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Chilli (Capsicum annum L.) is a common spice crop grown in India. During 2018, twig blight symptoms were first observed in experimental plots of chilli in ICAR-IIHR-Central Horticultural Experimental Station, Odisha, India. High incidence of twig blight to the tune of 23% was documented during 2018 Kharif crop of chilli. There was no occurrence of twig blight in rabi and summer crops. This study reported the detailed cultural and micro-morphological characteristics of Choanephora cucurbitarum infecting chilli and pathogen identity was confirmed by molecular phylogenetic analyses. Pathogenicity tests were repeated twice with same results. To best of our knowledge, C. cucurbitarum infecting chilli is the first report in the state of Odisha, India.

Butternut squash (Cucurbita moschata Duch.) is a fruit vegetable, and the hardy nature of fruit enables it resistant to many pests. However, flowers and young fruits are vulnerable to diseases. In the Northern Province of Sri Lanka, soft rot symptoms were noted in flowers and young fruits in some fields during Maha season in 2018. The fungus isolated from infected tissues produced cotton-like white mycelium and black spore masses on PDA. The internal transcribed spacer (ITS) section of the ribosomal DNA sequences was amplified using PCR, and the generated sequences had > 99 % similarity with the nucleotide sequences of Choanephora cucurbitarum in the BLAST search. In phylogenetic analysis, the isolates clustered in a clade of C. cucurbitarum with a high bootstrap value. Based on the results of morphological studies and the ITS region’s molecular analysis, the fungal identity was confirmed as C. cucurbitarum. The symptoms were reproduced in inoculation tests. The present findings confirm the first report of fruit rot of butternut squash caused by C. cucurbitarum in Sri Lanka.

This study aimed at identification and characterisation of the wilting, leaf spot, stem canker and fruit-blight diseases of okra. Pathogen infected plant samples were sterilised with 2% sodium hych...

The present investigation was carried out to characterize the pathogen responsible forChoanephorafruit rot disease in bitter gourd along with find out appropriate control measure using biological resources. The pathogenic fungal strain was isolated from the disease infected fruit part of bitter gourd. It was subjected to four different types of growth media named potato dextrose agar (PDA), Czapeck Dox agar (CDA), nutrient agar (NA) and Sabouraud dextrose agar (SDA) were used to note down the differences regarding to various characteristics appeared due to dissimilation of culture medium. Thereafter, the growth environment and medium contents were changed to evaluate the effect of temperature, pH, carbohydrate, sugar concentration, salt, organic acid etc. on the mycelial growth of the concerned fungus. In the experiment, these parameters significantly influenced the growth pattern of the fungus. For proper identification of the fungus, advanced molecular technique was used. The PCR product of the internal transcribed spacer (ITS) region of the fungi showed nearly 650 bp size of clear band followed by sequencing of the PCR product of the fungus. The sequenced data showed 99% similarities with the original sequence ofChoanephora cucurbitarum.In search of suitable biocontrol measure, the antifungal activities of the seven plant extracts namelyHibiscus rosa-sinensis,Ficus racemosa,Moringa oleifera,Psidium guajava,Azadirachta indica,Cassia alataandCoccinia grandiswere assayed whereAzadirachta indica(42.12%) andMoringa oleifera(35.71%) had promising inhibitory effects against the isolated fungus. The outcome of the experiment would be benevolent for the detection and development of eco-friendly control approach of the detrimental disease of the bitter gourd.

Okra (Abelmoschus esculentus (L.) Moench) is an economically important vegetable crop that is widely cultivated in the tropics for its nutritional properties. There are concerns, though, that in Ch...

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated “choanepins.” Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors. IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.

Pinellia ternata (Thunb.) Makino ex Briet. (banxia, crow-dipper) is a perennial herbaceous plant native to China, Japan, and Korea. A member of the family Araceae, it is considered an invasive weed...

