artificially inoculated plants showed the same symptoms similar to those observed in the plantation, like yellow leaves, dark brown and rotten roots, meanwhile, the roots of control plants displayed healthy. From symptomatic roots, the pathogen was reisolated which satisfying Koch's postulates. F. fujikuroi causes root rot of soybean and Reineckia carnea (Detranaltes et al. 2021, Sun et al. 2018).To the best of our knowledge, this is the first report of F. fujikuroi causing root rot of Torreya grandis in China.

First Report of Diaporthe eres Causing Leaf Spot on Pseudocydonia sinensis in China.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and β, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 μm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 μm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The β-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

First report of branch blight caused by Diaporthe ueckerae on Eucalyptus citriodora in China.

Eucalyptus citriodora is a wood and oil dual-purpose tree with strong growth adaptability and high ornamental value. Recent years, it has been widely planted in Guangxi in China. In Nov. 2021, branch blight was found to be widespread on E. citriodora in Qinzhou in China (21°57'57"N, 108°42'6"E). The occurrence area was over 7000 m2, and the disease incidence was 23% (23/100). Twigs were withered in most of infected plants, and only top branch died in few plants. The lesions started from branch tips, then expanded and caused 5-20 cm branches died in final. The lesions were tiny and brown at early stage, then turned dark brown or black. Ten diseased branches were sampled randomly in field and were cut into 1 cm pieces. After surface disinfecting with 75% ethanol for 3 min, 0.1% HgCl2 for 5 min, washing with sterile water three times, samples were placed onto Potato Dextrose Agar (PDA) medium. Hyphae appeared after incubating for 5 days at 28 ℃. The diameter of colonies reached 64-71 mm after 7 days incubation. The colonies were white and felt-like, and then turned yellowish gradually with flourish aerial hyphae. Two weeks later, pycnidia appeared, which were nearly spherical, initial pale yellow and later black. Sometimes secretions overflowed from the aperture on pycnidia. There were 2 types of conidia (α and β type), which were unicellular and hyaline. The α type was spindle to ellipse and had 1-2 oil globule, with 5.56±0.50 × 2.67±0.39 μm (n=100) in size. The β type was linear and one end bent in hook shape, with 17.10±2.54 × 1.55±0.32 μm (n=50) in size. Three isolates (LEQZ01, LEQZ02, LEQZ03) were selected for further study. The internal transcribed spacers (ITS) region of rDNA, translation elongation factor 1-α (tef1 α) and β-tubulin (tub2) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner et al. 2005) and Tub1/ Tub2 (Chauhan et al. 2007), respectively. BLASTn searches showed that the ITS (OM339849, ON075781, ON075782), tef1 α (ON093807-ON093809) and tub2 (ON093810-ON093812) sequences had the highest similarity with Diaporthe ueckerae strains, with 99% (543/549, 544/549, 544/549) identity for ITS (NR 147543.1), with 99% (350/351, 353/353, 349/350) identity for tef1 α (KY569388.1), with 99% (754/754, 753/754, 754/754) identity for tub2 (MW514128.1). A neighbor-joining tree constructed by combining 3 sequenced loci. Three isolates clustered in the D. ueckerae clade with 100% bootstrap support. Based on morphological (Yi et al. 2018) and molecular evidences, the pathogen was identified as D. ueckerae. In a pathogenicity test, 20 healthy E. citriodora seedlings were divided into 4 groups. Before inoculation, twigs were surface disinfected with 75% ethanol followed by washing 3 times using sterile water. Tiny artificial wounds at 5 cm below the seedling top were inoculated by hyphae taken from colonies incubated for 7 days at 25 ℃ in the dark, and covered with damp cotton in 1-3 groups (Yi et al. 2018). Yet the wounds were covered with damp cotton in control group. Two days later, wounds started to turn brown in test groups, and symptoms similar to field were obtained after 10 days. But no lesion emerged in control group. Then germs were re-isolated from symptomatic twigs and identified as D. ueckerae following the methods above. To our knowledge, this is the first report of branch blight caused by D. ueckerae on E. citriodora in China. Further researches on disease epidemiology would help to prevent spread to more locations.

