Morpho-molecular Identication and Chemical control of Alternaria alternata,causing leaf blight of Gardenia jasminoides Eills in China

Gardenia jasminoides Eills is a traditional aromatic and medicinal plant that is widely cultivated in China. In 2020, a severe leaf disease on Gardenia was observed in a growing herbal medicine area in Jian County, of Jiangxi Province, China. The causal agent was identified as Alternaria alternata (Fr.) Keissler. by amplification and sequencing the internal transcribed spacer (ITS) region, followed by phylogenetic analysis. Koch's postulates were confirmed by a pathogenicity test conducted with healthy gardenia, including reisolation and identification. To our knowledge, this is the first report of leaf blight caused by Alternaria alternata on Gardenia jasminoides Eills in JiangXi, China. PDA plate bacteriostatic experiment results showed that: Hexaconazole had the best inhibiting effect, which the EC 50 was 14.45 ppm. Therefore, the results are preliminary but promising for future field applications.


Introduction
Gardenia jasminoides Eills, belonging to the family Rubiaceae, is a famous ornamental plant widely distributed worldwide. The central officinal part of G. jasminoides is its dried ripe fruit. Chemical composition analysis finds multiple secondary metabolites in the fruit of G. jasminoides, like iridoid glycosides, crocin and crocetin, which have broad-spectrum anti-inflammatory properties, anti-cancer effects even can treat cerebral diseases . The crocus sativus glycoside in gardenia fruit can still be used to produce gardenia red, gardenia yellow, gardenia blue, three kinds of pigment, as a natural colourant widely used in the food industry (Wu et al. 2021). There is a great demand for gardenia in China due to its wide application in the medicine and food industry. As wild gardenia was not enough to meet the demand of many people, people began to cultivate gardenia on a large scale to produce more gardenia for people to use. However, the large-scale planting of gardenia resulted in the decline of disease resistance and the frequent occurrence of disease, which seriously affected the yield and quality of gardenia.
Once it happens, it is easy to break out and become a disaster, which seriously affects the growth and yield of plants and causes great economic losses. This is the first time that the leaf blight was observed in the gardenia planting base in Jiangxi province. The disease begins to infect the leaf margin, with irregular spots and later a large area of dry up, leading to plant death in severe cases. At present, the task is to identify the pathogen causing leaf blight and search for drugs to control the disease.

Isolates Used and Morphologica l Observation
Survey of herbal medicine growing areas in Jian country (114 • 09'E 27 • 07'N) was carried out to assess the prevalence of the disease. The fungi from the infected samples were isolated using routine tissue isolation method. Leaves were disinfected with 0.5% NaClO for 5 min and washed twice with sterile distilled water. Small pieces from the edges of diseased tissue were placed on potato dextrose agar (PDA) medium. The isolated pathogens were purified using hyphal tip technique. After culturing for two days at 30℃, transferred to a new PDA plate followed by incubation at 30℃ for five days. Emerging colonies were transferred several times by the hyphal tipmethod until and pure cultures were obtained. After five days, the morphological features of the culture of each isolate were studied on PDA at 30 ℃ . The sterile cultures were stored at 4 ℃ in a refrigerator.

Pathogenicity test
The purified fungi were inoculated on healthy leaves and cultured in darkness in a constant temperature incubator at 30 ℃ , with control leaves instead of being inoculated using fungi-free PDA. After 48 hours, the incidence was observed, recorded the symptoms and characteristics of the injury were and took photos were for preservation. To complete Koch's postulates, fungi were isolated from diseased parts of inoculated Leaves, using the same method as when pathogenic bacteria were isolated. The morphological characteristics of the isolated microorganisms were observed and recorded.

Molecular identification
Mycelium was used to extract the genomic DNA by following the CTAB

In vitro fungicide testing
Fifteen commercial chemical fungicides were considered. Before putting PDA into Petri dishes, fungicides were added in five different concentrations of the active ingredients: 10, 30, 50, 70, 100ppm.
Fungicide and active components (ai) are listed in Table 1.
At the end of the trials, the percentage of mycelial growth reduction (GR%) was calculated using theformula: GR % =RC−RA/RC x 100% RC: colony radius (mm) of the control strain.
RA: colony radius (mm) of the strain indual culture with the pesticides in amended PDA.

