Pythium aphanidermatum causing seedling damping-off of Hylocereus megalanthus in China.

Hylocereus megalanthus (family Cactaceae), commonly known as bird's nest fruit (Yanwo fruit), was a new tropical plant cultivated commercially in south China because of its high nutritional content and sweet taste. In August 2023, damping-off disease of approximately 60% of seedlings was observed at a nursery in Zhanjiang, Guangdong Province (E110°17'46″ N21°9'2″). Stems of infected seedlings exhibited symptoms of water-soaked tissue which caused collapse at the base of the stem and sloughing of necrotic root cortex tissue was observed (Figure 1). White aerial mycelia were visible on the surface of the stem and soil at a high relative humidity. Diseased tissues about 0.5 cm2 were taken from the infected roots and stems, surface disinfected with 75% ethanol and 3% hydrogen peroxide solution, each for 1 min, subsequently rinsed in sterile water, and placed on potato dextrose agar (PDA). Plates were incubated at 25 to 28℃ in the dark for 3 days. Coenocytic hyphae grew from all infected roots and stems. Hyphal tip transfers were completed twice, and twelve isolates with the same morphological characteristics were obtained. The colony growth on PDA was ample. Main hyphae are up to 9.5 µm wide. Sporangia were terminal, inflated, branched or unbranched. Encysted zoospores were 7.5 µm in diameter. Oogonia were terminal, globose, smooth and of 16.8 to 27.4 µm (average 21.5 µm) diameter. Oospores were typically spherical, thick-walled, yellowish, 19.7 to 26.3 µm (average 21.1 µm) diameter, wall 1 to 2 µm thick. Antheridia were mostly intercalary, sometimes terminal, broadly sac-shaped, 15.0×19.0 µm (Figure 2). The morphological features were very similar to those of Pythium spp. (Toporek and Keinath 2021). For further identification, the LSU and ITS regions of isolate CCAS-YWGCD (stored in Agricultural Culture Collection of China, ACCC 35633) were amplified and sequenced with using primer pairs LROR/LR7 and ITS1/ITS4, respectively (Gao et al. 2017; White et al. 1990). The resulting sequences were deposited in GenBank (ITS: OR775664; LSU: OR775667). BLASTn results showed 100% sequence similarity with reference sequences of Pythium aphanidermatum (AY598622 for ITS and HQ665084 for LSU). Phylogenetic tree generated from maximum likelihood analysis based on combined LSU and ITS sequence data with MEGA 10.1.8, clustered the oomycete in P. aphanidermatum clade with 100% bootstrap support (Figure 3). Therefore, the oomycete was identified as P. aphanidermatum. To confirm Koch's postulates, six three-month-old seedlings of H. megalanthus (height about 15 cm) were transplanted to 15 cm pots. Six-mm-diameter mycelial plugs obtained from 7-day-old cultures at 25℃ in the dark were buried adjacent to the stem of three unwounded healthy seedlings. Another three seedlings inoculated with PDA agar served as controls. The plants were covered with plastic bags, kept at about 30℃, and watered regularly to keep the soil moisture content high. All inoculated seedlings exhibited symptoms of stems rot and damping-off, Symptoms did not develop on the control seedlings. P. aphanidermatum by morphological and molecular analysis was reisolated from the stems. P. aphanidermatum had been reported worldwide causing disease in many agricultural crops (Qi et al. 2021; Kim et al. 2020), but this is the first report causing damping-off of H. megalanthus seedling in China as well as worldwide, and this disease should be monitored in nursery seedlings.

Occurrence of Cassava (Manihot esculenta) Leaf Spot Disease Caused by Diaporthe ueckeri in China.