Dimorphism and microcycle conidiation of Pseudocercospora pseudostigmina‐platani conidia from American sycamore

AbstractIn this study, the micromorphology and surface details of Pseudocercospora pseudostigmina‐platani conidia were investigated on American sycamore (Platanus occidentalis) leaves. Sooty films were collected from diseased leaves for fungal DNA extraction and observed using light and field‐emission scanning electron microscopy. Two types of conidia were present on the leaves: (i) hyaline and curved Cercostigmina‐like conidia and (ii) brown and straight Stigmina‐like conidia. The fungal pathogen was identified as P. pseudostigmina‐platani based on morphological characteristics and internal transcribed spacer region sequences. Some non‐glandular trichomes were found near the centre of the conidial masses on the abaxial surface of the naturally infected leaves. Leaf ultrasonication to dislodge conidia revealed sporodochia erupting through the stomata. Hyphae from sporodochia grew on the leaf surface and entered host tissues through the stomata. Conidiogenous cells had terminal rings and tapered towards the flat region where a conidium was attached. The conidia produced secondary conidia directly without hyphae. After dislodging the conidia by ultrasonication, scars were observed. These results indicate that the fungus has dimorphic conidia and microcycle conidiation for simple and rapid asexual reproduction.

First Report of Pleurostoma richardsiae Associated with Twig and Branch Dieback of Olive Trees in Spain.

Pleurostoma richardsiae has been described as an olive tree pathogen causing decline and brown wood streaking (Carlucci et al., 2013). A survey was carried out in plots under olive cultivation (Olea europaea L., cv. Picual; 10 year-old plants) at La Garrovilla, (Spain) in September 2020, in which a putative Verticillium wilt had been visually diagnosed. In Plot 1 (2.6 ha; 741 plants), 20.4% of the plants exhibited wilt, foliar browning and leaf drop, twig, and branch dieback. While the level of incidence in plots 2 (4.8 ha; 1368 plants), 3 (3.20 ha; 912 plants), and 4 (1.85 ha; 527 plants) was 25.0%, 19.5%, and 42.9% respectively, which meant for that harvest an average reduction in olive production, and an economic loss, of 30.2%. Three trees from each plot were uprooted and analyzed. Five out of 12 intriguingly showed brown streaking under the bark extending from the root system and ascending up the trunk, a symptom that is never associated with Verticillium dahliae wich does not produce necrosis and cankers in the wood (López-Escudero and Mercado-Blanco, 2011). Samples from the 5 tree trunks showing necrosis were taken to the lab and surface sterilized. Small pieces of discolored wood were placed onto malt extract agar plates containing chloramphenicol (0.25 g/L) and incubated for 21 days at 25°C in darkness. The growing fungal colonies were then transferred to potato dextrose agar (PDA). Isolates were identified by micromorphological characteristics, according to Vijaykrishna et al. (2004), as P. richardsiae. Colonies on PDA were cottony, brown with whitish edge, and produced abundantly two types of conidia: brown (spherical or subglobose), or hyaline (allantoids to cylindrical) that appeared on septated and inconspicuous phialides respectively. Identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al., 1990), and partial sequencing of the β-tubulin gene using T1 (O'Donnell and Cigelnik, 1997) and Bt2b (Glass and Donaldson, 1995) primers. ITS sequence showed a 99.82% identity with that of P. richardsiae IFM51337 (CBS406.93 type strain; GenBank AB364703.1), whereas β-tubulin sequence exhibited a 99.77% identity with P. richardsiae CBS406.93 β-tubulin gene (GenBank MT501304.1). ITS and β-tubulin sequences were deposited in GenBank (MZ519916 and MZ542764 respectively). The P. richardsiae isolate has been deposited in the Spanish Type Culture Collection (CECT 21196). Pathogenicity tests were conducted on 1-year old potted olive plants cv. Picual, maintained in a growth chamber at 25ºC and 12-h dark/12-h light. Twelve plants were inoculated in a wound made in the stem with a scalpel, and mycelial plug (5 mm diameter) from 15-day-old PDA plates were inserted into the wound. Another set of 12 plants were inoculated with sterile agar plugs and used as negative control plants. Four months after inoculation, 66% of the plants inoculated with mycelia plugs, showed wilting, necrosis under the bark, or even had died. P. richardsiae was successfully reisolated from necrotic areas in 75% of the plants inoculated with mycelia plugs. A total of 10 reisolates were identified as P. richardsiae by the above molecular techniques to confirm Koch's postulates. No symptoms were observed in the negative control plants and the pathogen was not re-isolated from them either. To our knowledge, this is the first report of P. richardsiae associated with twig and branch dieback of olive trees in Spain.

