Identiﬁcation and Characterization of Colletotrichum fructicola and Colletotrichum siamense Causing Anthracnose on Luffa Sponge Gourd in China

: Luffa sponge gourd ( Luffa cylindrica ) is an important cucurbitaceous vegetable and is known as the source of loofah. From 2020 to 2021, a leaf disease occurred on luffa leaves in the Hunan Province of China. Symptoms were displayed as oval to irregular chlorotic lesions surrounded by yellow halos. The pathogens were isolated from the affected leaves. According to morphological characterization and molecular identiﬁcation using multi-locus phylogenetic analysis of the internal transcribed spacer (ITS), actin ( ACT ), chitin synthase ( CHS-1 ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), β -tubulin ( TUB2 ), and partial mating type (Mat1-2) gene ( ApMAT ) regions, the pathogens were identiﬁed as two Colletotrichum species: Colletotrichum fructicola and C. siamense . Koch’s postulates were identiﬁed by a pathogenicity test and re-conﬁrmation. To the best of our knowledge, C. fructicola and C. siamense are two new species associated with luffa sponge gourd anthracnose.


Introduction
Luffa cylindrica (L.) Roem (Luffa sponge gourd), belonging to the Cucurbitaceae family, is an important horticultural crop. This plant is widely cultivated in tropical and subtropical areas globally, including countries in Asia, America, and Africa [1]. The sponge gourd is a commercial crop since it has important vegetable and medicinal values [2,3]. In recent years, luffa sponge gourd planting has had good economic benefits, and the cultivation area is expanding. However, many diseases have been recorded in the luffa sponge gourd. For example, cucurbit downy mildew caused by Pseudoperonospora cubensis [4,5], gray mold caused by Botrytis cinerea [6,7], Fusarium wilt caused by Fusarium oxysporum. sp. luffa [8], stem rot caused by Sclerotium rolfsii [9], phytophthora fruit rot caused by Phytophthora capsica [10], and fruit rot caused by Stagonosporopsis cucurbitacearum [11]. In addition, anthracnose is a common disease in Cucurbitaceae crops [12]. Colletotrichum orbiculare has been recorded as the pathogen of L. cylindrica [13]. However, until now, there has been no reports of other Colletotrichum species causing luffa anthracnose.
In August of 2020 and 2021, we observed the occurrence of a leaf spot disease on sponge gourd in some private cultivation farms in Hunan Province of China. Almost 90% of the surveyed leaves were infected. Disease symptoms mainly occurred in the leaf of sponge gourd, including water soaking, round, light yellow, chlorosis spots, and extended or coalesced to oval or irregular lesions. The spots were brown at the margin and grayish white in the center, with clear yellow halos surrounding the outside. When grayish white in the center, with clear yellow halos surrounding the outside. When it was dry, the center of the spots were easily perforated and ruptured (Figure 1). At a later stage, lesions expanded further and caused leaf blight and wither. In this study, the pathogen responsible for the disease was determined by examination of its morphological and molecular characteristics, and through pathogenicity testing to fulfill Koch's postulates.

Fungal Isolation
A total of 10 fungal isolates were obtained from 10 affected sponge gourd leaves that were collected from 10 cultivation farms in Changsha of Hunan Province. The information of the isolates are shown in Table 1. Based on the colony morphology, the fungal strains were primarily identified as Colletotrichum species. Four isolates, which could be putatively divided into two types, were selected for further morphological and molecular identification.

Fungal Isolation
A total of 10 fungal isolates were obtained from 10 affected sponge gourd leaves that were collected from 10 cultivation farms in Changsha of Hunan Province. The information of the isolates are shown in Table 1. Based on the colony morphology, the fungal strains were primarily identified as Colletotrichum species. Four isolates, which could be putatively divided into two types, were selected for further morphological and molecular identification.
Plants 2022, 11, x FOR PEER REVIEW 3 of 9 described in Table 2. A combination of the morphological characteristics and phylogenetic analysis confirmed that CSSGY1, CSSGY5-1, and CSSGY5-2 strains belonged to C. siamense while CSSGY4 belonged to C. fructicola.