Periwinkle or vinca (Catharanthus roseus (L.) G. Don) is widely planted as a flowering annual in home and business landscapes in hardiness zones 9 and 10 and the southernmost portions of zone 8. Many improved selections, such as those in the Pacifica, Tropicana, and Cooler cultivar series, are currently available. Dark gray flower spots that often coalesced and led to blighting of flowers under conditions of high humidity, were observed on all of 18 periwinkle cultivars in a variety trial at Burden Research Plantation in September 1997. The disease affected only the flowers and was also observed on periwinkle in commercial landscape plantings around Baton Rouge. Fungi were isolated from flower spots by plating necrotic tissue on acidified potato dextrose agar (PDA). Fungal isolates were maintained on PDA and 20% V8 juice agar under continuous fluorescent light. A fungus that produced cottony, white mycelium and black spore masses was consistently isolated from diseased tissue. Pathogenicity tests were conducted by misting a spore suspension (70,000 per ml produced on V8 juice agar) onto detached flowers held in a moist chamber and to flowers on intact plants. Inoculated plants were held for 24 h in a dew chamber at 25°C. Typical irregular dark spots appeared on attached and detached flowers within 18 h after inoculation and flowers held for longer periods under high humidity developed a soft rot. Koch's postulates were fulfilled by reisolating the fungal pathogen, which was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (1). This is the first report of the occurrence of C. cucurbitarum on periwinkle. Reference: (1) P. M. Kirk. Mycol. Pap. 152:1, 1984.

A blight (wet rot) of petunia (Petunia ×hybrida Hort. Vilm.-Andr.) was observed in a wholesale propagation nursery in Baton Rouge, LA in September 2002. The grower reported that plants wilted and then completely rotted. The disease occurred during a period of hot, humid, and cloudy weather. Approximately 100 flats of flowering-age plants of cvs. Rose and White Madness were destroyed. No fungal sporulation was noticed on dead plants, but occasional strands of white mycelium were observed. The grower's use of azoxystrobin, iprodione, and thiophanate methyl plus mancozeb fungicides during current and past outbreaks of this disease did not prevent disease spread, but disease activity stopped after temperature and humidity dropped in early October. A fungus that produced white aerial mycelia that later developed light yellow areas and also black aerial spore masses was consistently isolated from diseased tissue placed on acidified potato dextrose agar (APDA). The fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. on the basis of cultural and morphological characteristics (3). Sporangiola were ellipsoid, pale brown to reddish brown with distinct longitudinal striations and measured 15 to 20 × 9 to 14 μm. Sporangiospores were broadly ellipsoid, pale brown to reddish brown, indistinctly striate with fine, hyaline polar appendages, and measured 16 to 34 × 7 to 12 μm. Spore measurements were within the range previously given for C.cucurbitarum (3). Pathogenicity tests were performed by misting a mixture of sporangiola and sporangiospores (25,000 to 70,000 per ml of water taken from 7- to 10-day-old cultures grown on APDA) on flowering-age petunia plants (cvs. Rose Madness, White Madness, and Dreams Pink). Tests were repeated twice. Inoculated plants and uninoculated control plants (2 to 4 of each treatment in each test) were held in a dew chamber at 28°C for 48 h and then moved to a greenhouse. Within 48 h after inoculation, plants developed water-soaked lesions on flowers, leaves, and stems, then wilted and rotted. Uninoculated plants remained disease free except for several that developed disease symptoms in the first test, apparently from the presence of natural inoculum on healthy-appearing plants that were obtained from the nursery where the disease was found. Koch's postulates were completed by reisolation of the pathogen from diseased inoculated plants. C. cucurbitarum (1) and C. infundibulifera (Curr.) Sacc. (2) have been reported to cause flower blight of petunia in the United States and whole plant blight (wet rot) of petunia in Japan (4). To our knowledge, this is the first report of C. cucurbitarum causing whole plant blight of petunia in the United States. References: (1) M. L. Daughtrey et al. Choanephora wet rot of poinsettia. Page 15 in: Compendium of Flowering Potted Plant Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (3) P. M. Kirk. Mycological Paper 152:1, 1984. (4) J. Takeuchi and H. Horie. Jpn. J. Phytopathol. 66:72, 2000.

