First report of Colletotrichum siamense as a causal agent of anthracnose on wax apple (Syzygium samarangense) in China.

Wax apple (Syzygium samarangense) is an important fruit tree widely cultivated in China. Yield losses are usually serious due to different diseases among which anthracnose (Colletotrichum spp.) is one of the most damaging (He et al, 2019). This disease was found in Yunnan, China and an average incidence of 56.7% diseased leaves was recorded in21 orchards surveyed in July2021. The disease lesions on leaves were circular, angular or oval (7.2-15.6 mm), with whitish center and brown outer area surrounded by a yellow halo; irregular spots or blight areas formed later. It can also infect fruits forming pale-brown, circular and sunken spots before harvest and rot of stored fruits. Diseased leaves were sampled from orchards in Ximeng (N117.78oE39.89o) and Ninger (E101.04oN23.05o) counties of Yunnan for fungal isolation; three and five pure isolates were recovered from Ximeng (LWTJ1-LWTJ3) and Ninger (LB4-LB8) samples, respectively, by plating disinfested tissue (surface-sterilized with 2% NaClO3) on potato dextrose agar (PDA) followed by hyphal tip purification and incubation at 25oC. Two repeated tests following Koch's postulates were conducted to verify pathogenicity of the eight isolates. In each test, three healthy seedlings per isolate were sprayed with conidia suspenson (2.26×105cfu/mL) until runoff from leaves while control plants were sprayed with sterile water. The plants were kept in the dark at RH100 for 24 h in a black box and then in a growth chamber (28oC, RH>90% and lighting 12h/d). Detached fruits were inoculated with mycelial discs on the puncture-wound surface. Anthracnose symptoms developed on all seedlings and fruits inoculated with LWTJ2 or LB4 isolates, which were re-isolated from lesions of inoculated leaf/fruit, completing Koch's postulates. Control plants were healthy and symptomless. LWTJ2 and LB4 isolates were morphologically the same: the colonies on PDA were circular, pale-white, with cottony surface and readily forming orange conidium masses. The hyphae were hyaline, septate, branched mostly in near right angles. The conidia were hyaline, one-celled, smooth-walled, cylindrical with round ends, 9.8-17.5 (av.13.8) µm×4.4-6.5 (5.6) µm. The teleomorph was not observed in culture or on orchard trees. The morphological characters were consistent with those of C. siamense described by Weir et al (2012). The internal transcribed spacer region (ITS) was amplified from the two isolates by PCR and sequenced (1990) and were 545 bp in length (OL963924 & OL413460). BLAST analysis showed that both were 100% identical and they shared 99.08% identity with C. siamense WZ-365 from the ITS region (MN856443).The Tub2 (788 bp, ON637119) and Cal (768 bp, ON622249) genes (Weir et al, 2012) of LB4 were also obtained and they shared closest identity (99.45% & 100%) with those of C. siamense WZ-365 as well. Phylogenetic tree (neighbor-joining) analysis of the concatenated sequence of ITS, Tub2 and Cal genes of LB4 and those of related Colletotrichum spp. showed that LB4 clustered IN the same end-branch with C. siamense ICMP18578 (Bootstrap sup. = 98%). Thus, C. siamense was identified as the pathogen of wax apple anthracnose in Yunnan. It caused anthracnose on other crops as oranges and cacao (Azad et al, 2020). Also, C. fructicola and C. syzygicola were identified as pathogens of wax apple anthracnose in Thailand (Al-Obaidi et al, 2017). To our knowledge, this is the first report of C. siamense causing wax apple anthracnose in China.

First report of Aloe root and stem rot caused by Phytophthora palmivora in Yunnan Province, China.