Cassava (Manihot esculenta) is a perennial crop of the family Euphorbiaceae, widely cultivated due to its phytopharmacological and economic values in China. In November 2022, a leaf spot disease on cassava was observed in Zhanjiang, Guangdong, China (21.17° N, 110.18° E), with 100% disease incidence. About 80 % of leaves were covered with spots on the infected plants. Typical symptoms initially appeared as irregular water-soaked lesions that became brown and whitish with the progress of the disease, lesions gradually expanded and coalesced, causing leaf withering, drying and final fall. Tissues (4 to 5 mm) were excised from the margin of lesions, sterilized in 3% H2O2 solution for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium (containing 50mg/L penicillin), and incubated at 25-28 °C. Ten single hypha isolates with similar morphology were obtained and further purified as single conidium subcultures. The colony was grey whitish with sparse aerial mycelium and colony diameter reached 70.4 mm after four days incubation at 25-28℃ in the dark. Black pycnidia occurring as clusters were spherical or irregular, erumpent at maturity, often with a creamy whitish, conidial cirrus extruding from ostiole after 30-days incubation. Conidiophores were hyaline, smooth, unbranched. Alpha conidia were bi-guttulate, hyaline, ellipsoidal, aseptate, with dimensions of 5.1~7.5×1.9~3.4µm (mean 6.2×2.8 µm, n>50). Beta conidia were abundant, filiform, hyaline, smooth, curved in a hooked shape, with a truncate base and dimensions of 18.5-26.4 × 0.6-1.2µm (mean 23.4 × 1.0 µm, n= 40) . Gamma conidia were not observed. The morphological characteristics were similar to those of Diaporthe ueckeri (Udayanga et al. 2015). The internal transcribed spacer (ITS) region, large subunit (LSU) rRNA sequence, actin (ACT), calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α), and ß-tubulin (TUB) genes of a representative isolate CCAS-MS-6 (ACCC 35497) were amplified and sequenced using primer pairs: ITS5/ITS4, LR0R/LR5, ACT-512F/ACT-783R, CAL228F/CAL737R, CYLH3F/ H3-1b, EF1-728F/ EF1-986R and Bt2a/Bt2b (Gao et al 2017；Udayanga et al 2014). All sequences were deposited in GenBank (OR361671, OR361672, and OR365605-9). BLAST search showed high similarities with sequences of Diaporthe ueckeri (Tab 1). Maximum likelihood analyses of the concatenated data of CAL, HIS, ITS, TEF and TUB using Mega 11 placed CCAS-MS-6 in the D. ueckeri clade. Thus, the fungus was identified as D. ueckeri. Three one-year old healthy plants were used for pathogenicity tests in pots. Two 15-day old leaves of each plant were cleaned with 75% alcohol, three sites on each leaf were wounded, and sites on one of the leaf were covered with fungal plugs from 15-day-old cultures on PDA, and sites on the other leaf with PDA plugs as a control. All plants were kept at ambient temperature (about 28℃) and covered with plastic bags containing sterile wet cotton to maintain the humidity. Seven days after inoculation, all inoculated sites showed symptoms of necrosis, while control sites showed no symptoms. The same fungus identified on the basis of morphological and molecular criteria was reisolated from symptomatic inoculated leaves. In China, D. ueckeri had been reported to cause diseases on Eucalyptus citriodora, Camellia sinensis, and Michelia shiluensis (Gao et al 2016; Liao et al 2023; Yi et al 2018), this is the first report on M. esculenta. The definition of the disease etiology is a prerequisite to develop effective management strategies.

Neoscytalidium dimidiatum causing stem canker on Hylocereus megalanthus in China.

Hylocereus megalanthus (syn. Selenecereus megalanthus), commonly known as Yanwo fruit (bird's nest fruit), is an important tropical fruit, which is popular and widely planted due to its high nutritional and economic value in southern China. In September 2022, a serious stem and fruit canker was observed on Ecuadorian variety of Yanwo fruit plant in a 0.2 ha orchard in Guangdong (N21°19'1.24" E110°7'28.49"). Almost all plants were infected and disease incidence of fruits and stems was about 80% and 90% respectively. Symptoms on the stem and fruits were small, circular or irregular, sunken, orangish brown spots that developed into cankers (Fig 1 A, B and C). Black pycnidia were embedded under the surface of the cankers at the initial stage, subsequently they became erumpent from the surface, and the infected parts rotted. Five symptomatic stems from five plants were collected, 0.2 cm2 tissues adjacent to cankers were surface sterilized and placed on potato dextrose agar (PDA) to incubate at 25 to 28 ℃. Fungal isolates each with similar morphology grew from 100% of the tissues. Colonies covered with aerial mycelium were grayish white, and then gradually turned to grayish black. Septate hyphae were hyaline to brown and constricted into arthroconidial chains. The arthroconidia were variously shaped and colored, orbicular to rectangular, hyaline to dark brown, thick-walled, and zero- to one- septate, averaging 7.7 × 3.6 µm (n>50) (Fig 1 D, E, F and G). To identify the fungus, the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), histone H3 (his3) and chitin synthase (chs) gene of isolate ACCC 35488 and ACCC 35489 (Agricultural Culture Collection of China) were amplified and sequenced with primer pairs: ITS1/ITS4 (White et al. 1990), EF1-728F/EF2-rd (Carbone & Kohn 1999; O'Donnell et al.1998), TUB2Fd/ TUB4Rd（Aveskamp et al 2009）, CYLH3F/H3-1b (Crous et al. 2004) and CHS-79F/CHS-345R (Carbone & Kohn 1999) (ITS: OQ381102 and PP488350; tef1: OQ408545 and PP510454; tub2: OQ408546 and PP510455; his3: OQ408544 and PP510453; chs: OQ408543 and PP510452). Sequence Blastn results showed above 99% identical with those of Neoscytalidium dimidiatum ex-type strain CPC38666. Phylogenetic tree inferred from Maximum Likelihood analysis of the combined ITS, tub2 and tef1 sequences revealed two isolates clustered with N. dimidiatum (Fig 2). Pathogenicity was tested on healthy one-year-old cuttings and fruits of Ecuadorian variety at room temperature. Six sites were pin-pricked on each stem and fruit. Both wounded stems and fruits were inoculated with spore suspensions (106 spore/ml) and 6-mm fungal plugs respectively. Sterile water and agar were used as control. The test was repeated twice. Stems and fruits were enclosed in plastic boxes with 80% relative humidity. Symptoms described above were observed on inoculated stems and fruits at five days post inoculation (Fig 1 H and I). No symptoms developed on the controls. Neoscytaliudium dimidiatum was reisolated from the cankers with a frequency of 100% via morphological and molecular analysis. This is first report of stem and fruit canker caused by N. dimidiatum on H. megalanthus in China and this disease represents a serious risk of Yanwo fruit yield losses. This fungus is widespread occurring throughout the world causing diseases on a wide variety of plants. The finding will be helpful for its prevention and control.