Withania somnifera (Ashwagandha) is native to India and commercially cultivated for the production of root withanolides that have anticarcinogenic properties. A disease appeared on plantings of W. somnifera during the 2010 monsoon at the CIMAP and in adjoining areas of northern India. Symptoms first appeared as water-soaked lesions on leaves and stems that progressed to a wet rot. Mature lesions harbored black fructifications of the suspect pathogen. Pathogen isolations were done by placing pieces of infected tissues on potato dextrose agar. A fungus tentatively identified as a Choanephora sp. that produced white aerial mycelia that later turned pale yellow was consistently isolated from infected plant parts. Mycelia were hyaline and nonseptate. Sporangiophores bearing sporangiola were erect, hyaline, unbranched, apically dilated to form a clavate vesicle from which arose dichotomously branched distally clavate secondary vesicles. Sporangiola were indehiscent, ellipsoid, brown to dark brown with distinct longitudinal striations, and measured 12 to 20 × 6 to 12 μm. Sporangia were multispored, spherical, initially white to yellow and pale brown to dark brown at maturity, and measured 40 to 160 μm. Sporangiospores from sporangia were ellipsoid to broadly ellipsoid, brown to dark brown, indistinctly striate with fine hyaline polar appendages, and measured 16 to 20 × 8 to 12 μm. On the basis of the cultural as well as morphological characteristics and description in the monograph by Kirk (2), the fungus was identified as a Choanephora sp. The identification was also confirmed by IMTECH, Chandigarh, India with Accession No. MTCC-10731. The species was later characterized as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt (GenBank Accession No. AB470642) by using universal primers ITS-1 and ITS-4. Its sequence comprising of 18S rRNA partial, complete ITS 1, 5.8S rRNA, ITS 2, and 28S rRNA partial was submitted to NCBI GenBank with Accession No. JN639861. Pathogenicity of the fungus was established on five healthy plants by artificial inoculation with spray of an aqueous spore suspension containing 106 spores/ml. Plants sprayed with sterile distilled water were used as controls. Both inoculated and control plants were kept in a humidity chamber (96%) for 3 days and thereafter placed in the glasshouse at 28 ± 2°C. Initial symptoms developed in 2 to 3 days while typical disease symptoms appeared on all the inoculated plants after 7 to 10 days. Control plants were free from infection. The reisolation from artificially infected plants again yielded a Choanephora sp., thus fulfilling Koch's postulates. W. somnifera cultivation has been affected by root rot and wilt caused by Fusarium solani and leaf spot caused by Alternaria dianthicola (3). The occurrence of a Choanephora sp. was reported on periwinkle, petunia (1), and Boerhavia diffusa (4). However, to our knowledge, incidence of this pathogen on W. somnifera has not been reported so far. Thus, wet rot of W. somnifera caused by C. cucurbitarum is a new report from India and worldwide. References: (1) G. E. Holcomb. Plant Dis. 87:751, 2003. (2) P. M. Kirk. Mycol. Pap. 152:1, 1984. (3) C. K. Maiti et al. Plant Dis. 91:467, 2007. (4) N. Singh et al. New Dis. Rep. 23:29, 2011.

Hosta plantaginea (Lam.) Asch. is an herbaceous perennial plant with ornamental value. In August 2013, water-soaked spots and wet rot were found on flowers of H. plantaginea in a garden bedded out for landscaping in Hongcheon County, Korea. Symptoms initially appeared as water-soaked spots at the tips of flowers. Dark gray spots on flower petals often coalesced and led to rotting of flowers, with abundant sporulation. However, no symptoms were found on the leaves. Approximately 30% of the flowers were affected in the landscape bed. A fungal isolate was obtained by plating surface-disinfested diseased flower tissue on potato dextrose agar (PDA). Fungal colonies covering the plate (diam. 90 mm) in 48 h were white at first, with abundant aerial mycelia, but later turned pale yellow with abundant sporangiola. Sporangiophores bearing sporangiola were aseptate, hyaline, and usually arose from infected tissue. Sporangiola were ellipsoid to ovoid, indehiscent, brown to dark brown, pediculate, 7 to 12 μm wide and 9 to 20 μm high, and showed longitudinal striations at high magnification. Sporangia were few-spored to multispored, pale brown to brown, and 50 to 150 μm. Sporangiospores from sporangia were broadly ellipsoid, brown to pale brown, with hyaline polar appendages, 8 to 10 μm wide and 15 to 22 μm high. Zygospores were not observed. The morphological and cultural characteristics, especially based on shape and striation of sporangiola, were identical with those of Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (2,3). A representative specimen was deposited in the Korea University Herbarium (KUS-F27540). Genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). The primers ITS1/ITS4 and NL1/LR3 were used to amplify the internal transcribed spacer (ITS) region of rDNA and the D1/D2 region of the large subunit (LSU), respectively (4). The PCR products were purified and directly sequenced. The resulting 594-bp ITS and 680-bp D1/D2 sequences were submitted to GenBank (Accession Nos. KM200034 and KM200035). A GenBank BLAST search of the fungal database showed that the sequences of ITS and D1/D2 regions matched those of C. cucurbitrarum (JN943006 and JN939195) with 100% similarity. A pathogenicity test was conducted by spraying three healthy potted plants (2 months old) with a sporangiola suspension (2 × 104 conidia/ml). Another three potted plants of the same age were treated with sterile water and served as controls. The plants were kept in humid chambers for 2 days and placed in a greenhouse (28°C and 60 to 80% RH). After 4 to 5 days, water-soaked spots were evident on the flowers of inoculated plants. No symptoms were observed on control plants. A pathogenicity test was conducted twice with the same results, fulfilling Koch's postulates. C. cucurbitarum has a wide host range but has not been previously reported to cause disease on H. plantaginea (1). To our knowledge, this is the first report of C. cucurbitarum on H. plantaginea globally as well as in Korea. Choanephora rot of flowers is an issue under high-moisture conditions, so allowing for adequate airflow and a dry plant canopy should aid in disease suppression. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab. Online publication, ARS, USDA, retrieved July 11, 2014. (2) P. M. Kirk. Mycol. Pap. 152:1, 1984. (3) A. Saroj et al. Plant Dis. 96:293, 2012. (4) G. Walther et al. Persoonia 30:11, 2013.