Aloe genus plants are perennial evergreen herb belonging to Liliaceae family which is widely used in food, medicine, beauty, and health care (Kumar et al. 2019). In August 2021, symptoms of root and stem rot was observed in approximately 20% of Aloe vera plantings in Yuanjiang County, Yunnan Province, China (23° 64' 53" N, 101° 99' 84" E). The most typical symptoms were stem and root rot, browning and necrosis of vascular tissues, gradual greening, and reddish-browning of leaves from bottom to top, abscission, and eventual plant death (Fig. S1). Therefore, to isolate and identify the pathogen, the plants showing the above symptoms were collected. The plant tissues were cut from the edges of root and stem lesions, followed by disinfection with 75% ethanol for 1 min, rinsed three times with sterilized distilled water, and cut into 3 × 3 mm small squares after excision of marginal tissues. The tissues were transferred to the oomycetes selective medium (Liu et al. 2022) and incubated at 28 °C in the dark for 3~5 days, and suspected colonies were purified. The colonies were then inoculated onto potato dextrose agar (PDA), V8-juice agar (V8), and oatmeal agar (OA) medium plates for morphological characteristics. Finally, 18 isolates with the same colonial and morphological characteristics were obtained from 30 lesioned tissue and one of them was named as ARP1. On PDA, V8 and OA medium plates, the ARP1 colonies were white. On PDA plate, the mycelia were dense and the colonies were petal-like; on V8 plate, the mycelia were cashmere and the colonies were radial or star-like. Whereas, on OA plate, the mycelia were cotton-like and the colonies were fluffy and radial (Fig. S2 A~C). Mycelium did not have septum with high branching and swelling. Sporangia were abundant, semi-papillate, varying in shape from ovoid-ellipsoid to long-ellipsoid, 18-26 × 45-63 µm (average: 22 × 54 µm, n = 30), sporangia released numerous zoospores from the papillate after maturation. The chlamydospores were spherical, 20-35 µm in diameter (average: 27.5 µm, n = 30) (Fig. S2 D~F). These morphological features were like those of the pathogenic species of the oomycetes (Chen et al. 2022). For the molecular characterization, the genomic DNA of the isolate was extracted using the cetyl trimethyl ammonium bromide method, and the translation elongation factor 1α (tef-1α) (Stielow et al. 2015), ß-tubulin (ß-tub) (Kroon et al. 2004) and internal transcribed spacer (ITS) (White et al. 1990) of isolated strain ARP1 were amplified using primer pairs EF1-1018F/EF1-1620R, TUBUF2/TUBUR1 and ITS1/ITS4, respectively. The tef-1α, ß-tub genes and ITS region of ARP1 were directly sequenced and their sequence information was deposited in GenBank under accession numbers OQ506129, OQ506127 and OQ449628. ARP1 was clustered on the same evolutionary branch with Phytophthora palmivora (Fig. S3). To confirm the pathogenicity of ARP1, the main root of A. vera was wounded to 1 cm long and 2 mm deep with a scalpel blade followed by inoculation with 50 ml suspension of ARP1 zoospores at a concentration of 1 × 106 spores / ml per potted plant, and an equal volume of water as control. All inoculated plants were placed in the greenhouse at 28°C, 12 h / 12 h light / dark. After 15 dpi, the inoculated plants showed typical symptoms of wilted and drooping leaves and stem and root rot, same as observed in the field condition (Fig. S4). After inoculation with ARP1, a strain with the same morphological and molecular characteristics as the original isolate was re-isolated, confirming Koch's postulates. To our knowledge, this is the first report of P. palmivora causing root and stem rot of A. vera in the study region. This disease could be a potential risk for aloe production and therefore appropriate management measures should be taken.

First report of a new leaf blight disease of bletilla (Bletilla striata) caused by Curvularia reesii in China.

