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Abstract A severe leaf spot disease on patchouli， was observed in Guangdong， China. Early symptom appeared as circular or irregularly shaped and dark brown spots along the leaf margins and tips. As the disease progressed， the spots enlarged and with masses black， sessile conidiomata were observed in lesions. The results of morphology， molecular biology and pathogenicity test indicate that the causal agent of leaf disease of patchouli is Diaporthe arecae.
Key words Patchouli; Phomopsis leaf spot; Diaporthe arecae
Patchouli [Pogostemon cablin （Blanco） Benth.] is mainly cultivated in China and Southern Asia as an important traditional medicinal plant and a source of patchouli oil used in perfumery. In 2019， leaf spot symptoms （up to 70% of 13 plants were infected） were observed on P. cablin in a garden （23.9°N and 113.23°E） in Guangzhou， China. Affected leaves had circular or irregularly shaped spots along the leaf margins and tips. Lesions were dark brown in color， then enlarged and with masses black， sessile conidiomata were observed in lesions.
Experimental Part
Lesion tissues were surface sterilized and cultured on potato dextrose agar medium （PDA）， then using hyphal tipping method to obtain pure cultures. Finally， 16 isolates were obtained， as shown in Table 1. Colonies on PDA medium started as white mycelium， then grew into white tufted aerial mycelium， and concentric rings of mycelium was observed as following colonies grew. Alpha conidia were abundant in culture， 6.0-7.6 μm×2.0-2.8 μm [mean （6.7±0.3） μm×（2.4±0.2） μm， n=40]， hyaline， aseptate， biguttulate and tapering at two ends. Beta conidia were filiform or hamate， 23.1-24.5 μm×1.6-1.8 μm [mean （24.1±0.4）×（1.7±0.1） μm， n=30]. Gamma conidia were not observed. These morphological characteristics were typical to fungus Diaporthe spp.[1].
For molecular identification， PCR was conducted using DNA extracted from fungal colony to amplify rDNA internal transcribed spacer region （ITS; primers ITS1/ITS4）， part of the translation elongation factor 1-alpha gene （EF1-α; primers EF1-728F/EF1-986R）， β-tubulin gene （tub-2; primers Bt2a/Bt2b）[2-3]， histone H3 gene （HIS; primers HISdiaF/HISdiaR）[4] and calmodulin gene （CAL; primers CL1F/CL2A）[5]. The obtained sequences were deposited in NCBI GenBank. The identification of the isolate as D. arecae was validated by conducting phylogenetic tree and it was with high congruence to the D. arecae CBS 161.64 and D. arecae CBS 535.75[4]. Koch’s postulates were verified by inoculating leaves of P. cablin with the representative isolate GDTCMF6， and control leaves was drenched with sterile water. All leaves were covered with plastic bags and incubated at 25 ℃ for monitoring symptom development. After 10 d， inoculated leaves showed brown leaf spots， while control leaves remained symptomless. Results and Discussion
The fungus was re-isolated from the symptomatic tissue and showed identical morphology compared with the initial isolate， thus confirming Koch’s postulates that D. arecae is the causal agent of leaf disease of patchouli. To our knowledge， this is the first report of D. arecae causing leaf spot of patchouli in the worldwide. Diaporthe spp. are important plant pathogens causing branch cankers and fruit stem-end rot diseases[6]， and this report will assist in monitoring the severity of this pathogen and conducting control measures in patchouli production.
References
[1] UDAYANGA D， CASTLEBURY L， ROSSMAN A， et al. Species limits in Diaporthe： molecular re-assessment of D. citri， D. foeniculina and D. rudis with a new species on Citrus[J]. Persoonia，2014， 32（1）： 83-101.
[2] GLASS N， DONALDSON G. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J]. Applied and Environmental Microbiology， 1995， 61（4）： 1323-1330.
[3] VALESKA C， STEPHAN P， CLAUDIA W. Multi-xenobiotic-resistance a possible explanation for the insensitivity of bivalves towards cyanobacterial toxins[J]. Toxicon， 2008， 52（8）： 936-943.
[4] YAN D， SONG X， LI H， et al. Antifungal activities of volatile secondary metabolites of four Diaporthe strains isolated from Catharanthus roseus[J]. Journal of Fungal， 2018， 4（2）： 65.
[5] SANTOS L， ALVES A， ALVES R. Evaluating multi-locus phylogenies for species boundaries determination in the genus Diaporthe[J]. PeerJ， 2017， 5（e3120）：e3120.