Crotalaria breviflora (Fabaceae) is used as green manure crop because of its nitrogen fixation and nematode control (Nascimento et al. 2020). In April 2018, leaf wilting, flower rot, and stem necrosis symptoms were observed on C. breviflora with 100% incidence, in Sorriso (12° 33' 31″ S, 55o 42' 51″ W), Santa Carmem (11° 55' 52″ S, 55o 16' 47″ W), and Sapezal (12o 59' 22″ S, 58o 45' 52″ W) counties in the state of Mato Grosso, Brazil. Three monosporic isolates were isolated from symptomatic leaves, cultivated in potato dextrose agar (PDA) medium, and deposited at the Cultures Collection of the University of Brasilia (codes CCUB 1293, CCUB 1667, CCUB 1668). Colonies on PDA were white and cottony with presence of hyaline and coenocytic hyphae. The mycelia later became pale yellow with abundant reproductive structures. Sporangiophores were hyaline, aseptate, unbranched, and apically dilated to form a clavate vesicle, which produced secondary vesicles bearing sporangiola. Secondary vesicles were clavate, light brown, and 37 to 51 µm in diameter. Sporangia were brown to dark brown, globular to ellipsoid, 115 to 140 µm long, and 96 to 122 µm wide. Sporangiospores (n=30) were brown to reddish-brown, ellipsoid to ovoid, with longitudinal striae, 14 to 19 µm long, and 8 to 12 µm wide. Some with hyaline appendages at both ends. Their morphological characteristics were consistent with the descriptions of Choanephora cucurbitarum (Kirk 1984). To confirm the identity, the DNA of the three isolates was extracted and the sequences of Small Subunit (SSU), Large Subunit (LSU), and complete Internal Transcribed Spacer (ITS) of rDNA were amplified using V9G, ITS3, and LR5 primers (GenBank acc. no: MN897836, MN897837 and MN897838). The sequences were aligned with the MAFFT software. The alignment matrix was subjected to Maximum Likelihood (ML) analysis using RAxML v. 8 and Bayesian Inference performed in MrBayes v.3.1.2. The tree was edited in the FigTree software. The sequences showed 100% identity with the sequences from C. cucurbitarum found on the GenBank. To confirm pathogenicity, a suspension at 5.4 ×106 spores/ml was prepared from a 15-day-old culture grown at 25°C and sprayed on asymptomatic plants of C. breviflora. Sterilized water was sprayed as the control. Plants were kept in a humid chamber at 20°C for 48 h. Initial symptoms were visualized 16 days after inoculation. Complete necrosis of leaves and stems with spore mass on infected tissue was observed 19 days after inoculation. To satisfy the Koch's postulates, the fungus was successfully reisolated from the infected tissues. No symptoms were observed on the control plants. In Brazil, this pathogen has been reported on Brassica oleracea var. capitata, Capsicum annuum, Crotalaria spectabilis, Cucurbita sp., and Vigna unguiculata (Alfenas et al. 2018; Mendes and Urben, 2019). C. cucurbitarum has been reported to have a wide range of hosts (Farr and Rossman, 2020). It can infect the crops grown in rotation or in succession, including common bean, corn, cotton, quinoa, soybean, and sunflower. Therefore, this pathogen is of epidemiological importance and poses a threat to the croplands where environmental conditions are conducive to the disease to develop and spread. To our knowledge, this is the first report of C. cucurbitarum causing leaf and flower wilt, and stem rot on C. breviflora in the world. Acknowledgment We thank the Environmental Sciences Graduate Program, Federal University of Mato Grosso, University of Brasilia, PROPeq/PROPG-UFMT, EMBRAPA, CODEX/UFMT, Institute of Agricultural and Environmental Sciences (ICAA)/UFMT and CAPES for providing the Master's scholarship. References Alfenas, R. F., et al. 2018. Plant Dis.102:1456. https://doi.org/10.1094/PDIS-10-17-1610-PDN, Google Scholar. Farr, D. F., and Rossman, A. Y. 2020. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved May 26, 2020 from https://nt.ars-grin.gov/fungaldatabases/, Google Scholar. Kirk, P. M. 1984. Mycol Paper. 152:1. Google Scholar. Mendes, M. A. S., and Urben, A. F. 2020. Fungos relatados em plantas no Brasil, Retrived May 26, 2020 from http://pragawall.cenargen.embrapa.br/aiqweb/michtml/fgbanco01.asp, Google Scholar. Nascimento, D. D. et al. 2020. Bioscience Journal. 36:713. https://doi.org/10.14393/BJ-v36n3a2020-42248, Google Scholar.