Pathogenicity Test
The four representative isolates were selected for a pathogenicity test. Luffa potted sponge gourd plants were in vivo inoculated with conidial suspensions. Five days after inoculation, all the inoculated sponge gourd leaves developed symptoms, showing yellow halos in the inoculated sites. Later, leaf spots occurred, which were enlarged, and in some case were perforated, which was similar to those observed in the field. No symptoms were observed on the control plants ( Figure 4). All four strains were pathogenic to the luffa sponge gourd plants and caused similar symptoms. The pathogenic fungi were recovered from the inoculated symptomatic leaves, and identified by morphological observation and molecular sequencing using the methods described above, thus fulfilling Koch's postulates. CSSGY4, the GAPDH, ACT, CHS-1, TUB2, and ApMAT sequences had a 99.59% to 100% identity with the corresponding sequences of C. fructicola strains.
For further phylogenetic analysis, a neighbor-joining phylogenetic tree based on concatenated datasets of ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT was constructed, which included the 4 strains and 10 referenced Colletotrichum strains. Phylogenetic analysis revealed that the strains CSSGY1, CSSGY5-1, and CSSGY5-2 were clustered with the C. siamense clade, including the type strain CBS: 125378, while the strain CSSGY4 was grouped with the C. fructicola clade, which was consistent with the homology search results that were conducted using BLASTn (Figure 3).  Table 3.   Table 3.

Pathogenicity Test
The four representative isolates were selected for a pathogenicity test. Luffa potted sponge gourd plants were in vivo inoculated with conidial suspensions. Five days after inoculation, all the inoculated sponge gourd leaves developed symptoms, showing yellow halos in the inoculated sites. Later, leaf spots occurred, which were enlarged, and in some case were perforated, which was similar to those observed in the field. No symptoms were observed on the control plants ( Figure 4). All four strains were pathogenic to the luffa sponge gourd plants and caused similar symptoms. The pathogenic fungi were recovered from the inoculated symptomatic leaves, and identified by morphological observation and molecular sequencing using the methods described above, thus fulfilling Koch's postulates.

Discussion and Conclusions
In this study, the fungal pathogens of the leaf-spot-infected luffa sponge gourd were isolated and identified by cultural and microscopic examination; phylogenetic analysis was carried out using the concatenated datasets of ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT; and pathogenicity tests were carried out on potted luffa sponge gourd plants. The pathogens were confirmed to be two Colletotrichum species: C. siamense and C. fructicola. Therefore, this disease in luffa sponge gourd was identified as anthracnose. To our

Discussion and Conclusions
In this study, the fungal pathogens of the leaf-spot-infected luffa sponge gourd were isolated and identified by cultural and microscopic examination; phylogenetic analysis was carried out using the concatenated datasets of ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT; and pathogenicity tests were carried out on potted luffa sponge gourd plants. The pathogens were confirmed to be two Colletotrichum species: C. siamense and C. fructicola. Therefore, this disease in luffa sponge gourd was identified as anthracnose. To our knowledge, this is the first report of C. siamense and C. fructicola causing anthracnose in luffa sponge gourd. The identification of this new disease and its causal agent provides an important basis for the diagnosis and control of diseases in luffa sponge gourd.
Colletotrichum species is a kind of fungal group that cause diseases in a wide range of plant hosts. Some species in the Colletotrichum genera are difficult to distinguish using sole morphological observation and single gene locus analysis; thus, multi-gene phylogenetic analysis is considered necessary for the identification of individual Colletotrichum isolates [14]. In our study, based on the concatenated ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT six-gene phylogenetic analysis, the four representative isolates that were pathogenic to luffa sponge gourd were classified into two Colletotrichum species: C. siamense and C. fructicola. This is the first time these species have been recorded as pathogens that cause luffa anthracnose, which is another aspect that broadens our understanding of various plant diseases caused by the two Colletotrichum species.
C. fructicola is a species established from the C. gloeosporioides species complex. It has a worldwide distribution with a wide host range, including some important fruit crops, such as apple, pear, chili and strawberry, and other cash crops and vegetables, such as oil tea [15], tea [16], and chili [17]. The disease of particular concern caused by C. fructicola is the Glomerella leaf spot of the apple, which could lead to severe defoliation of the infected plants [18,19]. C. siamense is also in the C. gloeosporioides species complex, which was first recorded on coffee berries [20]. C. siamense has been reported as a worldwide pathogen of anthracnose on a variety of economically important plants, including fruits, ornamental plants, and vegetables, such as the mango, custard apple, papaya, tea plants, chili pepper, persimmon, orange, avocado, guava, rubber plant, and jasmine [21][22][23][24][25][26]. Until now, reports of disease caused by C. fructicola and C. siamense in cucurbitaceous vegetables have rarely been reported.
The anthracnose disease can damage leaves, often leading to wilting and defoliation. Therefore, it might seriously affect the photosynthesis of the plants, which in turn influences the yield of luffa sponge gourd fruit. Since C. siamense and C. fructicola also have a relatively wide host range, it might be possible that the pathogen of luffa sponge gourd can also infect other vegetables grown nearby. In this study, anthracnose of luffa sponge gourd occurred in almost 90% of the plants. Therefore, to mitigate further possible damage caused by this disease, studies in global prevention and management strategies should be conducted. Considering food and environmental safety, resistant breeding might be an ideal method to control this disease. In addition, the development of molecular detection methods is also important for early diagnosis and prevention of the disease.
In conclusion, the causal pathogens of emerging anthracnose on luffa sponge gourd were identified and characterized. Fungal isolates were isolated and identified by morphological characteristics and multi-gene sequencing and phylogenetic analysis. The pathogenicity of the representative isolates was tested. Identification of these pathogens might provide important insights for the diagnosis and management of disease on luffa sponge gourd.

