Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense

doi:10.3969/j.issn.1004-1524.20230435

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (4): 837-845.DOI: 10.3969/j.issn.1004-1524.20230435

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Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense

HE Jiawei(), Huang Leqin, LU Zhenyu, FANG Jin, LEI Ziyang, ZHANG Huijuan, JIANG Ming()

College of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China

Received:2023-04-06 Online:2024-04-25 Published:2024-04-29
Contact: JIANG Ming

Abstract

Abstract:

Rhododendron huadingense is a rare and endangered plant species endemic to Zhejiang Province, and leaf spot is an important disease of this plant. R. huadingense leaves infected with leaf spot disease were used as materials. Based on pathogen isolation, purification, and pathogenicity identification, both morphological and molecular biology methods were applied to determine the fungal pathogen. The results showed that one fungal strain designated HDDJ was isolated from the diseased leaves. Using Koch’s postulates, the fungus caused leaf spot disease, and the characteristics of the lesions were consistent with those observed in the field. The colonies of HDDJ were white, then gradually turned gray, and finally became black with fluffy aerial mycelium. The hyphal characteristics of HDDJ were identical to those of Botryosphaeria dothidea. Fragments of actin and tubulin genes were cloned by using PCR, and phylogenetic analysis results revealed that both were clustered with B. dothidea. According to the morphological and molecular identification results, it was confirmed that the fungal pathogen causing R. huadingense leaf spot disease was B. dothidea. Isolation and identification of R. huadingense leaf spot disease laid a foundation for this disease control in the future.

Key words: Rhododendron huadingense, leaf spot disease, Botryosphaeria dothidea, molecular identification

CLC Number:

HE Jiawei, Huang Leqin, LU Zhenyu, FANG Jin, LEI Ziyang, ZHANG Huijuan, JIANG Ming. Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense[J]. Acta Agriculturae Zhejiangensis, 2024, 36(4): 837-845.

Figures/Tables 8

Fig.1 Normal and diseased leaves of Rhododendron huadingense A, Normal leaves; B, Diseased leaves.

Fig.2 Pathogenicity detection A, Front side of leaf at 5 d after inoculation with pathogen; B, Back side of leaf at 5 d after inoculation with pathogen.

Fig.3 Morphological characteristics of pathogenic fungus HDDJ A, Colony morphology of HDDJ after 2 days of cultivation; B, Colony morphology of HDDJ after 5 days of cultivation; C, Morphology of mycelium under a light microscope.

Fig.4 PCR products of different gene fragments M, Middle DNA Marker II; 1, Tubulin gene fragment; 2, Actin gene fragment.

Fig.5 Multiple alignment of HDDJ actin gene sequence and its homologous sequences AY972118, Botryosphaeria dothidea strain CMW8000; HDDJ, B. dothidea; AY972117, B. dothidea strain CMW7779; KF951843, Macrophomina phaseolina strain CPC21388; MH712507, M. euphorbiicola strain McBr39; MK134684, Sphaeropsis ulmicola strain PB_11f; AY624264, Diplodia scrobiculata strain CMW5870; AY624265, D. scrobiculata strain MW4900; MK574917, Lasiodiplodia theobromae strain HNWN02; MZ723784, L. iranensis strain SZ-YAM2.

Fig.6 Multiple alignment of HDDJ tubulin gene sequence and its homologous sequences HDDJ, Botryosphaeria dothidea; MN370930, B. dothidea strain SX03; MG761773, B. dothidea strain PPO-46523; MK482382, B. dothidea strain JZB310177; KX505929, Neofusicoccum luteum strain CAA628; ON773422, N. parvum strain LSCKS8-7; KU976452, Diplodia seriata strain Mz-F44; OL405580, Lasiodiplodia mahajangana strain 17b; MW315842, L. euphorbiaceicola strain GB49.

Fig.7 The phylogenetic tree based on HDDJ actin gene fragment and it homologous sequences

Fig.8 The phylogenetic tree based on HDDJ tubulin gene fragment and it homologous sequences

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Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense

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