Isolation and identification of strawberry anthracnose and fungicide screening in Henan province

SUN Wenbin; ZHANG Jingjing; SONG Yanhong; ZHAO Xia; LI Gang; ZHOU Houcheng; LIU Lifeng

doi:10.13925/j.cnki.gsxb.20240633

Abstract：[Objective]Strawberry anthracnose,caused by fungi of the genus Colletotrichum,presents a significant threat to strawberry cultivation in Henan Province,China.The rapid proliferation of this dis-ease has led to considerable yield losses and a decline in fruit quality.The primary objective of this com-prehensive study is to identify the species and predominant strains of pathogenic fungi associated with strawberry anthracnose.Additionally,the research aims to evaluate the efficacy,safety,and overall ef-fectiveness of various fungicides in managing this disease.By identifying the most suitable fungicides,agricultural practitioners in Henan province can implement improved disease management strategies that enhance both sustainability and productivity within their strawberry farming operations.[Methods]To achieve the objectives outlined above,a systematic and multifaceted methodology was employed.This research specifically focused on strawberry anthracnose attributed to Colletotrichum.Initially,sam-ples exhibiting symptoms of strawberry anthracnose were collected from three major strawberry cultiva-tion regions in Henan province including Zhengzhou,Xinxiang,and Mengzhou.Tissue isolation tech-nique was utilized to extract and purify pathogens from these samples,resulting in pure cultures of the pathogenic fungi.The primary biological characteristics of these isolates were examined with an empha-sis on how various environmental factors,such as temperature,pH levels,carbon sources,and nitrogen sources,influenced mycelial growth.Inoculation experiments were conducted to determine the pathoge-nicity of isolated strains,which involved inoculating detached leaves,petioles,and fruits of strawberries as well as applying spore suspensions onto living strawberry leaf surfaces and integrating spore suspen-sions into substrates.The concentration of the spore suspension employed in these experiments was standardized at 1.0 × 105 spores·mL-1 to ensure consistency and reproducibility of results.Given the lack of effective agents specifically designed for controlling strawberry anthracnose in agricultural pro-duction,the mycelial growth rate method was utilized for indoor virulence testing and to evaluate the ef-ficacy of ten selected fungicides.This approach enabled an assessment of the sensitivity of isolated strains to various fungicides,thereby providing valuable insights into potential agents that may be effec-tive in managing this disease.[Results]The findings indicated that all the five isolated strains derived from strawberry anthracnose samples exhibiting characteristic symptoms from the specified regions were classified as Colletotrichum siamense.The conidia size was(15.95±1.06)μm by(4.49±0.38)μm.Among the five strains,designated ZG-2 to ZG-6,strain ZG-2 exhibited the highest level of pathogenici-ty.Inoculated leaves with strain ZG-2 displayed rot after one week,while the lesion areas caused by the other strains were less than 50％,indicating a lower pathogenicity in comparison to ZG-2.The C.sia-mense ZG-2 strain was found to be highly pathogenic to strawberry plants,affecting various plant parts including leaves,fruits,petioles,stem bases,and roots.The infectivity of strain ZG-2 varied across dif-ferent plant components,petioles and stem bases experienced the most severe damage,followed by leaves,fruits,and roots.Notably,distinct orange spores were observed on detached leaves approximate-ly seven days post-infection,leading to leaf rot.During live inoculation experiments,lesions on the ab-axial surface of the leaves were larger and more numerous than those on the adaxial surface,indicating a higher susceptibility of the abaxial side to infection by this strain.Following root infection,plants ex-hibited inhibited growth characterized by a marked decrease in both leaf and root quantity as well as re-duced root lengths,brown lesions appeared on root surfaces compared to controls.Specifically recorded measurements for root length(3.72 cm),fresh weight(0.452 g),and dry weight(0.082 g)reflected sig-nificant reductions of 40.7％,43.7％,and 64.6％respectively when compared with healthy control groups.The occurrence of stem base rot could ultimately lead to mortality in strawberry plants.The bio-logical characteristics of the ZG-2 strain were distinctly notable,as it demonstrated the ability to grow within a temperature range of 15 ℃ to 35 ℃,with the most rapid mycelial growth rate of 6.2 mm per day observed at 27 ℃.The optimal pH for growth was determined to be 8,and the strain exhibited resil-ience across a pH range of 4 to 11,indicating strong adaptability to varying acid-base conditions.Solu-ble starch and peptone were identified as the most effective sources of carbon and nitrogen,respective-ly.The antibacterial efficacy of the ZG-2 strain was assessed with relative accuracy.Among the ten agents tested,propiconazole,cyproconazole,and benzoic-prochloraz displayed effective antibacterial properties,with EC50 values recorded at 0.035 μg·mL-1,0.048 μg·mL-1,and 0.089 μg·mL-1,respective-ly.Tebuconazole at a concentration of 430 g·L-1 exhibited superior control against the pathogen.[Con-clusion]Strawberry anthracnose has been documented in Henan Province,particularly in Zhengzhou,Xinxiang,and Mengzhou,throughout the entire growth cycle of strawberry plants.This disease results in significant blight and mortality rates that considerably hinder development of the strawberry industry in this region.To elucidate the primary pathogenic fungi affecting strawberries in the Henan Province,DNA was extracted from each isolated and purified fungal strain,subsequently,PCR amplification was performed using the ITS-1F/ITS-4R primers.The obtained sequences were analyzed via BLAST on the NCBI database,revealing strains with 99％similarity to known species.The results indicated that the predominant pathogenic fungal genera in this region include Colletotrichum,Fusarium,Botrytis,Clado-sporium,Alternaria,Phytophthora,Pilidium,and Oomycetes.Notably,Colletotrichum,the causative agent of strawberry anthracnose,was identified as a common strain across the three representative re-gions.A total of fifty-two isolates of the pathogen were collected from strawberry samples in Henan Province.Through polygenic analysis coupled with associated infection studies,C.siamense ZG-2 was identified to be closely related to Anthracis siamensis.It exhibited a self-spreading rate of approximate-ly 72％.Disease resistance evaluation were conducted on seven commercially cultivated strawberry va-rieties,as well as three advanced breeding lines(designated as zx.19.3,zx.60.11,and zx.32.17)selected by the research group.The results revealed that the fungal strain C.siamense ZG-2 exhibited pathoge-nicity across all tested strawberry varieties.Moreover,significant variations in resistance levels were ob-served among the different cultivars,each demonstrating distinct capacities to resist pathogen infection.This strain has been classified as a pathogenic species responsible for causing strawberry anthracnose in Henan Province.C.siamense is recognized as a major pathogen affecting various crops throughout Chi-na.The findings suggest that pentazolol demonstrates strong inhibitory effects on strawberry anthrac-nose and may serve as an effective agent for managing this disease.