Bletilla striata is an important Chinese herbal plant grown widely in southwest China (Qian et al. 2021). Leaf blight was found on cultivated bletilla crops in Yunnan in 2021. The disease infected bletilla leaves and it was present in the field from April to November with the highest incidence (86% plants diseased) recorded in early September in Puer area. Foliar lesions were circular (Φ0.5-1.8 cm) or oval, with pale-gray center and narrow gray-brown outer area surrounded by a yellow halo. The lesions coalesced later to form large irregular spots or blighted areas on leaves. Symptomatic bletilla leaves were sampled from fields in Jiangcheng (E101.8672o, N22.5803o) and Simao (E109.7816o, N22.7891o) counties, Yunnan in July 2021. Seven fungal isolates were obtained from (BJ01-BJ04) and Simao samples (HBJ05-HBJ07) via lesion-tissue culture and hypha-tip purification on PDA medium. A pathogenicity test following Koch's Postulates (Grimms et al. 2006) was conducted using each isolate by inoculating 45-day old bletilla plant (n=30, Zihua cultivar) in a greenhouse through spraying hypha-spore suspension (3.25×104 CFU/mL) prepared with 14 d fresh DNA culture. Non-inoculated plants (n=30) were used as controls. The experiment was repeated once. The isolates BJ02 and HBJ06 (deposited in Yunnan Agric. Univ. Microbes Herbarium) were shown pathogenic to bletilla since similar lesions formed on seedlings 7 d post inoculation and pure fungal cultures with the same colony morphology as those of BJ02 and HBJ06 were re-isolated from leaf lesions 14 dpi. Isolates BJ02 and HBJ06 produced identical colony and conidium morphology after they were incubated at 25oC for 7 d on PDA. Colonies were circular, pale brown, Φ5.5-7.5cm, with villous surface and abundant aerial hyphae. Mycelia were septate, colorless, Φ3-4 µm and with acute-angled branches. Conidiophores developed from hyphae were erect, septate, pale-brown colored and 60-200 µm long. Conidia (produced scarcely and ripened slowly) were long-oval or petaloid, straight or slightly curved, brown, sized 28-45×10-14 µm. Most conidia were divided into 4 cells by 3 septa; the middle two were bigger than the basal and apex cells. Both BJ02 and HBJ06 were identified as Curvularia sp. based on their morphological characters (Tan et al. 2018). The rDNA-ITS, TEF1α and GAPDH genes (Tan et al. 2018) were amplified from these isolates with PCR (White et al. 1990) and sequenced. ITS sequences of the two isolates were both 574 bp (acc. no. OL587997 & OL336480) and 100% (574/574 bp) identical shown by blast comparison. Further blast analyses of ITS (574 bp, OL587997), TEF1α (532 bp, ON637120) and GAPDH (881 bp, ON637121) from isolate BJ02 showed that they were 99.27% (547/551 bp), 100% (842/842 bp) and 99.8% (507/508 bp) identical respectively with those of Curvularia reesii BRIP4358 (MH414907). The 3 genes of BJ02 were concatenated and phylogenic analysis (Tamura et al, 2013) of the concatenated sequence with those of Curvularia spp. showed that BJ02 was clustered with C. reesii BRIP4358 on the same end-branch of the tree with 100% confidence. Therefore, BJ02 and HBJ06 are the same species identified as Curvularia reesii and it is the pathogen causing bletilla leaf blight. C. reesii was first isolated from the air in Australia in 1963 and was named by Tan et al. in 2018. It has not been reported as a plant pathogen elsewhere. This is the first record of this fungus causing bletilla leaf blight in China. Keywords: Bletilla striata; leaf blight; Curvularia reesii; disease symptoms; pathogen morphology; multigene identification References (1) D.J. Grimes. Microbes, 1(5): 223-228, 2006. (2) L.H. Qian et al. Jiangshu Agric. Sci. 49(19): 64-71, 2021. (3) K. Tamura et al. Mol. Bio. & Evol. 30 (12): 2725- 2729, 2013. (4) Y. P. Tan et al. MycoKeys, 35: 1-25. 2018. (5) T.J. White et al. In: PCR Protocols: A Guide to Methods and Applications (eds. M.A. Innis et al.), Acad. Press, Inc. New York. 315-322, 1990.