Expression of Enhanced Thermostability 42 kDa Chitinase from Trichoderma asperellum SH16 in Escherichia coli

ilamentous fungi of the genus Colletotrichum and its teleomorph Glomerella are considered major plant pathogens worldwide. They cause significant economic damage to crops in tropical, subtropical, and temperate regions. Cereals, legumes, ornamentals, vegetables, and fruit trees may be seriously affected by the pathogen (3). Although many cultivated fruit crops are infected by Colletotrichum species, the most significant economic losses are incurred when the fruiting stage is attacked. Colletotrichum species cause typical disease symptoms known as anthracnose, characterized by sunken necrotic tissue where orange conidial masses are produced. Anthracnose diseases appear in both developing and mature plant tissues (4). Two distinct types of diseases occur: those affecting developing fruit in the field (preharvest) and those damaging mature fruit during storage (postharvest). The ability to cause latent or quiescent infections has grouped Colletotrichum among the most important postharvest pathogens. Species of the pathogen appear predominantly on aboveground plant tissues; however, belowground organs, such as roots and tubers, may also be affected. In this article, we deal in particular with methods used to identify and characterize Colletotrichum species and genotypes from almond, avocado, and strawberry, as examples, using traditional and molecular tools. The three pathosystems chosen represent different disease patterns of fruitassociated Colletotrichum. Multiple Species on a Single Host Numerous cases have been reported in which several Colletotrichum species or biotypes are associated with a single host. For example, avocado and mango anthracnose, caused by both C. acutatum and C. gloeosporioides, affect fruit predominantly as postharvest diseases (25,40,41). Strawberry may be infected by three Colletotrichum species, C. fragariae, C. acutatum, and C. gloeosporioides, causing anthracnose of fruit and other plant parts (31). Almond and other deciduous fruits may be infected by C. acutatum or C. gloeosporioides (Table 1) (1,5,46,50). Citrus can be affected by four different Colletotrichum diseases (61): postbloom fruit drop and key lime anthracnose, both caused by C. acutatum, and shoot dieback and leaf spot, and postharvest fruit decay, both caused by C. gloeosporioides. Additional examples of hosts affected by multiple Colletotrichum species include coffee, cucurbits, pepper, and tomato. Single Species on Multiple Hosts It is common to find that a single botanical species of Colletotrichum infects multiple hosts. For example, C. gloeosporioides (Penz.) Penz. & Sacc. in Penz. (teleomorph: Glomerella cingulata (Stoneman) Spauld. & H. Schrenk), which is considered a cumulative species and forms the sexual stage in some instances, is found on a wide variety of fruits, including almond, avocado, apple, and strawberry (Table 2) (6,15,31,46). Likewise, C. acutatum J.H. Simmonds has been reported to infect a large number of fruit crops, including avocado, strawberry, almond, apple, and peach (1,5,16,25,27). Examples of other species with multiple host ranges include C. coccodes, C. capsici, and C. dematium (14,56).