Dalbergia odorifera, a new host of Colletotrichum tropicale causing anthracnose in China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is an endangered, wild and second class key protected tree species in China. It has been cultivated widely in south China because of its valuable rosewood and pharmacological uses. During January and September 2022, anthracnose was observed on D. odorifera in Chikan, Zhanjiang, Guangdong (N21°15' 9.4" E110°22' 25.9"). All of the D. odorifera plants were infected and the leaf disease incidence was above 30%. The lesions were 0.35 to 0.65 cm in size, greyish-white, and gradually expanded into brown black circular spots surrounded by a yellowish halo. Later, the centers of spots were white with dark-brown borders. In severe cases, the leaves turned yellow, withered and fell off. To isolate the pathogen, about 5×5 mm tissues at the disease-health junction of spots were sterilized with 75% ethanol for 30s and 3% hydrogen peroxide for 3 min, rinsed with sterile water three times, and plated onto PDA for incubation at 25-28℃ in the dark. Isolates were obtained by picking the tip of hypha growing from the tissues, further purified by coating the diluted spore suspension to obtain single-spore isolates. Colonies with abundant aerial mycelium were white to light gray, 4.0 to 5.0 cm in diameter after 5 days at 25 to 28℃ in the dark. Conidiogenous cells were hyaline, cylindrical and monoblastic. Conidia were unicellular, subcylindrical, hyaline, guttulate, 3.5 to 5.6×11.1 to 16.8 (av. 4.6±0.48 × 14.0±1.25) µm (n>50). Appressoria were dark brown subglobose, clavate, fusiform, occasionally lobed, terminal, 7.8 to 16.3×4.6 to 8.9 µm. No swollen cells and setae were observed. To further confirm identity of the fungus, partial regions of six genetic loci of isolate ACCC 35246 (stored in Agricultural Culture Collection of China) were amplified and sequenced: actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), histone (HIS), beta-tubulin (TUB) and internal transcribed spacer (ITS) utilizing the primer pairs (Damm et al. 2012; Weir et al. 2012; White et al 1990) (OP314900-OP314904 and OP269660, respectively). Based on a BLAST analysis, the six sequences were about 99% identical to those of Colletotrichum tropicale holotype strain CBS124949 (ACT: JX009489 270/272; CHS: JX009870 295/299; GPDH: JX010007 274/279; HIS: KY856395 638/670; ITS: JX010264 546/548 and TUB: JX010407 494/497, respectively) (Weir et al. 2012). Phylogenetic analyses, using a combined dataset of ACT, CHS, GPDH, ITS and TUB were carried out in MEGA X using the maximum likelihood method, placed the fungus within the C. tropicale clade. Based on morphogical and molecular analyses, the fungus was identified as C. tropicale (Rojas et al 2010). To test the pathogenicity, five healthy leaves of 1 year-old potted plants were stab-wounded with a sterile needle and inoculated with 10 µL of spore suspension (105 conidia/mL). Another five healthy leaves were inoculated with sterile water as the control. Symptoms expressed by the inoculated leaves, 10 days after inoculation, were the same as those of the diseased plants, while the non-inoculated leaves remained asymptomatic. The same fungus based on morphogical and molecular criteria was re-isolated from the spots. As an endophyte or plant pathogen, C. tropicale inhabits a wide host range of plant species belonging to 31 genera in 25 families (Farr and Rossman 2022), D. odorifera is a new host of C. tropicale causing anthracnose in China. This work will be helpful for the diagnosis and control of this disease.

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

First report of Brown blotch disease caused by Diaporthe ueckeri on Hyophorbe lagenicaulis in China.