Results
Multiple fungal strains were isolated and purified from gardenia leaf blight collected in the field. The pathogenicity was determined according to Koch's rule, and one of them was determined to be the pathogen of the disease, which was temporarily named GD1.

Morphological characterization and identification
Morphological characteristics of the causal pathogens were studied both on the host and artificial culture medium to identify the associated pathogen, which was studied on the PDA medium. The pure culture of the GD1 initially produced white colonies (Fig. 1A), which gradually turned to light brown in 2 days and attained a growth of 9 cm in 7 days.
Microscopic observations revealed that the hyphae are smooth and septate, the conidia are chain, the base is blunt and round, the other end is pointed, and there are vertical and horizontal septa. Generally, there are 1-3 transverse septa, 1-6 mediastinum, the spore size is 9.9-35.2μm ×5.9-12.5μm, the average size is 18.8μm×7.9μm (Fig. 1B).
These characteristics are consistent with the morphological characteristics of Alternaria alternata (Fr.) Keissler.

Pathogenicity and Symptomatology
Leaves inoculated with GD1 mycelium showed similar symptoms to naturally occurring leaf blight after two days of moisturizing at room

Molecular Characterization and phylogeny
Pathogen template DNA and universal primers were used for rDNA-ITS sequence amplification of pathogen GD1, and a single band between 500-750bp was obtained, as shown in Figure 2C. After sequencing the PCR product and analyzing it with BLAST, our sequence showed 96%-100% sequence homology with GenBank sequences. GD1 Sequence was submitted to NCBI GenBank, and an accession number was obtained (OK041346). Phylogenetic analysis revealed that our isolates clustered along with other submitted Leaf blight isolates from GenBank. The sequences of GD1 formed the same cluster with Alternaria sp. (Fig. 3).
Based on morphological and phylogenetic analysis, the pathogen GD1 was identified as Alternaria alternata (Fr.) Keissler.

In vitro fungicide testing
Laboratory virulence tests of 15 tested fungicides against Alternaria alternata. showed that the inhibition effects of different fungicides were significantly different, as shown in  (Table 2).

Discussion
Gardenia fruits are rich in natural pigments and geniposide, usually used as medicinal materials and natural dyes. And gardenia flowers smell fragrant, can be used as a landscaping plant. Alternaria alternata has been reported to cause leaf spot on Rumex dentatus in Pakistan (Siddiqui et al. 2009) and leaf blight of tomato reported from parts of Pakistan ( Akhtar et al. 2010) and leaf spot on Yucca gloriosa in China. But Alternaria alternata has not been yet reported to cause gardenia leaf blight in Jiangxi province.
At present, the main measures of plant disease control still rely on pharmaceutical control. In the process of pesticide use, the effects of pesticide economy, safety and efficiency should be considered comprehensively to maximize the effectiveness of pesticides and minimize adverse factors. Comparing the bacteriostatic effects of 5 biological agents and 10 chemical agents on pathogenic bacteria, it was found that the control effect of biological agents was generally lower than that of chemical agents. The EC 50 of streptomycin, oxytetracycline and oligosaccharide were all above 400ppm, which were not suitable for the control of large area diseases. The main characteristics of biopesticides are environmental protection, low toxicity and low pesticide residue rate. Chemical pesticides have better effects but are not conducive to environmental protection, and the pesticide residue rate is high. Therefore, multiple aspects need to be considered comprehensively in the choice of pesticide application. Plant growth promoting rhizobacteria is an effective and eco-sustainable solution to protect crops against phytopathogens. Bacillus safensis STJP (NAIMCC-B-02323) from the rhizospheric soil of Stevia rebaudiana showed strong biocontrol activity against phytopathogen, Alternaria alternata (Prakash et al. 2021). As a biopesticide, biological control bacteria have the advantages of environmental protection and sustainability, and may become the next hot spot of plant protection research.