Mango (Mangifera indica L.) is one of the world's most significant economic fruit crops, and China is the second-largest producer of mango (Kuhn et al., 2017). Postharvest mango anthracnose is caused by Colletotrichum species and reduce the self-life of mature fruit (Wu et al., 2020). Colletotrichum species also cause postharvest anthracnose and fruit rot disease of Apple, Banana and Avocado (Khodadadi et al., 2020; Vieira et al., 2017; Sharma et al., 2017). In July 2019, mango fruits cv. 'Jin-Hwang' were observed at different fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district, Beijing, China, exhibiting typical symptoms of anthracnose including brown to black lesions in different size (≤ 2 cm) with identified border on the mango fruit surface. Later, the lesions were coalesced and extensively cover the surface area of the fruit. The lesions were also restricted to peel the fruit and pathogen invaded in the fruit pulp. About 30% of mango fruits were affected by anthracnose disease. The margins of lesions from infected mango fruits (n=56) were cut into 2 × 2 mm pieces, surface disinfected with NaClO (2% v/v) for 30 s, rinsed thrice with distilled water for 60s. These pieces were placed on PDA medium and incubated at 25°C for 7 days. Pure culture of fungal isolates was obtained by single spore isolation technique. Initially, the fungal colony was off white, and colony extended with time, turning light gray at the center. The morphological examination revealed that conidia were hyaline, oblong, and unicellular. The conidia were measured from 10 days old culture and dimensions varied from 13.3 to 15.8 µm in length and 4.6 to 6.1 µm in width. For molecular identification, a multi-locus sequence analysis; the Internal Transcribed Spacers (ITS) region, partial actin (ACT) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and chitin synthase (CHS-1) gene were amplified by using the primer sets ITS1/4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF1/GDR1 (Guerber et al. 2003) and CHS1-79F/CHS-1-354R (Carbone and Kohn 1999) respectively. The partial sequences of MTY21 were deposited to GenBank accessions (MT921666 (ITS), MT936119 (ACT), MT936120 (GAPDH) and MT936118 (CHS-1). All obtained sequences showed 100% similarity with reported sequences of Colletotrichum alienum ICMP.18691 with accessions numbers JX010217 (ITS), JX009580 (ACT), JX010018 (GAPDH) and JX009754 (CHS-1) which represented the isolate MTY21 identified as C. alienum by constructing Maximum Likelihood phylogenetic tree analysis using Mega X (Kumar et al., 2018). For the confirmation of Koch's postulates, the pathogenicity test was conducted on 36 fresh healthy mango fruits for each treatment. Fruits were punctured with the help of a sterilized needle to create 2mm2 wounds and inoculated with 10µL inoculum (107 spores/mL) of MTY21. Control mango fruits were inoculated with 10µL sterilized distilled water and incubated at 25 °C with 90% relative humidity. The lesions appeared at the point of inoculation and gradually spread on the fruit surface after 7 days post inoculation. The symptoms were similar to the symptoms on original fruit specimens. The re-isolated fungus was identified as C. alienum based on morphological and molecular analysis. Mango anthracnose disease caused by several Colletotrichum species has been reported previously on mango in China (Li et al., 2019). Liu et al. (2020) reported C. alienum as the causal organism of anthracnose disease on Aquilaria sinensis in China. C. alienum has been previously reported causing mango anthracnose disease in Mexico (Tovar-Pedraza et al., 2020) To our knowledge, this is the first report of C. alienum causing postharvest anthracnose of mango in China. The prevalence of C. alienum was 30% on mango fruit which reflects the importance of this pathogen as a potential problem of mango fruit in China.

Lifestyles of Colletotrichum acutatum.

Mango Anthracnose: Economic Impact and Current Options For Integrated Managaement.

Effect of Chitosan to control Colletotrichum capsici on chili fruits in post harvest period was investigated. Chitosan is an anti-fungi from crab or shrimp shells. Chitosan is a poly-cation polyglucosamine (the one component of fungi cell wall). The author investigated Anthracnose disease (%) and relative inhibition of anthracnose disease measured by scoring from AVRDC. Spraying chitosan on chili fruit (harvesting time) covered the surface of chili from external factors including C. capsici . Other research investigated that chitosan inhibited conidia germination, conidia development and destroyed cell wall of hyphae of C. capsici. Chitosan layer on chili fruits surface inhibited penetration process by C. capsici. Effect chitosan on relative inhibition of anthracnose disease from all treatments of chitosan were significant with control (P≤ 0.05). Chitosan 0.75% was the optimal concentration to inhibit anthracnose disease on chili fruits in post harvest period. Chitosan is a physically barrier and an antifungal for C. capsici. Chitosane 0.75% inhibited anthracnose disease 97.58%. The mechanism of chitosane may offer the better strategy in post

The literature on the entomopathogenic fungi in the genus Cordyceps describing its use in the agricultural area as a biocontrol agent is limited. In this study, a total of 47 isolates of entomopathogenic fungi were isolated from dead cicada nymphs obtained from various locations in the northeast of Thailand. These isolates were primarily screened for antagonistic activity to inhibit the mycelial growth of one isolate of Colletotrichum gloeosporioides and one isolate of C. capsici. The screen revealed that five isolates of entomopathogenic fungi showed good inhibitory effects on the fungal mycelial growth and were chosen for further confirmation of their antagonistic effects against five isolates of C. gloeosporioides and five isolates of C. capsici by the dual culture method. After investigation, the isolate Cod-NB1302 had the best inhibitory effect. Moreover, the mycelium extract and culture filtrate of isolate Cod-NB1302 also had inhibitory effects on the mycelial growth and conidial germination of all isolates of plant pathogenic Colletotrichum spp. under in vitro conditions. Interestingly, the mycelium extract and culture filtrate effectively reduced the size of the disease lesion and disease severity on chili fruits after inoculation with the plant pathogenic fungi. However, the mycelium extract exhibited greater antifungal activity than the culture filtrate. Finally, the isolate Cod-NB1302 was identified as Ophiocordyceps sobolifera based on the sequence of three ribosomal nuclear DNA genes and two protein-coding genes. These findings suggest that the isolate Cod-NB1302 is a potential candidate, with antagonistic activity, for use as a source of antifungal agents to control anthracnose disease caused by the plant pathogenic Colletotrichum spp.