Bottle palm (Hyophorbe lagenicaulis) is a picturesque evergreen plant in the family Aceraceae, ubiquitously cultivated as an ornamental tree throughout the tropics and subtropics due its attractive shape and small stature. During 2016-2022, brown spots were observed on the leaves of bottle palm on both sides of a campus road in Mazhang, Zhanjiang, Guangdong province (N21°8' 59.9" E110°17' 51.4"). Symptoms appeared as circular, light yellow to brownish red, slightly sunken spots, and brown to black in the center (Fig 1 A-C). The spots expanded to big irregular blotches, which finally led leaves to wither. 0ne hundred percent of 50 plants were infected and 90% of the leaves each plant were covered with brown patches of different sizes. Tissues (5 × 5 mm) from the diseased-healthy junction of the leaf spots were surface disinfected with 75% ethanol for 30 s and 3.5% hydrogen peroxide solution for 5 min, rinsed three times with sterile water and placed on potato dextrose agar (PDA) at 25-28℃ in the dark for 3 days. Fungi with the similar morphology grew from 100% of these inoculated tissues. Hyphal tips from the colony edges were transferred to PDA. Single isolates were obtained by plate dilution method after sporogenesis. Colonies with sparse aerial mycelium were flat, grey. Conidiomata were superficial, black, solitary, scattered. Conidiophores were cylindrical, densely aggregated. Alpha conidia with bi-multiguttulate were hyaline, smooth, ellipsoidal, aseptate, 4.7-7.9×1.6-3.4 µm (Av. 6.4×2.3µm, n>50). Beta conidia were filiform, slightly curved, 15.9-28.3× 0.7-1.1 µm (20.1 × 0.9 µm, n>50) (Fig 1 E-H). Gamma conidia were not observed. The internal transcribed spacer (ITS) regions, large subunit (LSU) rRNA sequence, translation elongation factor (TEF), actin (ACT), calmodulin (CAL), histone H3 (HIS), and beta tubulin (TUB2) genes were amplified using the primers ITS4/ITS1 (White et al. 1990), LR0R/LR5, EF1-728F/EF-2, ACT-512F/ACT-783R, CAL228F/ CAL737R, CYLH3F/ H3-1b, and TUB2Fd /TUB4Rd (Aveskamp et al. 2009; Carbone and Kohn 1999; Crous et al. 2004; O'Donnell et al. 2000), respectively. All seven sequences of the isolate CCAS-JPYZ-4B (ACCC 35493) were submitted to NCBI (OR430112-3, OR451702-6). BLAST search result showed high sequence identity with several Diaporthe ueckeri isolates (Tab1). Phylogenetic analysis of the concatenated data of CAL, HIS, ITS, TEF and TUB genes using Maximum Likelihood method placed CCAS-JPYZ-4B in the D. ueckeri clade. Thus, the isolate was identified as D. ueckeri (Udayanga et al. 2015). Pathogenicity tests were performed on three 5-year-old plants, one healthy new leaf per plant and 10 sites on per leaf were slightly wounded and inoculated with 5-mm mycelial plugs from 10-day-old culture on PDA. The control sites were treated with PDA plugs. Each inoculated leaf was covered with a plastic bag to maintain high humidity and kept at natural temperatures (25-28 ℃). The experiment was repeated once. Symptoms appeared as those described as above 5 to 10 days after inoculation (Fig 1 D). Controls were asymptomatic. The fungus was reisolated from diseased leaves and identified as D. ueckeri. D. ueckeri may infect Arachis hypogaea, Cucumis melo, Camellia sinensis, Glycine max, Mangifera indica and Michelia shiluensis, and this is first report causing brown blotch on bottle palm in China. This disease occurred all year round, which seriously affected plants growth and ornamental value; it is necessary to develop an effective management strategy.

Erysiphe elevata causing powdery mildew on Eucalyptus urophylla × E. camaldulensis in China.