Isolation and identification of Esteya vermicola and its potential for controlling pinewood nematode

AbstractPine wilt disease is a devastating forest disease worldwide caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus). Esteya vermicola is widely recognized as a promising bio‐control agent effective against PWN. During a survey of associated microorganisms of Pinus yunnanensis in Southwest China, a novel isolate of E. vermicola was obtained. This isolate produced two types of conidia. One type of conidia was hyaline, unicellular, bacilloid, and non‐adhesive. The second type of conidia is solitary, hyaline, unicellular asymmetric elliptic or lunate, concave inward, ending slightly apiculate, adhesive on the concave surface, and contained an ovoid endospore‐like structure. Only the lunate conidia showed infection activity against PWN. This novel isolate produced a high proportion of infective lunate conidia and exhibited high adhesion and infection activity against PWN by adhering to the nematode within 48 h and killing 74.5% of the tested PWN individuals within 4 days. Given that the isolate was obtained from P. yunnanensis, it may be well adapted to the endogenous environment of pine trees. The isolate shows potential as a bio‐control agent against a plant‐parasitic nematode in the field.

Brown Culm Rot of Dendrocalamus latiflorus Munro Caused by Diaporthe guangxiensis in Sichuan Province, China.

Dendrocalamus latiflorus Munro, the most widely cultivated bamboo species in southern China, has high ornamental value used in gardens, while culms are also used for buildings and as fibers and edibles (Gao et al. 2011). In June 2020, brown culm rot of bamboo was observed in Yibin city, Sichuan Province, in an area of approximately 1000 hectares. Disease incidence was approximately 60%, of which 30% of the plants had died. At the end of June, the lesions expanded but did not surround the base of the culm. From the end of June to the beginning of September, the lesions expanded upward and formed a streak, of which the color gradually deepened to purple-brown and black-brown. At the same time, the disease spots at the base of the culm also expanded horizontally. After the spots surrounded the base of the culm, the diseased bamboo died. Ten culms showing typical symptoms were collected and cut into 5×5 mm pieces at the junction of infected and healthy tissues. The tissues were sterilized for 1 to 2 min in 3% sodium hypochlorite, decontaminated in 75% alcohol for 3 to 5 min, placed on modified potato glucose agar (PDA) with streptomycin sulfate (50 μg/ml), and incubated at 26°C. Two isolates were obtained by the single-spore method (Sivan et al. 1992). The isolates both produced white round colonies similar to Diaporthe guangxiensis and two types of conidia: one was α type (5.5 to 8.2×1.0 to 2.8 µm, n=30), colourless, single-celled, undivided, and oval, containing two oil droplets; and β type (21.1 to 30.2×0.8 to 1.4 µm, n=30), colourless, single celled and hook shaped. Genomic DNA was extracted from the two isolates by using a fungal genomic DNA extraction kit (Solarbio, Beijing). The products were amplified by polymerase chain reaction (PCR) with primers for the internal transcribed spacer 1 (ITS) region (White et al. 1990), calmodulin (CAL) gene (Carbone and Kohn 1999), translation elongation factor 1-alpha (TEF) gene (Glass and Donaldson 1995) and beta-tubulin (TUB) gene (Soares et al. 2018). The amplified products were sequenced and blasted in GenBank (accession numbers MW380383, MW431318, MW431317 and MW431316 for ITS, CAL, TEF, and TUB, respectively). The ITS, CAL, TEF, and TUB sequences showed 100%, 99.33%, 100%, and 99.80% identity to D. guangxiensis JZB320094 (accession numbers MK335772.1, MK736727.1, MK523566.1, MK500168.1 in GenBank), respectively. To evaluate the pathogenicity of the isolates, five plants were each inoculated with two isolates. The cortex of potted bamboo were injured locally with sterilized needle, and the bamboo culms were inoculated with 100 μl of conidial suspension (105 cfu/ml). The surface of the inoculation wound was covered with gauze soaked with sterilized water. Five plants inoculated with sterile water were used as controls. The treated plants were maintained in a greenhouse at a temperature of 22 to 29°C and relative humidity of 70 to 80%. One month later, of all inoculated plants showed similar symptoms as those observed in the field. D. guangxiensis was re-isolated from all inoculated plants. The pathogenicity test was repeated three times with similar results. This is the first report of D. guangxiensis causing brown culm rot of D. latiflorus in China. These results will facilitate an enhanced understanding of factors affecting bamboo and the design of effective management strategies of the pathogenic species on bamboo and thus to develop corresponding control measures.

