灰葡萄孢菌引起的向日葵叶斑和盘腐及热胁迫对病菌菌核形成的影响

Leaf blotch and head rot caused by Botrytis cinerea on sunflower and effect of heat stress on sclerotium formation of the pathogen

摘要:

【背景】 2022年8−9月，从甘肃省兰州市种植的食用向日葵田中采集到零星发病的叶斑和盘腐病害标样。【目的】 明确叶斑病和盘腐病的病原及其生物学特性。【方法】 采用单孢分离法进行病原菌分离；通过Koch’s法则明确分出病菌的致病性；采用形态学和分子生物学方法对病原菌进行种类鉴定；通过平板法测定试验菌株的适宜生长温度；观察经30 ℃ (生长抑制温度)培养后试验菌株产菌核特性的变化。【结果】 分离得到12株菌落形态不同的葡萄孢属菌株。试验菌株SKY-A−SKY-D接种食用向日葵离体叶片和花盘可引起与自然发病相似的症状，接种发病的叶片和花盘病组织上原接种菌的分出率达100%。试验菌株在PDA平板上15 ℃和20 ℃培养10−40 d，大型分生孢子无色至淡褐色，单胞，卵圆形、长椭圆形、球形、近球形、棒状或不规则形，[6.0−14.2 (−20.1)] μm×[6.0−10.4 (−14.9)] μm；菌核表生，黑色，球形、近球形、椭圆形或不规则形，(0.5−11.1) mm×(0.5−5.0) mm；Ⅰ型微菌核红褐色至黑褐色，表生或埋生，(26.9−492.5) μm×(14.9−149.3) μm，微菌核的组成细胞大小为(12.7−35.0) μm×(11.9−25.3) μm；Ⅱ型微菌核近黑色，埋生，(35.8−373.1) μm×(23.9−229.9) μm，微菌核的组成细胞大小为(8.2−16.4) μm×(8.2−14.9) μm；假微菌核由特化的附着胞构成，暗褐色至近黑色，埋生，(32.0−447.8) μm×(19.4−358.2) μm。试验菌株的菌丝适宜生长温度为20−25 ℃；菌核产生温度为5−25 ℃。30 ℃培养7 d后转至20 ℃培养14 d，可诱导2个不产菌核的菌株SKY-B和SKY-C产生菌核，突变株在继代培养时保持其产菌核能力。BLASTn分析结果显示，试验菌株SKY-A–SKY-D的rDNA-ITS序列(国家微生物科学数据中心登录号为NMDCN00038NP–NMDCN00038NS)与已知灰葡萄孢菌(Botrytis cinerea)序列的相似性达99.8%–100.0%。g3pdh、hsp60和rpb2多基因系统发育分析结果显示，4个试验菌株被聚在B. cinerea的不同亚群里。【结论】 引起向日葵叶斑和盘腐的病原菌被鉴定为B. cinerea，这是灰葡萄孢菌引起向日葵叶斑病和盘腐病在我国西北地区的首次报道。首次发现灰葡萄孢菌可以产生2种类型的微菌核。30 ℃热胁迫处理可诱导不产菌核的灰葡萄孢菌产生菌核，突变株的产菌核能力可遗传。

关键词:向日葵;叶斑;盘腐;灰葡萄孢菌;菌核;微菌核;假微菌核;热胁迫

Abstract:

[Background] In August and September 2022, the plant samples of leaf blotch and head rot were collected from sporadic diseased fields of confectionery sunflower (Helianthus annuus) in Lanzhou, Gansu, China. [Objective] To identify and characterize the pathogens of leaf blotch and head rot. [Methods] We isolated the pathogens by the single spore isolation method, measured the pathogenicity of the isolates based on Koch's postulates, and identified the isolates by morphological observation and molecular biological methods. Furthermore, we determined the optimum growth temperatures of the isolates by the plate culture method, and the changes of sclerotium formation characteristics of the tested isolates were observed after cultured at 30℃ (growth inhibition temperature). [Results] Twelve Botrytis isolates with different colony morphology were isolated from the diseased plant samples. The inoculation of four isolates, SKY-A to SKY-D, on detached sunflower leaves and heads induced the symptoms similar to those of natural diseases in the field, and the re-isolation rates of inoculated isolates from the infected leaves and heads were 100%. When the four isolates were cultured on PDA plates at 15℃ and 20℃ for 10–40 d, macroconidia were pale to light brown, unicellular, oval, oblong, spherical, subspherical, clavate or irregular shaped, (6.0–14.2 (–20.1)) μm×(6.0–10.4 (–14.9)) μm. The sclerotia were superficial, black, spherical, subspherical, ellipse or irregularly shaped, (0.5–11.1) mm×(0.5–5.0) mm. Type-I microsclerotia were reddish brown to dark brown, superficial or submerged, (26.9–492.5) μm×(14.9–149.3) μm, with the cell sizes of (12.7–35.0) μm×(11.9–25.3) μm. Type-II microsclerotia were nearly black, submerged, (35.8–373.1) μm×(23.9–229.9) μm, with the cell sizes of (8.2–16.4) μm×(8.2–14.9) μm. Pseudo-microsclerotia consisting of specialized appressoria were dark brown to nearly black, submerged, (32.0–447.8) μm×(19.4–358.2) μm. The optimal temperature range for mycelial growth and the temperature range for sclerotium formation were 20–25℃ and 5–25℃, respectively. After incubation at 30℃ for 7 d and then at 20℃ for 14 d, SKY-B and SKY-C originally uncapable of forming sclerotia formed sclerotia. Moreover, the mutants retained the ability of forming sclerotia in subculturing. BLASTn analysis showed that the rDNA-ITS sequences of SKY-A to SKY-D (National Microbiology Data Center Acc. No. NMDCN00038NP–NMDCN00038NS) had the similarity of 99.8%–100.0% with those of Botrytis cinerea strains in GenBank. The phylogenetic analysis based on the glyceraldehyde 3-phosphate dehydrogenase gene (g3pdh), heat shock protein 60 gene (hsp60), and DNA-dependent RNA polymerase subunit II gene (rpb2) showed that the four isolates were clustered in different subgroups of B. cinerea. [Conclusion] The pathogens causing leaf blotch and head rot on confectionery sunflower were identified as B. cinerea. This is the first report of leaf blotch and head rot caused by B. cinerea on confectionery sunflower in the Northwest China. For the first time, we discover that B. cinerea can produce two types of microsclerotia. The heat stress at 30℃ induced the B. cinerea isolates uncapable of forming sclerotia to form sclerotia, and the sclerotium-forming ability of the mutants could be inherited.

Key words:sunflower (Helianthus annuus);leaf blotch;head rot;Botrytis cinerea;sclerotium;microsclerotium;pseudo-microsclerotium;heat stress