Eucalyptus urophylla × E. camaldulensis, named Chiwei eucalypt, is a hybrid species widely used in China. Many of its clones are cultivated for afforestation due to cold tolerance, high yield, high strength and disease resistance. Clone LH1 is planted extensively for its high stability and machinability in South China. In December 2021, severe powdery mildew signs were observed on clone LH1 in Zhanjiang, Guangdong (N28°8'29"; E110°17'5"). Whitish powder principally appeared on both abaxial and adaxial leaf surfaces. All plants were infected within about a week and above 90% leaves were diseased, which resulted in abnormal growth and shrinkage of leaves. Hyphae with single, lobed appressoria were hyaline, septate, branched, 3.3-6.8 µm (ave. 4.9 µm, n>50) wide. Conidiophores with a straight to flexuous foot-cell (14.7-46.1×5.4-9.7 µm, ave. 25.8×7.9 µm, n>30) were erect, hyaline, 2-septate, unbranched, 35.4-81.8 × 5.7-10.7 (ave. 56.7×8.7 µm, n>50). Conidia were solitary, hyaline, cylindrical to elliptical, 27.7-46.6 ×11.2-19.0 (ave. 35.7×16.6 µm, n>50). Chamothecia were not found on infected trees. The further identification was confirmed by partial sequences of internal transcribed spacer (ITS), large submit rRNA gene (LSU), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and RNA polymerase II second largest subunit (RPB2) gene. A very small amount of mycelia and spores from voucher specimens CCAS-ASBF-1 and CCAS-ASBF-2 were deposited in the herbarium of Guangdong Ocean University. Specimens were PCR amplified and sequenced with primer pairs ITS1/ITS4 (White et al 1990), LROR/LR7 (Moncalvo et al 1995), PMGAPDH1/PMGAPDH3R, GSPM2/GSPM3R and PmRpb2_4/ PMRpb2_6R (Bradshaw, et al. 2022), respectively. BLASTn results showed that ITS (OP270019 and OQ380937), LSU (OP270018 and OQ380938), GAPDH, GS and RPB2 (OQ414445- OQ414450) were above 99% identical with those of E. elevata on Catalpa bignonioides (ITS: AY587013) (Cook et al 2004), Plumeria rubra (ITS: MH985631) (Yeh et al 2019), Cerbera manghas (ITS: MZ379159; LSU: MZ379160) (Mukhtar et al 2022), and Eucalyptus camaldulensis (LSU: LC177375-6) (Meebon et al. 2017), and above 99% identical with those of Erysiphe vaccinii FH00941201 on Vaccinium corymbosum (ITS: ON073869; RPB2: ON119159; GS: ON075687) and FH00112205 on V. vacillans (ITS: ON073870; GAPDH: ON075646) (Bradshaw et al 2022). This is the first sequence data for non rDNA of E. elevata. In an ITS tree phylogenetic analysis with Maximum likelihood (ML) method showed the fungus clustered in a highly supported clade with E. elevata and E. vaccinii. In a multi-locus tree, E. elevata grouped in a sister position to E. vaccinii FH00941201. Thus, the pathogen was identified as E. elevata based on morphology, DNA BLASTn and phylogenetic analysis (Braun and Cook 2012). Pathogenicity tests were conducted on healthy leaves of 1-year-old potted plants. Ten leaves were cleaned with sterile water, inoculated by gently dusting conidia from single lesion on the naturally infected leaves, and then covered with plastic bags containing wet absorbent cotton. Non-inoculated leaves served as controls. Symptoms developed on all inoculated leaves 3 to 5 days after inoculation, and the fungus was identical to the original fungus on the infected leaves, whereas control plants remained symptomless. This is the first report of powdery mildew caused by E. elevata on Eucalyptus sp. from China. This finding is helpful for land managers to diagnose and control the disease.

Leaf blast, a new disease on Pennisetum sinese caused by Pyricularia pennisetigena in China.

Pennisetum sinese Roxb, named king grass or 'sugarcane grass', belonging to the Poaceae family, is widely cultivated in China because of its use for livestock feed, bioenergy, water-soil conservation and phytoremediation. In September 2018, leaf blast on P. sinese was observed in a pasture in Zhanjiang, Guangdong province (21.15°N, 110.30°E ). The lesions initially were round, water-soaked, oval or spindle-shaped, then later turned to typical eye-shaped, whitish in the center with brown-black necrotic borders surrounded by a yellow halo (Fig. S1). Almost all of plants were infected and most of the spots appeared on the blades in the lower part of the plants. The leaves gradually turned yellow and withered, which affected the growth and quality of the herbage. Diseased leaves were surface-sterilized with 75% ethanol for 30s, followed by 3% hydrogen peroxide solution for 3 mins, and rinsed three times with sterile water, then placed in a hermetic container with moistened filter paper, and kept at 21-24℃ to stimulate conidiogenesis. After conidiation, conidial suspensions were prepared with sterile water and evenly spread onto PDA, and incubated at 25 ℃ in the dark to collect single-spore isolates. Colonies on PDA were fuscous black with grey centers, broad white and flat. Conidiophores were solitary, erect, straight or curved, unbranched, medium brown, smooth, 2 septate, and sometimes up to 5 septate. Conidia with a protruding and truncating hilum on basal-cells were pyriform to obclavate, 2 septate, 22.2-28.8 × 7.1-9.6 µm ( 24.9±1.7 × 8.5±0.6 µm) (Fig S2). The internal transcribed spacer region (ITS), nrRNA gene large subunit (LSU), actin gene (ACT) and RNA polymerase II largest subunit gene (RPB2) were PCR amplified, and the amplicons were sequenced by Sangon Biotech (Shanghai) Co., Ltd. (ITS: OM294657, LSU: OM294656, ACT: OM304642, RPB2: OM304643). BLASTn result showed above 99% nucleotide identity with the ITS (KM484935 465/465 bp), LSU (MH412641 862/863 bp), ACT (XM_029891619 418/421 bp) and RPB2 (XM_029892603 894/898 bp) of Pyricularia pennisetigena strains. Maximum likelihood (ML) analysis based on ITS using MEGA revealed that the fungal isolate clustered in P. pennisetigena clade with 99% bootstrap support (Fig S3). Based on morpho-molecular criteria, the fungus was identified as P. pennisetigena (Klaubauf et al., 2014; Gómez Luciano et al, 2019; Wang et al., 2019). Four healthy leaves of a 2-month-old plant were surface disinfected, and 2 leaves were wounded with a sterile needle (3 wound sites each leaf), then one leaf was inoculated with 10 µl 105 conidia/ml suspension on each site, and on the other leaf, sterile water was used as the control inoculation. The similar inoculations were conducted on the other 2 unwounded leaves. Pathogenicity tests were repeated three times. Seven days later, all inoculated leaves (wounded and unwounded) showed typical symptoms, while no symptoms were observed with the controls on both the wounded and unwounded leaves. The same fungus was recovered and confirmed by morphology and the ITS and LSU sequences. P. pennisetigena is pathogenic on a wide range of Poaceae plants in Brazil, Japan, Mali, Philippines, United States, China (Klaubauf et al., 2014; Reges et al., 2016, Wang et al., 2019). Han et al (2020) reported bacterial leaf blast on king grass while this is first report of P. pennisetigena causing leaf blast. This finding is a warning to prevent and control of this disease and its infectivity to Poaceae plants.