Persistence of several antagonistic fungus on chilli and its potential to suppress anthracnose disease caused by Colletotrichum gloeosporioides. Anthracnose disease caused by C. gloeosporioides is one of the important diseases on chilli because its directly gives negative impact on chilli production. The aim of this research was to obtain the superior antagonistic fungi that have ability to persist on chili fruit and potential to control anthracnose disease caused by C. gloeosporioides The experiment consist of two units: 1. Testing of persistence ability of antagonistic fungi on chilli fruit, 2. Testing the potential of antagonistic fungi to suppress anthracnose disease on chilli fruit. Both of the test used Randomized Block Design (RBD) with 10 treatments and 4 replication, each of replication consist of 4 chilies fruit. Those treatments were Trichoderma-PP1, Trichoderma-PP3, Trichoderma-AG2, Trichoderma-PYK3, Paecilomyces-PP6, Paecilomyces-PP7, Paecilomyces-AG4, Paecilomyces-PYK4, Aspergillus PP2 and without antagonistic fungi (control). The result indicated that all antagonistic fungi isolate could persist on chili fruit. The highest persistence were Trichoderma-PP3 and TrichodermaAG2 (95.83%) and the lowest belonged to Paecilomyces-PP7 (50%). Trichoderma-PP3 and Trichoderma-AG2 were the best isolates for suppressing anthracnose disease caused by C. gloeosporioides.

Oligochitosan (OC) with molecular weight Mw of 5000 g/mol was prepared by gamma Co-60 ray irradiation of chitosan solution. Nanosilica (nSiO2) with the size of 10 - 30 nm was synthesized by calcination of acid treated rice husk. The mixture of 1% OC - 1% nSiO2 was prepared by dispersion of nSiO2 in OC solution. The morphology of nSiO2 in the mixture of OC-nSiO2 was measured from images of transmission electron microscopy (TEM). The effect of foliar application of the mixture of OC-nSiO2 on the induction of resistance against anthracnose disease caused by Colletotrichum gloeosporioides fungus on chili fruits was investigated. Results indicated that foliar application of OC-nSiO2 with the concentration of 60 mg/l - 60 mg/l was found to be as the optimal treatment that reduced the disease severity on chili fruits to 22.2% compared with 90.0% of the control. Thus, OC-nSiO2 hybrid material could be considered as an effective biotic elicitor to prevent anthracnose disease infection for chili fruits. Furthermore, the prepared OC-nSiO2 hybrid material can also be used as an environmentally friendly agrochemical product for sustainable development of agriculture.

Anthracnose disease is still a threat to avocado fruit quality, and the use of fungicide (Plochloraz®) for its control has generated safety concerns that necessitate the search for alternatives. Therefore, the efficiency of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against Colletotrichum gloeosporioides was investigated in this study. Weissella cibaria 21 (LAB 21), Leuconostoc pseudomesenteroides 56 (LAB 56), Weissella confusa 17 (LAB 17), Lactiplantibacillus plantarum 75 (LAB 75), and Lactiplantibacillus plantarum 171 (LAB 171) were evaluated in vitro as potential biocontrol agents to replace the Prochloraz® that is currently used in susceptible avocado (Persea americana Miller) Fuerte fruit. To confirm the biocontrol activity of the selected LAB strains, the antagonistic growth, spore germination, LAB recovery, nutrient competition, acid tolerance, and biofilm formation were assessed. In fruit treated with a LAB cell suspension, curatively inoculated with C. gloeosporioides, or naturally infected avocado cv Fuerte fruit, the epicatechin content and expression of defense-related genes (PAL, LOX, AVFADl 2–3, AVFAEL, and FLS) were compared with Prochloraz® and sterile water (control) treatments. With LAB 56, LAB 75, and LAB 21, significant inhibition of radial mycelial growth (MGI) (>90%) and spore germination (100%) was observed similar to those due to Prochloraz®. The MGI increased with a reduction in nutrient concentration. LAB strains reduced anthracnose disease incidence and severity compared with Prochloraz® and were the highest in LAB 21 and LAB 56. The LAB 21 and LAB 56 strains produced strong biofilms against C. gloeosporioides. In contrast to LAB 56, the control, and Prochloraz®, and LAB 21 had the highest epicatechin content (406 mg/g) and upregulated the PAL, AVFADl 2–3, AVFAEl, and FLS genes, thereby reducing the incidence of anthracnose in avocado fruit. As a result, LAB 21 suspensions can be used as an alternative to Prochloraz® in the control of anthracnose disease.