[6] GAO Y， LIU F， DUAN W， et al. Diaporthe is paraphyletic[J]. IMA Fungus，2017， 8（1）：153.
Key words Patchouli; Phomopsis leaf spot; Diaporthe arecae
Patchouli [Pogostemon cablin （Blanco） Benth.] is mainly cultivated in China and Southern Asia as an important traditional medicinal plant and a source of patchouli oil used in perfumery. In 2019， leaf spot symptoms （up to 70% of 13 plants were infected） were observed on P. cablin in a garden （23.9°N and 113.23°E） in Guangzhou， China. Affected leaves had circular or irregularly shaped spots along the leaf margins and tips. Lesions were dark brown in color， then enlarged and with masses black， sessile conidiomata were observed in lesions.
Experimental Part
Lesion tissues were surface sterilized and cultured on potato dextrose agar medium （PDA）， then using hyphal tipping method to obtain pure cultures. Finally， 16 isolates were obtained， as shown in Table 1. Colonies on PDA medium started as white mycelium， then grew into white tufted aerial mycelium， and concentric rings of mycelium was observed as following colonies grew. Alpha conidia were abundant in culture， 6.0-7.6 μm×2.0-2.8 μm [mean （6.7±0.3） μm×（2.4±0.2） μm， n=40]， hyaline， aseptate， biguttulate and tapering at two ends. Beta conidia were filiform or hamate， 23.1-24.5 μm×1.6-1.8 μm [mean （24.1±0.4）×（1.7±0.1） μm， n=30]. Gamma conidia were not observed. These morphological characteristics were typical to fungus Diaporthe spp.[1].
For molecular identification， PCR was conducted using DNA extracted from fungal colony to amplify rDNA internal transcribed spacer region （ITS; primers ITS1/ITS4）， part of the translation elongation factor 1-alpha gene （EF1-α; primers EF1-728F/EF1-986R）， β-tubulin gene （tub-2; primers Bt2a/Bt2b）[2-3]， histone H3 gene （HIS; primers HISdiaF/HISdiaR）[4] and calmodulin gene （CAL; primers CL1F/CL2A）[5]. The obtained sequences were deposited in NCBI GenBank. The identification of the isolate as D. arecae was validated by conducting phylogenetic tree and it was with high congruence to the D. arecae CBS 161.64 and D. arecae CBS 535.75[4]. Koch’s postulates were verified by inoculating leaves of P. cablin with the representative isolate GDTCMF6， and control leaves was drenched with sterile water. All leaves were covered with plastic bags and incubated at 25 ℃ for monitoring symptom development. After 10 d， inoculated leaves showed brown leaf spots， while control leaves remained symptomless. Results and Discussion
The fungus was re-isolated from the symptomatic tissue and showed identical morphology compared with the initial isolate， thus confirming Koch’s postulates that D. arecae is the causal agent of leaf disease of patchouli. To our knowledge， this is the first report of D. arecae causing leaf spot of patchouli in the worldwide. Diaporthe spp. are important plant pathogens causing branch cankers and fruit stem-end rot diseases[6]， and this report will assist in monitoring the severity of this pathogen and conducting control measures in patchouli production.
References
[1] UDAYANGA D， CASTLEBURY L， ROSSMAN A， et al. Species limits in Diaporthe： molecular re-assessment of D. citri， D. foeniculina and D. rudis with a new species on Citrus[J]. Persoonia，2014， 32（1）： 83-101.
[2] GLASS N， DONALDSON G. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J]. Applied and Environmental Microbiology， 1995， 61（4）： 1323-1330.
[3] VALESKA C， STEPHAN P， CLAUDIA W. Multi-xenobiotic-resistance a possible explanation for the insensitivity of bivalves towards cyanobacterial toxins[J]. Toxicon， 2008， 52（8）： 936-943.
[4] YAN D， SONG X， LI H， et al. Antifungal activities of volatile secondary metabolites of four Diaporthe strains isolated from Catharanthus roseus[J]. Journal of Fungal， 2018， 4（2）： 65.
[5] SANTOS L， ALVES A， ALVES R. Evaluating multi-locus phylogenies for species boundaries determination in the genus Diaporthe[J]. PeerJ， 2017， 5（e3120）：e3120.
[6] GAO Y， LIU F， DUAN W， et al. Diaporthe is paraphyletic[J]. IMA Fungus，2017， 8（1）：153.