First report of anthracnose on Ophiopogon jaburan caused by Colletotrichum liriopes in China.

Ophiopogon jaburan (Liliaceae), named white lilyturf, is widely cultivated as an ornamental plant in south China. During 2017-2019, leaf spots on O. jaburan were observed all year in Zhanjiang, Guangdong, China (N21°9'3"; E110°17'47"). Almost all plants were infected and the disease incidence on affected leaves was about 80% in the field. Initially, spots were brown, round or oval, and gradually enlarged to irregular shapes. The color of the spots changed from rusty-brown to grayish-white with rusty-brown borders. Subsequently, the spots expanded until the leaves withered and died. Infected tissues were surface-sterilized with 75% ethanol for 30s followed by 1% NaClO solution for 1 min, then rinsed thrice with sterile water, before placed on potato dextrose agar (PDA) containing 50mg/L ampicillin, and incubated in darkness at 25℃ with 90% relative humidity. Colonies growing on PDA were cushion-like, pale greenish grey to grayish black on the front side and clearly dark gray on the reverse. Colony diameter was av. 86.0 mm (n = 15) grown in the dark at 25 ℃ for 10 days. Conidia with oil droplets were colorless, hyaline, smooth-walled, aseptate, slightly curved, and tapered gradually to each end, 12.3-28.9 × 2.2-6.6 µm (av. 20.9×4.2µm, n=200). Setae were brown to dark brown, 2-4 septate, with the base slightly inflated, and measured 40.0-130.3 × 2.2-5µm (av. 84.3 × 3.3µm, n=23). On PDA, scattered or loosely clustered appressoria were elliptical or irregular, smooth-walled, aseptate, and dark brown. To confirm the identification, partial regions of the internal transcribed spacer (White et al. 1990), beta-tubulin (Aveskamp et al. 2009) and actin (Carbone et al 1999) were amplified and sequenced (MW989743, MZ014461 and MZ014462). The blast results showed these sequences had >99.59% homology with sequences of Colletotrichum liriopes holotype strain CBS 119444 (NR_111449, GU228098 and GU227902). Maximum likelihood analysis and Bayesian inference were performed from concatenated sequences using RAxML v.1.0.0 and MrBayes v.3.2.1 software respectively. Several C. liriopes strains clustered in the same clade. Based on morphological-molecular characteristics, the fungus was identified as C. liriopes (Damm et al 2009; Chen et al. 2019). To confirm pathogenicity, healthy leaves were surface disinfected with 75% ethanol and rinsed thrice with sterile water. On ten leaves, three sites were wounded by pricking with needles, and inoculated 20 µL of 106 conidia/ml suspension or mycelium in contact with blade surface using 6-mm mycelial plugs. Similarly, the inoculation was done for three unwounded sites each leaf. Sterile water and medium plugs (without fungus) served as controls. All leaves were incubated on sterile wet filter paper at 25-28℃ with 90% relative humidity. After 7 days, all the inoculated leaves showed symptoms similar to those of field diseases, whereas control leaves remained healthy. The fungus with morphological-molecular features identical to the original isolate was reisolated from the disease lesions. C. liriopes causes anthracnose on Bletilla ochracea, Eria coronaria, Hemerocallis fulva, Pleione bulbocodioides (Jayawardena et al 2016) and Liriope sp. (Yang et al 2020; Chen et al 2019) in China. This is the first report of C. liriopes causing anthracnose on O. jaburan in China. Anthracnose could greatly affect ornamental value of O. jaburan, and this work can alert gardeners to prevent and control of the disease.

Leaf Spots on bodhi tree (Ficus religiosa) caused by Diaporthe tulliensis.