First Report of Round Spot Disease of Zanthoxylum armatum DC Caused by Fusarium fujikuroi in China.

Zanthoxylum armatum DC, a deciduous tree in Rutaceae, has significant economic value as an important food condiment, spice, and medicine (Cao et al. 2019). Recently, an unknown round leaf spot disease has been found on Z. armatum in Meishan and surroundings areas of Sichuan province. The disease mainly affected the leaves, mostly on seedlings, with incidence of approximately 50%. Isolate HJYB-4 was isolated from typical diseased leaves and purified on potato dextrose agar (PDA). The isolate produced floccose white, magenta, or grey aerial mycelium. On the reverse side of the culture, the colony had the pigment of pale gray or magenta, with concentric rings of dark red and pale brown in the center. Morphological characteristics were recorded using a pure culture grown on PDA and Synthetic low nutrient agar (SNA). The hyphae of the isolate were colorless and septate. There were two types of conidia on SNA, microconidia, and macroconidia. Macroconidia were long and slender with parallel dorso-ventral sides, usually 3 to 5 septate, 21.89 to 47.21 × 3.33 to 4.73 μm. Microconidia were oval, melon-shaped, ovate, 0-1 septate, mostly no septate, 5.35 to 11.22 × 2.24 to 3.79 μm. A few pyriform microconidia and chlamydospores were observed. DNA of the isolate was extracted using the Column Fungal DNAout 2.0 (Tiandz Inc., Irvine, Beijing, China). PCR was performed using the following primers, ITS1/ITS4, TEF1/TEF2, TUBT1/TUBT2, fRPB2-5f2/fRPB2-7cr, LR0R/LR5, and NL1/NL4 to amplify the loci of the representative isolate in the ribosomal internal transcribed spacers (ITS), translation elongation factor EF-1α (TEF1), β-tubulin 2 (TUB2), RNA polymerase II largest-subunit (RPB2), the Large subunit (LSU) region of rDNA, 26S rDNA D1/D2 domain. The products were sequenced and Blasted. Blast analysisf the ITS, TFE, TUB2, RPB2, LSU, and NL amplicon revealed more than 99% of sequence identify with Fusarium fujikuroi. These sequences were submitted to GenBank and the GenBank accession numbers were as follows MT864359 (ITS), MT864358 (LSU), MT877222 (NL), MT902141 (RPB2), MT902140 (TEF), and MT902139 (TUB2). The phylogenetic tree was inferred from the combined datasets (TEF, TUB, and PRB2) from members of the F. fujikuroi species complex analyzed in this study (Jayawardena et al. 2019). The Phylogenetic tree revealed isolate HJYB-4 matched F. fujikuroi with a clade credibility value of 100%. According to the morphological characteristics and multi-gene phylogenetic analysis, the isolate was identified as F. fujikuroi. To complete pathogenicity tests, healthy leaves were needle-wound inoculated with mycelial plugs. The leaves that were inoculated with PDA-only plugs served as the control. After 3 days incubation at 26±2°C and 100% relative humidity, brown lesions that developed from inoculated leaves were similar to those in the field. No symptoms developed in the controlled leaves. Typical fungal cultures consistently isolated from symptomatic leaves, indicating that the fungus was responsible for the development of the disease. F. fujikuroi has been reported to cause root rot on Reineckia carnea (Sun et al. 2018), black rot on Macleaya cordata (Yu et al. 2019), and a wilt disease on sugarcane (Bao et al. 2020). This is the first report of F. fujikuroi attacking leaf of Z. armatum in China. The identification of this disease could provide the basis for the prevention and control of the disease at the seedling stage of Z. armatum.

