油茶炭疽病复合病菌鉴定及室内药剂评价

doi:10.11707/j.1001-7488.LYKX20220324

Abstract

Abstract:

Objective: This study aims to identify the compound pathogens causing anthracnose of Camellia oleifera and to screen effective fungicides for compound pathogens on C. oleifera, in order to provide targeted strategy for controlling C. oleifera anthracnose. Method: Leaf samples with anthracnose symptoms were collected from C. oleifera plantations in Hunan, Guangxi, Guangdong, Jiangsu and Hainan Provinces, China. Tissue separation method was used to isolate and purify the pathogenic fungi, and the morphological characteristics combined with and multi gene phylogenetic analysis were used to identify species. The composite infection experiments were used to study the relationship between pathogenic fungi and pathogenicity. The mycelial growth rate method was used to determine the indoor virulence of fungicides such as prochloraz, difenoconazole, azoxystrobin, picoxystrobin, fluopyram and boscalid to pathogens of C. oleifera. Result: 1）The result showed that a total of 186 isolates were obtained from 106 symptomatic lesions, among which 130 isolates of Colletotrichum genus were the main pathogens, followed by 18 isolates in Pestalotiopsis genus, 28 isolates in Neopestalotiopsis genus, 4 isolates in Pseudopestalotiopsis genus and 6 isolates in Alternaria genus. There were 55 lesions from which only species of Colletotrichum genus were isolated, and 51 lesions from which both Colletotrichum species and other pathogens were isolated. There were 51 lesions from which multiple pathogens with different species in the same genus or from the different genus were simultaneously isolated. There were 7 combinations of different strains isolated: different species of Colletotrichum genus, Colletotrichum sp. and Pestalotiopsis sp. or Neopestalotiopsis sp. or Pseudopestalotiopsis sp. or Alternaria sp., Colletotrichum sp., Pestalotiopsis sp. and Neopestalotiopsis sp. or Alternaria sp. 2）Further co-infection experiment showed that the lesion size caused by inoculation with mixed strains was significantly larger than that caused by inoculation with single strain, indicating that there was synergistic effect of compound infection of Colletotrichum sp. and other pathogens in C. oleifera anthracnose lesions. 3）The virulence test results showed that difenoconazole and prochloraz had the strongest inhibitory effects on Colletotrichum, Pestalotiopsis and Neopestalotiopsis pathogens of C. oleifera, with EC₅₀ mean values of 0.011 to 0.555 μg·mL^?1. Azoxystrobin and picoxystrobin had the highest indoor toxicity to Pestalotiopsis and Neopestalotiopsis, with an average EC₅₀ from 0.007 to 0.020 μg·mL^?1, but had weak inhibitory effect on Colletotrichum. Boscalid and fluopyram had almost no inhibitory effects. Conclusion: The results indicate that there is compound infection of Colletotrichum sp. and other pathogens in anthracnose lesions of C. oleifera, and prochloraz and difenoconazole can be used as the main fungicides to control C. oleifera anthracnose caused by compound infection of pathogens.

Key words: Camellia oleifera, anthracnose, pathogens, co-infection, fungicide selection

CLC Number:

S763.11

Lingling Li,Yuanye Zhu,Shengpei Zhang,He Li. Identification of the Compound Pathogens Causing Anthracnose of Camellia oleifera and Evaluation of Fungicides in Laboratory[J]. Scientia Silvae Sinicae, 2023, 59(5): 100-108.

Figures/Tables 5

Table 1

Primer sequence and PCR cycling conditions"