Hibiscus syriacus, as a national flower of Korea, is most popularly used for ornamental purposes and includes numerous cultivars, and it is widely planted in temperate zones that feature hot summers. We investigated Choanephora flower rot on H. syriacus from 2012 to 2014 in Korea and Japan and confirmed Choanephora infection in several localities in both countries. Here, our objectives were to identify the main causal agent of Choanephora flower rot on H. syriacus and describe its morphological and molecular characteristics. We identified 44 out of 50 isolates as Choanephora cucurbitarum and the remainder as C. infundibulifera based on morphological characterization and phylogenetic analysis. The sequences of the internal transcribed spacer region (ITS) of ribosomal DNA and the D1/D2 region of the large subunit (LSU) rDNA of examined isolates were compared with sequences obtained from GenBank, and the analysis of the results revealed 100 % identity with the corresponding sequences of C. cucurbitarum and C. infundibulifera strains. Classification of the Choanephora species performed here according to the key described by Kirk (1984) corresponded with the results of the phylogenetic analysis of this study. Through intraspecific and interspecific mating tests, the characteristics of zygospore were described in details. Pathogenicity tests using both species showed the same symptoms, causing blossom blight and soft rot on the flowers, which were identical to those observed in the field. All identified causal agents of Choanephora rot were indeed Choanephora species, where C. cucurbitarum was identified in the majority, while the others were in the minority of examined samples.

Mexico is the fifth largest producer of papaya worldwide and has recently reported problems with mucoralean fungi in this crop. These fungi are considered saprophytes in the soil and are ubiquitous in nature. In this work, they were isolated from soil in regions of intensive papaya cultivation in Mexico. Collections were made in the states of Colima, Oaxaca and Veracruz in Apr 2016. A total of 72 mucorales fungal isolates was obtained and morphologically characterized and then molecular characterization (28S ribosomal region) of 25 representative isolates was carried out. Phylogenetic analysis of the sequences confirmed the presence of the species Gilbertella persicaria, Rhizopus oryzae, Rhizopus stolonifer, Mucor circinelloides and Mucor hiemalis, which cause soft rot in papaya fruits, therefore, spores of these fungi found in the orchard soils can be considered as a constant source of contamination that affects healthy fruits. Additionally, Choanephora cucurbitarum, Mucor ellipsoideus, Rhizopus homothallicus, Rhizopus microsporus, Rhizopus schipperae, Lichteimia ramosa, Gongronella butleri, Cunninghamella bertholletiae and Cunninghamella blakesleeana were identified which are considered to have agricultural, biotechnological and medical importance.

In August 2015, twig blight disease of ban tulsi (Croton bonplandianus Bail.) caused by Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. was observed for the first time, in the Gangetic alluvial region of West Bengal, India. A severe disease incidence (40-50%) showed twig blight symptoms starting with shoot apical meristem (SAM), leaf, and blossom blight symptoms. Typical symptoms were characterized by over-projecting black pin head like emerging sporangiola which formed mycelial cushion on the infected surface. The present report describes the identification of the causal pathogen as C. cucurbitarum based on its morphology and the internal transcribed spacer of its ribosomal DNA (rDNA - ITS) sequences with 100% identity of NCBI-GenBank published Choanephora database. Optimum temperatures, 28-30°C, coupled with high relative humidity (80-90%) during the monsoon season enhances the disease’s progress. To the best of our knowledge this is the first report of twig blight of C. bonplandianus, caused by C. cucurbitarum, in India as well as globally.