Stem Rot Disease of Juglans sigillata Dode Caused by Fusarium fujikuroi in China.

Juglans sigillata Dode is an endemic species in the southwest China, and is an important nut and woody oil tree. The shell of its fruit is hard and can be used to make various crafts. From 216 to 2019, typical stem rot symptoms of 8-year-old J. sigillata were observed in cultivated fields in a 600-ha orchard in Zigong, Sichuan province, China. At this orchard, approximately 35% of the trees have been seriously damaged over the past few years. The typical symptoms were water-soaking on the stem, rotting, wilting, and blighting, eventually leading to the death of the plant. In June, ten diseased tissues were collected and surface-sterilized by 3% NaClO and 75% alcohol. Morphological observations were made from the isolates grown on Potato dextrose agar (PDA) and incubated at 25°C for 3 to 9 days. Morphological characteristics were made on pure cultures grown on Synthetic low nutrient agar (SNA). Five isolates with similar morphology were isolated from single spores. Colonies on PDA reached 8.3 cm in diameter after 6 days at 25 °C, aerial mycelia were white to cream and wol-like, later turning violet and dark purple with age. The hyphae of the strain were colorless and septate. There were two types of conidia on SNA, microconidia and macroconidia. Microconidia (n = 50) were oval, elliptic or clavate, no septate, 2.2 to 3.8 × 7.6 to11.7 μm. Conidiophores were branched or unbranched, solitary or in groups, phialides cylindrical to flask-shaped, monophialidic and polyphialidic. Macroconidia (n = 50) were long slender with a curved apical cell and foot-like basal cell, 3 to 4 septate and 2.1 to 3.9 × 26.2 to 53.4 μm. For molecular identification, the internal transcribed spacer (ITS), β-tubulin (TUB2), translation elongation factor (TEF1) and large subunit (LSU) were amplified with the corresponding primer pairs ITS1/ITS4 (White et al. 1990), BT2A/BT2B, EF1/EF2 (O'Donnell et al. 1997), and LROR/LR5 (Rehner and Samuels 1994), respectively. BLAST search results indicated that the ITS, TUB2, TEF1, LSU sequences (GenBank acc. nos. MT791384, MT786729, MN853324, and MT705246) showed 99 to100% identity with Fusarium fujikuroi sequences at NCBI (GenBank acc. nos. MG798789, MH398245, MK604519 and KJ954504). The results were confirmed by multilocus phylogenetic analysis. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as F. fujikuroi (Leslie and Summerell 2006). Koch's postulates were checked under controlled conditions. Fifteen 2-year-old healthy potted J. sigillata were inoculated by pricking the epidermis of stem with a needle and applying 150 µl of a microconidial suspension (1 × 106 spores/ml) to the wounded surface with a brush. Sterilizd distilled water was used as the control. The experiment was repeated three times. All the plants were incubated at 25 ± 2°C after inoculation for daily observation of disease development. After 12 days, the inoculated plants showed the same symptoms as observed in the original diseased plants, while the control plants were asymptomatic. The fungus was re-isolated from the symptomatic stems and was completely identical to the isolates used to inoculate the plants. Thus, we confirmed that F. fujikuroi caused the stem rot of J. sigillata. To our knowledge, this is the first report of this fungus causing stem rot in J. sigillata in China. Our results can help identify stem rot disease of J. sigillata and develop control measures for the disease.

Micromelanconis kaihuiae gen. et sp. nov., a new diaporthalean fungus from Chinese chestnut branches in southern China.

Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.