Sample Collection and Fungal Isolation
From August to September 2020 and 2021, luffa sponge gourd leaves that exhibited visible leaf spots were collected from different vegetable gardens in Changsha, the Hunan Province of China. Symptoms of the diseased leaves were observed and recorded. Pieces of small sections in approximately 5 × 5 mm fragments were cut off from the junction of lesions, disinfected with 70% alcohol for 30 s, 0.1% mercuric chloride for 45 s, rinsed with sterile distilled water 3 times, and then placed on potato glucose agar medium (PDA) and cultured at 28 • C in the dark. Hyphal tips developed from the sample pieces were picked and transferred onto fresh PDA plates and incubated at 28 • C. Single-spore isolation was conducted to purify the fungal cultures. All single-spore cultures were stored at 4 • C in a refrigerator.

Morphological and Cultural Characterization
For morphological examination, mycelial plugs (8 mm diam) of the fungal strains were plated on PDA and cultured at 28 • C. After 7 days, the colony characteristics of the morphology and color were observed. The morphology of the conidia and appressoria was observed at 400× magnification using an optical microscope (life technology, EVOS)™ XL core imaging system). The length and width of the conidia and appressoria were determined by measuring about 50 randomly selected structures each.
The ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT sequences of our isolates and other similar Colletotrichum species were selected for phylogenetic analysis. The concatenated sequences of the ITS, ACT, CHS-1, GAPDH, TUB2, and ApMAT loci were aligned with ClustalX [33] and subjected to phylogenetic tree construction using the NJ method in MEGA 6, with the bootstrap values calculated by 1000 replications [34].

Pathogenicity Assay
To fulfill the Koch's postulates, the pathogenicity tests of the representative strains were carried out by in vivo inoculation in potted sponge gourd plants with conidial suspensions. The four strains were cultured on PD liquid medium at 28 • C in a shaker incubator (180 rpm/min) for 4 days, filtered with 4 layers of sterile gauze to harvest the conidial suspensions, and then they were adjusted to a final concentration of 1 × 10 5 spores/mL by a hemocytometer. Leaves of the luffa sponge gourd plants were disinfected with 70% alcohol. Inoculation was carried out by spraying 5 mL of conidial suspension on each plant while sterile water was inoculated on control plants using the same procedure. All the inoculated plants were placed in plastic chamber with 95% relative humidity at 26 • C under a 16/8 h light/dark cycle. All the inoculums were observed daily for symptom progression. The experiments were repeated twice, with three potted plants inoculated for each strain.
To confirm Koch's postulates, the pathogen fungal strains were re-isolated from the experimentally symptomatic leaves and identified according to the morphological and molecular characteristics as mentioned above.