Current study aims to explore natural anti-phytopathogenic compounds for plant disease management. Syzygium cumini essential oil and its isolated compounds were screened for their antifungal assay against two phytopathogenic fungi, Rhizoctonia solani AG 4HG-III and Choanephora cucurbitarum . The chemical composition of oil the isolated oil was identified by gas chromatography and gas chromatography/mass spectrometry. Compounds such as 7-hydroxycalamenene, 7-acetoxycalamenene, 1-epi-cubenol, and α-terpineol showed an inhibition, ca. 95%, 80%, 76%, and 82%, respectively, against R . solani whereas 95%, 70%, 92%, and 100% against C . cucurbitarum in contact phase. In addition, high ocimene rich column fractions showed 70% and 87% inhibition against R . solani and C . cucurbitarum , respectively, in volatile phase. The findings of the present study suggest that the isolated 7-hydroxycalamenene, 7-acetoxycalamenene, 1-epi-cubenol, α-terpineol, and ( Z )-β- & ( E )-β-ocimene have a potential to be used as antifungal agents for the effective control of fungal diseases in medicinal plants. We report 7-acetoxycalamenene as novel compound from natural source for first time.

In July 2016, wet rot was observed on leaves of Brassica chinensis in a private greenhouse in Hatyai city, Songkhla province, Thailand. The fungal pathogen infected young and expanded leaves. The fungus was identified by morphological characterization and molecular analysis as Choanephora cucurbitarum. Pathogenicity test revealed that C. cucurbitarum could infect B. chinensis.

A zygomycete fungus, Choanephora cucurbitarum is a plant pathogen that causes blossom rot in cucurbits and other plants. Here we report the genome sequence of Choanephora cucurbitarum KUS-F28377 isolated from squash. The assembled genome has a size of 29.1 Mbp and 11,977 protein-coding genes. The genome analysis indicated that C. cucurbitarum may employ a plant pathogenic mechanism similar to that of bacterial plant pathogens. The genome contained 11 genes with a Streptomyces subtilisin inhibitor-like domain, which plays an important role in the defense against plant immunity. This domain has been found only in bacterial genomes. Carbohydrate active enzyme analysis detected 312 CAZymes in this genome where carbohydrate esterase family 6, rarely found in dikaryotic fungal genomes, was comparatively enriched. The comparative genome analysis showed that the genes related to sexual communication such as the biosynthesis of β-carotene and trisporic acid were conserved and diverged during the evolution of zygomycete genomes. Overall, these findings will help us to understand how zygomycetes are associated with plants.

Choanephora cucurbitarum is a plant pathogenic fungus with a wide host range. The fungus was isolated from rotting fruits of Capsicum anuum, after it was observed that some vegetables were infected in the farm. The role of infection courts on severity was determined by inoculating the pathogen into soil, leaves and stems of the test plants. The test plants used were Abelmoschus esculentus, Amaranthus sp, Cucumis sativus and Vigna unguiculata. Determinants of pathogenicity were plant height, leaf reduction, fresh and dry weight. Results showed that the fungus was pathogenic to the test plants. A significant reduction in height, leaves, fresh and dry weight of the test plants was observed when compared with the control. The greatest effect of the pathogen was on the inoculated stem of the test plants with a mean of 42.94. The least effect was observed on infected soil with a mean of 92.99. Cucumis sativus showed the least effect by the pathogen with a mean of 84.18, while Abelmoschus esculentus showed the greatest effect with a mean of 44.59. The pathogen showed the greatest effect on leaves of Cucumis sativus with a mean of 20.45, while the least effect was on Vigna unguiculata with a mean of 36.77.Fresh weight ranged from 3.35g to 37.40g, Dry weight 2.15 to 7.90g as compared with the control which had a fresh weight of 7.0g to 57.25g and 3.8g to 11.90g for dry weight. Symptoms such as leaf blight, blight of the shoot apex, soft rot of stems, die back and decay of Vigna unguiculata pods were observed.

In July and August 2013, blossom blight and soft rot of pods were observed on okra in experimental fields in Iksan and Jeju, Korea. Infection started in fading flower petals, spread to entire flowers and young pods, resulting in blighted blossoms and soft rot of pods. Severe infection caused early falling of blossoms and fruit drop, reducing plant vigour in the summer season. On the basis of the morphological characteristics and phylogenetic analyses of two molecular markers ITS rDNA and D1/D2 region of the LSU, the fungus was identified as Choanephora cucurbitarum. A pathogenicity test was carried out to fulfil Koch's postulates. To our knowledge, this is the first report of C. cucurbitarum on okra in Korea.