Ficus religiosa L., known as bodhi tree, is an ornamental tree and widely planted as an avenue and roadside tree due to ovate-rounded leaves with narrow, elongated tips. During 2018-2021, circular to oval-shaped leaf spots with pale white centers and brown-black edges surrounded by a chlorotic halo were observed on the leaves of more than 200 bodhi trees all year round in a park in Zhanjiang, Guangdong (N 21°15'22.29''; E110°23'1.03''). The leaf disease incidences were usually 15-80%, in severe cases, up to 100% in autumn and winter every year, and some trees shed all leaves(Fig S1). Repeated annual defoliation may weaken the tree and decrease the aesthetic value in the landscape. Diseased tissues (5 × 5 mm) of five symptomatic infected leaves were surface sterilized in 3% hydrogen peroxide solution for 3 min, rinsed thrice with sterile water, plated on potato dextrose agar (PDA) amended with ampicillin (50mg/L), and incubated at 25-28 ℃ in the dark for 3-7 days. Five strains with similar morphology were obtained by transferring hyphal tips of the colonies to fresh PDA and further isolating by single spore method. Fungal colonies were flat and spreading, with sparse, white aerial mycelium, and black pycnidial conidiomata semi-immersed in PDA after 30-days incubation at 25-28 ℃ in dark. Conidiophores were hyaline and α-conidia were single-celled, oval to fusiform, guttulate, 5.3 × 2.5 µm (n>50), similar to Diaporthe sp. (Crous et al. 2015), but no ß and γ -conidia were observed. The internal transcribed spacer(ITS), large subunit ribosomal RNA gene(LSU), calmodulin (CAL) and ß-tubulin(TUB) gene regions of a representative strain were amplified using specific primers reported by White et al. (1990), Gao et al (2017) and Gomes et al (2013), and submitted to GenBank (ITS: OM200852, LSU: OM228732, CAL: OM244761, TUB: OM244760). NCBI Blastn showed above 99% identity to D. tulliensis (anamorph: Phomopsis heveicola ) isolates of ITS (MT974186, MN393590 and KX457967), LSU (KR936131), CAL (MW759801), and TUB (KR936132 and MN399886), respectively (Crous et al. 2015; Huang et al. 2021; Gong et al. 2020; Bai et al. 2017). Based on the concatenated ITS, CAL, and TUB, a Maximum Likelihood phylogenetic analyses using MEGA 10.1.8 clustered the fungus with D. tulliensis in a clade with a 93% bootstrap support(Fig S2). Therefore, the fungus was identified as D. tulliensis based on morpho- molecular characteristics. Healthy detached leaves were sanitized thrice with 70% alcohol, and rinsed with sterile water. PDA plugs with actively growing 10-days-old mycelium were placed on predetermined sites, put into a sealed box with above 80% relative humidity and incubated at room temperature (25-28℃). Each isolate was inoculated at 25 needle-wounded and unwounded sites, PDA plugs without mycelium served as controls. Symptomatic spots appeared on all wounded leaves by 7 days post-inoculation (dpi) and on all unwounded leaves by 12 dpi. No symptoms appeared on controlled leaves. Pure cultures were recovered from inoculated leaves and showed identical morpho-molecular criteria to the original isolates. More than 70 pathogenic fungi are known to cause diseases on F. religiosa while there is no record of D. tulliensis infecting bodhi according to the U.S. National Fungus Collection (Farr and Rossman 2022). This report could provide basic understanding and alerting role for horticulturist in daily management.

First report of internal black rot on Carica papaya fruit caused by Fusarium sulawesiense in China.

