Paramicrosphaeropsis eriobotryae sp. nov. a New Agent of Loquat Canker in Iran

Loquat (Eriobotrya japonica) is a subtropical evergreen plant important in terms of both horticulture and green space in the urban environment. In recent years, symptoms of branch and trunk canker, dieback, and decline have been observed in the main cultivation areas of this tree in Iran. To study the etiology of loquat canker in Fars Province, branches and trunks of loquat trees with disease symptoms were sampled from 2018 to 2019. Several isolates of an unknown Paramicrosphaeropsis species (Didymellaceae, Pleosporales, Dothidiomycetes) were recovered from the trunks and branches of infected loquat trees. Phylogenetic analysis of the isolates based on a multi-gene phylogeny of the ribosomal internal transcribed spacer (ITS), partial beta-tubulin gene (tub2) and the second-largest subunit of RNA polymerase II (rpb2) loci revealed that the isolates belonged to a new taxon, Paramicrosphaeropsis eriobotryae sp. nov. Pathogenicity tests revealed that this species was able to cause canker, trunk flaking, dieback, decline, leaf yellowing, defoliation and growth reduction in loquat trees. A host range evaluation on the detached branches of some important horticultural trees occurring adjacent to the loquat tree orchards of Fars Province showed that this species might have a broader host range, as it was also able to infect almond and olive trees. 

Paramicrosphaeropsis eriobotryae B. Tavakolian & Mostowf. sp. nov.

Conidiomata: pycnidial, (242.4–1117.6) × (130.1–944.4) μm (av. 513.4±182.2 × 363.8±143.1 μm) globose to subglobose, mostly solitary, sometimes confluent, pale brown and semi-immersed. Ostiole: erumpent and somewhat papillate. Pycnidial wall: pseudoparenchymatous, multilayered, thin, of textura angularis, hyaline at the beginning, dark brown at maturity. Chlamydospores: in chains, (5.4–14.3) × (3.9–14.3) μm (av. 8.7±1.6 × 7±1.5 μm), brown, ellipsoidal to globose, smooth, thick-walled. Conidiogenous cells: (7.2–20.2) × (5.3–15.4) μm (av. 11.8±2.8 × 8.6±2.1 μm), hyaline, phialidic, smooth, subglobose, ampulliform or lageniform to doliiform. Conidia: (3.5–11.2) × (2.8–7.9) μm (av. 6.7±0.5 × 4.3±0.5 μm), solitary, variable in shape, ellipsoidal, subcylindrical, obpyriform, straight to slightly curved, aseptate, hyaline at the beginning and pale brown, yellowish brown or greenish brown with age. Colonies on PDA: reaching 6.9 mm after 14 d, aerial mycelium flat, white at the beginning and darkened over time, dark orange at the center, margin regular, some black pycnidia visible after 20-25 d; reverse white at the beginning and brown to dark brown with age. Colonies on MEA: reaching 6.8 mm after 14 d, aerial mycelium floccose, white at the beginning, brown with age, margin smooth, pycnidia visible after one mo if formed; reverse dark orange at the center and dark brown with age. Colonies on OA: 6.7 mm after 14 d, immersed mycelium flat, white and pale olivaceous at the beginning, dark olivaceous with age, forming growth circles and sector, margin wavy, pycnidia visible after 14-20 d at 25 °C, under fluorescent light (12 h light/12 h dark) like small black dots; reverse dark olivaceous. All isolates of this species had a minimum growth temperature of 5°C, an optimum of 25°C and a maximum of 30 °C on MEA in the darkness.