Essential oil extracted from aerial parts of two Ocimum species (O. canum and O. kilimandscharicum) was investigated using gas chromatography and gas chromatography/mass spectrometry techniques on DB-5 (5% diphenyl-95% dimethylpolysiloxane) and β-cyclodextrin (6-tertiarybutyldimethylsiliyl-2,3-diethyl-β-cyclodextrin) capillary columns. Essential oil extracted from O. canum contained camphor, limonene, camphene and myrtenol as most abundant constituents among monoterpenoids, whereas β-selinene, α-selinene, maaliol and β-caryophyllene were identified under sesquiterpenoids class. On contrary, two folds higher camphor was recorded in O. kilimandscharicum. Maaliol (6.4%) was characterized in O. canum for the first time with the help of extensive 1D and 2D-NMR experiments. Therefore, maaliol can be considered as a marker constituent to differentiate both the camphor rich oils. Further, when the oils were subjected to chiral analysis on β-cyclodextrin column; a high enantiomeric excess for (1R)-(+)-camphor was recorded. This revealed a close relationship between these two species in terms of biosynthesis of single enantiomer. Apart from analysis, oils were assessed for antifungal activity against Rhizoctonia solani and Choanephora cucurbitarum. O. kilimandscharicum exhibited a complete inhibition against R. solani and C. cucurbitarum while O. canum was only effective against R. solani.

Chiral compounds have shown antimicrobial activity, enantioselectively. Leaf oil composition of Cinnamomum camphora was studied using GC-FID, SPME-GC-FID, enantio-GC and GC/MS techniques. Camphor exists in two chiral forms and contributes major percentage to C. camphora essential oil (68.4–81.2%). The results showed (1R)-(+)-camphor as major enantiomer (ee > 99%). The biological potential of essential oil, (1R)-(+)- and (1S)-(−)-camphor was investigated against Choanephora cucurbitarum, a Withania wet rot pathogen. The activity was evaluated against fungal growth and morphological changes in the hyphae of C. cucurbitarum, after 18, 21 and 24 h of treatments. A significant growth inhibition in C. cucurbitarum was observed against (1R)-(+)-camphor. Further, in comparison to (1R)-(+)-camphor, C. camphora oil revealed better activity. Fungal growth inhibition by (1R)-(+)-camphor and plant essential oil was due to cytoplasm coagulation and hyphal lysis of C. cucurbitarum. Moreover, C. camphora essential oil could be used in the preparation of strong fungistatic agent against C. cucurbitarum infection.

Leaf rot of cauliflower caused by Choanephora cucurbitarum is reported for the first time in India. Koch’s postulates were fulfilled for the first time for this fungus-host association demonstrating that C. cucurbitarum, known to be the pathogen behind soft rot on numerous hosts is also a pathogen of cauliflower.