Papaya (Carica papaya L.) is a tropical fruit consumed worldwide due to its nutritional, medicinal and pharmacological properties. In China, papaya was widely planted in Guangdong, Guangxi, Hainan, Yunnan, Fujian and Taiwan provinces. From September to December in 2015-2020, fruit with internal black rot disease was observed in papaya plantation in Xuwen, Guangdong province (N20°20'9"; E110°14'45"), approximately 5% fruits on about 85% trees were infected every year. The infected fruits showed the symptom of 'false-ripening' and the pericarp color changed from green to yellow earlier than that of normal fruits. In the cavity of diseased fruits, the sarcocarp black rotted and conspicuous mycelia were observed. Mycelia and infected tissues from symptomatic fruits were picked up, placed on potato dextrose agar (PDA) with 50mg/L ampicillin and incubated at 25± 2 ℃ in the dark. The fungus was purified by spore dilution plate method. Fast-growing colonies with dense, floccose, cottony mycelium were initially white gradually becoming buff brown. Macroconidia were falcate, 3-5 septa with foot-shaped cell and 10.35-41.50 (av. 25.41±6.82) ×1.90-5.95 (av. 3.67±0.85) µm (n>140) in size after 7 days of incubation on carnation leaf agar (CLA). There were scarce microconidia. Chlamydospores were intercalary, solitary or in chains, globose or irregular, hyaline to light brown. The morphological characteristics of the fungus were similar to that of Fusarium sulawesiense (Maryani et al. 2019). The internal transcribed spacer region (ITS) (KU881904 and KY436233), translation elongation factor 1-alpha (tef1) (KU894408 and KY436232), and RNA polymerase second largest subunit (rpb2) (KU894409 and KY436231) were sequenced from two isolates to cofirm species identification. Blast analysis in the FUSARIUM-ID and the NCBI databases revealed above 99 to 100% identity match with the F. sulawesiense strains NRRL34056, NRRL34059, NRRL34004 and NRRL43730 (Xia et al 2019). Maximum likelihood (ML) analysis and Bayesian inference (BI) based on the concatenated sequences using RAxML v.1.0.0 and MrBayes v. 3.2.1 software revealed that the isolates were resolved in the same clade with the F. sulawesiense strains. Thus, the fungus was identified as F. sulawesiense based on morphological characteristics and molecular criteria. To confirm pathogenicity, five healthy fruits were injected with 200 µl of spore suspension (approximately 104 spores/ml) in the field and laboratory, and isovolumetric sterile water served as control. Each fruit was sealed with a plastic bag and kept at natural temperature (about 25-30 ℃). All the inoculated fruits developed typical symptoms after 30 days in the field and 15 days in the laboratory, whereas no symptoms were observed on the control fruits. F. sulawesiense was reisolated from inoculated fruits, but not from non-inoculated fruits. F. sulawesiense displayed a broad host which included Oryza sativa, Musa nana, Citrus reticulata, and Colocasia esculenta etc. in China (Wang et al. 2019). To our knowledge, this is the first report of F. sulawesiense causing internal black rot on papaya fruit. This work is important for papaya growers to prevent this disease in time.

First Report of Southern Blight of Manglietia decidua Caused by Sclerotium rolfsii in China.

Manglietia decidua, named 'Hua manglietia', belonging to the Magnoliaceae family, is one of the most important ornamental plant in China. In 2019 and 2020, an unknown disease caused 3- to 12-month plants of M. decidua to wither and die in the field in Zhanjiang, Guangdong province（N21°9'3";E110°17'47"）. Initially, the infected plants showed leaves dehydration, chlorosis and wilting with water-soaked lesions on stems at ground level. About 7 days later, the plants completely wilted, collapsed and died. Delayed and stunted growth with wilting of foliage continued through the whole year. Dense white mycelial mat and small white-to-brown spherical sclerotia were observed on the surface of the stalk lesion when weather conditions were warm and humid. Approximately 10% of plants were infected. Especially from July to October 2020, up to 30% of about 500 plants were infected and died. To identify the causal agents of the disease, infected tissue and sclerotia were collected, surface disinfected in 75% alcohol for 30s and 30% hydrogen peroxide solution for 5 min, and washed with sterile water for 1 min. The surface disinfected tissue and sclerotia were put on potato dextrose agar containing ampicillin (50mg/L) and kept in an incubator at 25°C in the dark. Fast growing fungus colonies with white mycelium and numerous sclerotia developed in the plates after 6 to 8 days of incubation. The hyphae were septate, hyaline and formed typically clamp connection after 10 days of growth. Sclerotia were initially white and became tan to dark brown over time and 1.0 to 3.0 mm (2.13 mm on average, n=124) in diameter at maturity. For molecular identification，the ITS region was amplified using primer pair ITS1/ITS4 (White et al. 1990). A 666 bp PCR product was sequenced (GenBank accession no. MW093622) and shared above 99% sequence identity with some Athelia rolfsii isolates (GenBank accession Nos. HQ895869, KX499470 and AB075290). Based on morphological and molecular characteristics, the fungus was identified as Sclerotium rolfsii (teleomorph A. rolfsii) （Paul et al. 2017，Xu et al. 2010. Pathogenicity tests were conducted by inoculating ten healthy 1-year-old M. decidua plants grown in pots. Five sclerotia and mycelial mat obtained from 15-day-old cultures were buried adjacent to the stem of each unwounded healthy plant. Non-inoculated plants served as controls. After inoculation, the plants were maintained in a 25-28 ℃ greenhouse and watered regularly to keep the soil moisture content at about 15%. Symptoms of southern blight were observed on all inoculated plants, which began to wilt 7 to 10 days after inoculation and died within 15 to 20 days. The control plants remained healthy. S. rolfsii was again isolated from the artificially inoculated plants, but not from non-inoculated plants. The pathogenicity test was repeated twice and the results were the same. S. rolfsii has an extensive host range worldwide and the common host ornamental plants are Iris, Chrysanthemum, CymbidiumTrifolium, Jasminum, Begonia, and Stevia etc. in China. To our knowledge, this is the first report of southern blight caused by S. rolfsiii on M. decidua in China. M. decidua is a horticultural plant which belongs to the protected and endangered tree species. This finding is important to alert growers to realize the proper management of this disease during species protection and cultivar extension.