枇杷（Eriobotrya japonica）是一种兼具园艺价值与城市绿化价值的亚热带常绿植物。近年来，伊朗该树种的主栽培区域陆续出现枝、干溃疡，枯梢及衰退病症状。为探究法尔斯省枇杷溃疡病的病原学，研究团队于2018至2019年间采集了带病症的枇杷枝干样本。从感病枇杷的枝干中分离得到多株未知的微小球壳属（Paramicrosphaeropsis）未定种，该类群隶属于双毛壳孢科（Didymellaceae）、格孢腔菌目（Pleosporales）、座囊菌纲（Dothidiomycetes）。基于核糖体内部转录间隔区（ribosomal internal transcribed spacer, ITS）、部分β-微管蛋白基因（partial beta-tubulin gene, tub2）以及RNA聚合酶II第二大亚基（second-largest subunit of RNA polymerase II, rpb2）位点的多基因系统发育分析，结果显示这些分离菌株隶属于一个全新的分类单元——枇杷微小球壳菌（Paramicrosphaeropsis eriobotryae sp. nov.）。致病性试验表明，该新种可诱发枇杷产生溃疡、树干剥落、枯梢、衰退、叶片黄化、落叶及生长受抑等病症。对法尔斯省枇杷果园周边常见的重要园艺树种离体枝条开展寄主范围测定后发现，该菌种的寄主范围可能更广，还可侵染扁桃与油橄榄。

枇杷微小球壳菌 新种（Paramicrosphaeropsis eriobotryae B. Tavakolian & Mostowf. sp. nov.）
分生孢子器：为分生孢子器型结构，尺寸范围为(242.4–1117.6) × (130.1–944.4) μm（平均值为513.4±182.2 × 363.8±143.1 μm），呈球形至近球形，多单生，偶尔聚生，浅褐色，半埋生。孔口：突出于表面，略显乳头状。分生孢子器壁：由拟薄壁组织构成，多层结构，质地较薄，呈角状组织形态，初期无色，成熟后转为深褐色。厚垣孢子：呈链状着生，尺寸范围为(5.4–14.3) × (3.9–14.3) μm（平均值为8.7±1.6 × 7±1.5 μm），褐色，椭圆形至近球形，表面光滑，细胞壁较厚。产孢细胞：尺寸范围为(7.2–20.2) × (5.3–15.4) μm（平均值为11.8±2.8 × 8.6±2.1 μm），无色，瓶梗型产孢，表面光滑，形态涵盖近球形、瓶形、长颈瓶形或桶形。分生孢子：尺寸范围为(3.5–11.2) × (2.8–7.9) μm（平均值为6.7±0.5 × 4.3±0.5 μm），单生，形态多样，包括椭圆形、近圆柱形、倒梨形，直立至微弯曲，无分隔，初期无色，后期逐渐变为浅褐色、黄褐色或绿褐色。
在马铃薯葡萄糖琼脂（Potato Dextrose Agar, PDA）培养基上的菌落：培养14天后直径可达6.9 mm，气生菌丝平坦，初期为白色，随培养时间延长逐渐加深，菌落中心呈深橙色，边缘规整，培养20至25天后可见黑色分生孢子器；菌落背面初期为白色，后期转为褐色至深褐色。
在麦芽提取物琼脂（Malt Extract Agar, MEA）培养基上的菌落：培养14天后直径可达6.8 mm，气生菌丝呈絮状，初期为白色，后期转为褐色，边缘平滑，若形成分生孢子器则需培养1个月后方可观察到；菌落背面中心呈深橙色，后期转为深褐色。
在燕麦片琼脂（Oatmeal Agar, OA）培养基上的菌落：培养14天后直径可达6.7 mm，埋生菌丝平坦，初期为白色至浅橄榄色，后期转为深橄榄色，形成生长环与扇区，边缘呈波浪状，在25℃、荧光光照（12 h光照/12 h黑暗）条件下，培养14至20天后可见呈小黑点状的分生孢子器；菌落背面呈深橄榄色。
所有该物种的分离菌株在黑暗条件下的麦芽提取物琼脂（MEA）培养基上均表现出以下生长特性：最低生长温度为5℃，最适生长温度为25℃，最高生长温度为30℃。