Quinoa (Chenopodium quinoa Willd.) has been cultivated for centuries as an important subsidiary grain crop. In early August 2015 and 2016, Choanephora stem rot symptoms were observed on plants of Quinoa cultivar Longli 1 in a field with 0.3 ha planting area in Yanqing district, Beijing, China. This disease caused about 70% yield loss of quinoa. Disease symptoms initially appeared as water-soaked spots on the stem that turned into brown to dark brown lesions covering the whole stem, often resulting in the infected part breaking off the rest of the plant. Serious infections often caused the plant to wither and die under high humidity. The infected tissues produced an abundance of sporangiophores bearing brown to black sporangiola. To isolate the causal agent, sections of diseased stem were surface disinfected with sodium hypochlorite, placed on acidified potato dextrose agar (APDA), and incubated at 25°C with a 12-h photoperiod. Two isolates were obtained (ZD61 and ZD62). The colonies of both isolates grew quickly on PDA and were initially white with abundant aerial mycelia. The mycelia later turned pale yellow and produced abundant sporangiophores bearing sporangiola. Sporangiophores were smooth, hyaline, and aseptate, erect, unbranched, and apically dilated to form a clavate vesicle, which produced dichotomously branched and distally clavate secondary vesicles bearing monosporous sporangiola. Monosporous sporangiola were broadly ellipsoid, indehiscent, pediculate, brown to dark brown, with distinct longitudinal striations, and were 7 to 15 μm wide and 11 to 24 μm long by measuring 30 sporangiola. Sporangia contained few or multiple spores. They were also spherical, initially white to yellow, and pale brown to dark brown at maturity and measured 63 to 160 μm. Single-celled sporangiospores were aseptate, ellipsoid, and brown to pale brown, with multiple hyaline polar appendages and striations on the wall. They were 6 to 11 μm wide and 9 to 22 μm long by measuring 50 spores. The morphological and cultural characteristics of both isolates were identical to those of Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk 1984). Genomic DNA was extracted from the isolates with the DNA Isolation Kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) regions of the two isolates were sequenced using primers ITS1 and ITS4 (White et al. 1990). The two resulting sequences were submitted into NCBI (GenBank MG650193 and MG650194), which were 99% identical to known C. cucurbitarum strains (GenBank KY080447, KY080446, KU877802, and KR873353) according to a BLAST analysis. Pathogenicity tests were conducted on hypocotyls of 4-week-old quinoa seedlings inoculated with 2-day-old mycelia plugs. Control plants were inoculated with sterile APDA plugs. Plants were incubated under humid conditions for 3 days and then transferred to a greenhouse (28°C). After 4 to 5 days, lesions were evident on the stems of inoculated plants but not on control plants. After 7 days, all inoculated plants had developed brown stem lesions and were wilted. Additionally, C. cucurbitarum was reisolated from the inoculated plants, fulfilling Koch’s postulates. The fungus C. cucurbitarum reportedly has a wide host range. In China, C. cucurbitarum was recently observed on Moringa oleifera causing seed pod rot (He et al. 2017). However, to our knowledge, this is the first report of C. cucurbitarum causing stem rot on C. quinoa in China or globally.

In vitro and in vivo experiments were carried out to determine the mechanisms involved in suppression of Choanephora cucurbitarum the causal pathogen of wet rot disease of okra by using the tea produced from agro-waste such as rice straw (RST) and empty fruit bunch (EFB) of oil palm composts. Non-sterilized, filter-sterilized and heat-sterilized compost teas were tested on the growth of C. cucurbitarum. The highest percentage of inhibition in conidial germination was obtained by both the non-sterilized RST and EFB compost tea with values of 84% and 79%, respectively. The inhibitory efficacy was reduced significantly ( P ⩽ 0.05) when the teas were subjected to millipore membrane filters or heat-sterilization. The mycelial growth of C. cucurbitarum was reduced by 100% in plates amended with both the non-sterilized compost tea. Light microscopy micrographs of mycelial samples of C. cucurbitarum exposed to non-sterilized RST and EFB compost tea showed alterations in mycelial morphology and lysis, resulting in inhibition in mycelial growth. Glass house trials indicated that induced host resistance was stimulated in okra plants treated with non-sterilized and filter-sterilized compost teas based on the detection of inducible resistance-related compounds. The mean values for inducible enzymes which have a role in disease resistance [peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL)] increased until day 8, which were significantly higher in okra plants pre-treated with non-sterilized tea in view of the high nutritive and microbiological properties. There was a further increase in inducible compounds when the pre-treated okra plants were challenged with C. cucurbitarum. Following the increase in total PO, PPO and PAL activities, development of Choanephora wet rot was delayed, confirming the possible involvement of induced resistance. However, resistance was not maintained since it decreased over time. This could be due to the highly stressed environment of the leaf surfaces which had a detrimental effect on the survival of the antagonists and subsequently affected the efficacy of compost tea. It was therefore suggested that plants should be sprayed at short intervals with the compost tea to sustain the suppressive effect.

The potential of water extracts produced from rice straw (RST) and empty fruit bunch of oil palm (EFB) composts fortified with Trichoderma harzianum for the control of Choanephora wet rot of okra caused by Choanephora cucurbitarum was studied under field conditions. Disease severity was lowest in plants treated with Trichoderma-fortified RST extracts (9.56%) with a disease index of 1, mancozeb (Dithane M-45® (2 g l−1 of water), Trichoderma-fortified EFB compost extracts, extracts of RST and EFB, and an aqueous suspension of T. harzianum recorded disease severity values of 10.25%, 19.38%, 37.56%, 53.71% and 56.36%, with a disease index of 1, 1, 2, 3 and 3, respectively. There was a reduction of 85.04% in Choanephora wet rot severity on okra treated with Trichoderma-fortified RST extracts during 12 weeks of assessment in the field, which was comparable to the conventional fungicide Dithane M-45®, suggesting that application of extracts produced from well-matured compost fortified with biocontrol agents could be an alternative control strategy.