基因 Gene	引物序列（5’-3’） Sequence	PCR扩增程序 PCR cycling conditions
核糖体转录间隔区 Internal transcribed spacers（ITS）	F: TCCGTAGGTGAACCTGCGG	95 ℃预变性5 min; 95 ℃变性30 s, 56 ℃退火30 s, 72 ℃延伸1 min, 32个循环; 72 ℃延伸10 min。 An initial denaturation at 95 ℃ for 5 min, followed by 32?cycles of denaturation at 95 ℃ for 30?s, annealing at 56 ℃ for 30?s and extension at 72 ℃ for 1 min, coupled with a final extension step of 10 min at 72 ℃.
核糖体转录间隔区 Internal transcribed spacers（ITS）	R: TCCTCCGCTTATTGATATGC
apn2和MAT1-2-1基因（ApMat）	F: TCATTCTACGTATGTGCCCG
apn2和MAT1-2-1基因（ApMat）	R: CCAGAAATACACCGAACTTGC
β-微管蛋白基因 β-tubulin（TUB2）	F: AACATGCGTGAGATTGTAAGT	95 ℃预变性8 min; 95 ℃变性30s, 55 ℃退火30 s, 72 ℃延伸1 min, 35个循环; 72 ℃延伸5 min。 An initial denaturation at 95 ℃ for 8 min, followed by 35?cycles of denaturation at 95 ℃ for 30?s, annealing at 55 ℃ for 30?s and extension at 72 ℃ for 1 min, coupled with a final extension step of 5 min at 72 ℃.
β-微管蛋白基因 β-tubulin（TUB2）	R: ACCCTCAGTGTAACCCTTGGC
延伸因子 Elongation factor-1α（EF-1α）	F: GTCGTYGTYATYGGHCAYGT	95 ℃预变性8 min; 95 ℃变性15 s, 55 ℃退火20 s, 72 ℃延伸1 min, 35个循环; 72 ℃延伸5 min。 An initial denaturation at 95 ℃ for 8 min, followed by 35?cycles of denaturation at 95 ℃ for 15?s, annealing at 55 ℃ for 20?s and extension at 72 ℃ for 1 min, coupled with a final extension step of 5 min at 72 ℃.
延伸因子 Elongation factor-1α（EF-1α）	R:ACHGTRCCRATACCACCRATCTT
组蛋白 Histone-3	F: ACTAAGCAGACCGCCCGCAGG	96 ℃预变性2 min; 96 ℃变性15 s, 55 ℃退火30 s, 72 ℃延伸35 s, 30个循环; 72 ℃延伸2 min。 An initial denaturation at 96 ℃ for 2 min, followed by 30?cycles of denaturation at 96 ℃ for 15?s, annealing at 55 ℃ for 30?s and extension at 72 ℃ for 35 s, coupled with a final extension step of 2 min at 72 ℃.
组蛋白 Histone-3	R: GCGGGCGAGCTGGATGTCCTT

Table 1

Fig.1

Fig.2

Table 2

Table 3

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杀菌剂 Fungicide	质量浓度 Mass concentration/（μg·mL^?1）	不同病原种类的菌丝生长抑制率 Inhibition rate of different species（%）
杀菌剂 Fungicide	质量浓度 Mass concentration/（μg·mL^?1）	果生炭疽菌 C. fructicola	胶胞炭疽菌 C. gloeosporioides	暹罗炭疽菌 C. siamense	山茶炭疽菌 C. camelliae	新拟盘多毛孢属 Neopestalotiopsis sp.
啶酰菌胺 Boscalid	50	20.0±0.00b	17.0±0.00c	12.7±1.86d	15.6±0.72c	25.5±1.02a
氟吡菌酰胺 Fluopyram	50	33.0±1.06c	31.0±1.00d	25.3±0.29e	36.0±0.40b	43.3±0.96a
嘧菌酯 Azoxystrobin	50	57.4±0.57c	51.2±0.25d	40.5±0.69e	72.2±1.49b	100.0±0.00a
啶氧菌酯 Picoxystrobin	50	60.9±0.31d	64.1±1.05c	49.5±0.47e	86.8±0.49b	100.0±0.00a

杀菌剂 Fungicide	不同病原种类的 EC₅₀ Median effective concentration of different species/（μg·mL^?1）
杀菌剂 Fungicide	果生炭疽菌 C. fructicola	胶胞炭疽菌 C. gloeosporioides	暹罗炭疽菌 C. siamense	山茶炭疽菌 C. camelliae	新拟盘多毛孢属 Neopestalotiopsis sp.	拟盘多毛孢属 Pestalotiopsis sp.
嘧菌酯 Azoxystrobin	－	－	－	－	0.011±0.001	0.007±0.001
啶氧菌酯 Picoxystrobin	－	－	－	－	0.011±0.003	0.020±0.004
咪鲜胺 Prochloraz	0.043±0.006	0.023±0.001	0.022±0.002	0.011±0.009	0.110±0.040	0.026±0.007
苯醚甲环唑D ifenoconazole	0.306±0.023	0.228±0.012	0.153±0.007	0.123±0.039	0.555±0.145	0.150±0.044

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Identification of the Compound Pathogens Causing Anthracnose of Camellia oleifera and Evaluation of Fungicides in Laboratory

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