Biocontrol effectiveness of Trichoderma asperelloides SKRU-01 and Trichoderma asperellum NST-009 on postharvest anthracnose in chili pepper

Chilli fruit are good antioxidant sources and hold promise as natural ingredients in functional foods. Many other types of carotenoids accumulate in the fruit during the ripening process, for example, β-carotene, violaxanthin, lutein, and zeaxanthin. The present experiment was carried out to Effect of bio control agents and botanical extracts on antioxidant activity in chilli fruits. Chilli seedlings were treated with individually and in combination with effective bio control agents and botanical extracts in field condition applied as seedling treatment and foliar spray. Efficacy of botanical extract and bio control agents on antioxidant activity of fruits of chilli plant applied seedling treatment and foliar spray with T. Viride + P. Fluorescens + Neem + Garlic showed higher β-carotene and capsaicin content in chilli fruits in comparison to control. Maximum β-carotene (3.56 mg/mL) and capsaicin (2831.67µg/g) content were recorded in chilli fruits respectively. These antioxidants sources are responses to disease resistant against pathogen and benefiting the digestive tracts, promote healthy heart, relieve joint pains, promote weight loss, mitigate migraine, reduce cancer risk, prevent allergies etc.

Anthracnose of chili (Capsicum spp.) causes major production losses throughout Asia where chili plants are grown. A total of 260 Colletotrichum isolates, associated with necrotic lesions of chili leaves and fruit were collected from chili producing areas of Indonesia, Malaysia, Sri Lanka, Thailand and Taiwan. Colletotrichum truncatum was the most commonly isolated species from infected chili fruit and was readily identified by its falcate spores and abundant setae in the necrotic lesions. The other isolates consisted of straight conidia (cylindrical and fusiform) which were difficult to differentiate to species based on morphological characters. Taxonomic analysis of these straight conidia isolates based on multi-gene phylogenetic analyses (ITS, gapdh, chs-1, act, tub2, his3, ApMat, gs) revealed a further seven known Colletotrichum species, C. endophyticum, C. fructicola, C. karsti, C. plurivorum, C. scovillei, C. siamense and C. tropicale. In addition, three novel species are also described as C. javanense, C. makassarense and C. tainanense, associated with anthracnose of chili fruit in West Java (Indonesia); Makassar, South Sulawesi (Indonesia); and Tainan (Taiwan), respectively. Colletotrichum siamense is reported for the first time causing anthracnose of Capsicum annuum in Indonesia and Sri Lanka. This is also the first report of C. fructicola causing anthracnose of chili in Taiwan and Thailand and C. plurivorum in Malaysia and Thailand. Of the species with straight conidia, C. scovillei (acutatum complex), was the most prevalent throughout the surveyed countries, except for Sri Lanka from where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was also common in Indonesia, Sri Lanka and Thailand. Pathogenicity tests on chili fruit showed that C. javanense and C. scovillei were highly aggressive, especially when inoculated on non-wounded fruit, compared to all other species. The existence of new, highly aggressive exotic species, such as C. javanense, poses a biosecurity risk to production in countries which do not have adequate quarantine regulations to restrict the entry of exotic pathogens.

Postharvest diseases represent a critical challenge for global agriculture, resulting in substantial economic losses and threatening worldwide food security. Species of the genus Colletotrichum stand out among the main phytopathogens for being responsible for up to 40% of postharvest losses in various crops, including Capsicum species. This study evaluated the antifungal activity of two Streptomyces strains isolated from Amazonian sediments against different Colletotrichum species, with a focus on C. scovillei, the causal agent of anthracnose in Capsicum chinense fruits. Multilocus phylogenetic analyses indicated that strain APUR 32.5 possibly represents a new species, while MPUR 40.3 was identified as Streptomyces murinus. Both strains exhibited in vitro antifungal activity against seven Colletotrichum species, with inhibition percentages ranging from 56.3% to 88.6%. In fruit bioassays, S. murinus MPUR 40.3 reduced the incidence of anthracnose by 95%, while Streptomyces sp. APUR 32.5 achieved a 39.25% reduction. Scanning electron microscopy revealed complementary mechanisms of antifungal action, with MPUR 40.3 acting during the early infection stages through germination tube lysis, while APUR 32.5 targeted established mycelial structures through hyphal degradation. Additionally, both strains demonstrated plant growth-promoting capacity and exhibited biotechnologically relevant characteristics, including production of hydrolytic enzymes, siderophores, and phosphate solubilization ability. These results highlight the biotechnological promise of these Amazonian isolates as multifunctional agents for the sustainable management of anthracnose in Capsicum peppers.

Colletotrichum is a major plant-pathogenic fungus responsible for anthracnose, a disease that significantly reduces crop yield and quality, thereby hindering commercial production. This study aimed to isolate, identify, and characterize Colletotrichum species associated with anthracnose in various fruits and ornamental plants in Thailand, using both morphological and molecular approaches. Subsequently, the medicinal plant extracts were evaluated for their antifungal activity against the isolated Colletotrichum species, and their bioactive compounds were profiled using GC-MS and LC-MS analyses. Eleven Colletotrichum isolates were obtained and identified as nine distinct species, namely C. asianum, C. brasiliense, C. fructicola, C. musae, C. nymphaeae, C. okinawense, C. orchidearum, C. pandanicola, and C. truncatum. Among the ten medicinal plants tested, only the ethanolic extract of clove (Syzygium aromaticum) exhibited strong antifungal activity against all fungal isolates. The extract exhibited minimum inhibitory concentrations as low as 12.5 mg/mL against C. okinawense and C. orchidearum, and 25 mg/mL against the remaining species. GC-MS profiling of the ethanolic clove extract revealed that 2-methoxy-4-prop-2-enylphenol (eugenol, 72.417%) was the predominant compound, followed by (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo [7.2.0] undec-4-ene (β-caryophyllene, 12.125%), and (2-methoxy-4-prop-2-enylphenyl) acetate (eugenyl acetate, 8.121%). Additionally, LC-MS profiling indicated that the extract contained several antifungal constituents, including quercetin, kaempferol, isorhamnetin, myricetin, gallic acid, ellagic acid, catechol, caffeic acid, p-coumaric acid, methyl trans-cinnamic acid, and resveratrol. These findings highlight the diversity among pathogenic Colletotrichum species in Thailand and establish that clove extract, which contains flavonoids, phenolics, terpenoids, and alkaloids, holds potential as an eco-friendly alternative for agricultural disease management.

Primary screening of epiphytic and endophytic yeasts, the majority yeast was found from rhizosphere soil (21 isolates, 31.81%), and 5 antagonistic yeasts can have inhibited the growth of mycelia of C. capsici up to 60%. Efficacy of five antagonistic yeasts were screened under plant nurseries conditions. The result found all isolates were tested and compared with control and chemical plots. The result of plant height showed that, yeast isolate KS34 had highest of plant height at 45.80 cm. For the result of branch per plant of chili plant found isolate KS37 showed highest of branch per plant at 16.60 branches and the flower of chili per plant presented that isolate KS37 had highest of flower at 18.60 flowers. The result of anthracnose disease control by antagonistic yeasts in disease incidence and category mean as plant resistance. These results found that, Isolates KS46 had a great disease incidence at 7.80% that mean this isolate prove chili plant to resistance to C. capsisci. Addition, isolate KS46 had highest of activity of chitinase enzyme at 170.72 U/ml. Isolate KS46 identified by D1/D2 domain of 26S ribosomal DNA sequencing. The sequence analysis showed maximum identity of 100% with Candida haemulonii. This assumed C. haemulonii was showed the best to control C. capsici. For confirmed this result, all isolates were determined the mode of action by studied in relationship between antagonist isolates. The action of yeast isolate could competition the nutrient with mycelia of pathogen. This mode effect to C. capsici loss nutrient caused the pathogen get stuck to braked and died later.