First report of Nigrospora sphaerica associated with leaf spot disease of Crossandra infundibuliformis in India.

Crossandra (Crossandra infundubuliformis (L.) Nees.) is one of the main floriculture crops in Karnataka. In 2020 (March-June), a characteristic leaf spot disease of unknown etiology with an incidence ranging from 10-12% (~30 ha area evaluated) was observed in Southern Karnataka (Mysore, Mandya). Initially, the symptoms developed as small specks (3 to 8 mm), characterized by circular to irregular shapes in the beginning and coalesced to form larger lesions. Ten samples were collected in polybags followed by the isolation of associated fungal pathogen on potato dextrose agar (PDA) medium amended with Chloramphenicol (60 mg/L). Briefly, small pieces of infected leaves were cut into small pieces and surface sterilized with 2% sodium hypochlorite (NaOCl) solution, rinsed three times with sterile distilled water (SDW), blot dried, then inoculated onto PDA medium, and incubated at room temperature (27 ± 2°C) for 3 - 5 days. Fungal colonies developed from the segments and were subcultured through hyphal tipping to fresh PDA plates to get pure cultures. A total of 12 pure cultures were obtained. Mycelia were initially white and eventually turned gray. The conidia were black, single-celled, smooth, spherical to subspherical, 9 to 18 µm in diameter (n=50), and borne singly on a hyaline vesicle at the tip of each conidiophore. The identity was initially established based on the cultural features and conidial morphology as Nigrospora sp. (Deepika et al., 2021). To confirm the identity of fungal isolates based on molecular sequence analysis was performed for two representative isolates (CIT1 & CIT2). ITS-rDNA, tub2 & EF-1α gene were amplified using primers ITS1/ITS4, T1/T22 & EF1-728F/986R (White et al., 1990; O'Donnel and Cigelnik, 1997; Carbone and Kohn, 1999), then purified and sequenced. The BLASTn analysis of ITS, tub2 and EF-1α gene showed 99-100% similarity with reference sequences from the GenBank database to Nigrospora sphaerica (ITS: 520bp, KX985935 - LC7312; MH854878 - CBS:166.26; tub2: 357bp, MZ032030 - WYR007, 350bp, KY019606 - LC7298, KY019522 - LC4278, KY019520 - LC4274; EF-1α: 472bp, KY019397 - LC7294, KY019331 - LC4241; MN864137 - HN-BH-3) and the sequences were deposited in GenBank (ITS: OL672271 & OL672272; tub2: OL782120 & OL782121; EF-1α: ON051604 & ON051605) (Wang et al., 2017). The associated fungal pathogen was identified as N. sphaerica (Sacc.) Mason (Chen et al. 2018; Deepika et al., 2021) based on the cultural, morphological, microscopic, and molecular characteristics. Further, pathogenicity tests were conducted on healthy plants (Crossandra cv. Arka; n=30) grown under greenhouse conditions (28±2 °C; 80% RH). Inoculations were made with conidial suspension (18 days old N. sphaerica isolate CIT1, 106 conidia/ml) prepared in SDW, and healthy plants sprayed with SDW (n=10) served as controls. All the plants were covered with polyethylene bags for 24-48 hr and observations were made at regular intervals. Typical necrotic lesions developed on 16 plants after 12 days after inoculation but no symptoms were observed on the control plants. The associated pathogen was re-isolated from diseased leaves and confirmed their identity based on morphology and cultural characteristics. Earlier, N. sphaerica was associated with various tree species as an endophyte, and recently several reports have appeared to cause disease on various crop plants (Deepika et al., 2021). However, there are no previous reports on the association of N. sphaerica causing leaf spot disease on C. infundibuliformis from India. Early diagnosis of this leaf spot disease will help the floriculturist adopt suitable management practices to avoid significant economic loss.

First report of Athelia rolfsii (=Sclerotium rolfsii) associated with foot rot disease of Chrysanthemum morifolium in India.

Chrysanthemum morifolium L. is an important flower crop grown in different parts of Karnataka for its striking cut flowers and international market value. During a field survey (Mysore district, Karnataka, February, 2022), chrysanthemum fields were found infected with foot rot disease. The presence of white mycelial structures with sclerotia were recorded near the stem-soil interface. The disease incidence ranged 10-12% measured in an area of approximately 10 hectares. The infected plants showed quick wilt, yellowing and toppling of the entire plant. Infected plants from Doddamaragowdanahally and Rayanahally (n=15) were collected and associated fungal pathogen isolated after surface sterilization with NaOCl (1%) on potato dextrose agar (PDA) amended with chloramphenicol (50 mg/L). Fungal mycelia developed from the infected tissues were inoculated on to fresh PDA plates to obtained pure cultures for further identification. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.284.2 mm) were observed after 15 days of incubation (28 ± 2°C). Sclerotia were white in the beginning and turned brown at maturity. The average number of sclerotia produced per plate ranged from 240 to >480 (n = 10). To further to confirm the identity of the isolates, two representative isolates (CmSr1 and CmSr2) was subjected to molecular identification based on ITS-rDNA sequences. Briefly, genomic DNA was isolated from 12 day old cultures using the CTAB method and ITS-rDNA was amplified using ITS1-ITS4 primers (White et al., 1990). An expected amplicon of >650 bp (ITS) was obtained and later sequenced from both the directions. The consensus sequences were analysed through nBLAST search which revealed that 100% sequence similarity with reference sequences of Athelia rolfsii (S. rolfsii) from GenBank database (MT127465, MN974137, KC292637; identity 656/656; 0 gaps). A phylogenetic tree obtained by the neighbor-joining method using MEGAX shared a common clade with the reference sequences retrieved and computed, thus confirming the identification based on sequence analysis and molecular phylogeny. The representative sequence of A. rolfsii isolates CmSr1 and CmSr2 isolates deposited in GenBank with Accession nos. ON456153 and ON456154, respectively. Based on etiology, morphological, cultural and molecular data the pathogen was identified as Athelia rolfsii (Curzi) Tu & Kimbrough (Syn: Sclerotium rolfsii Sacc.) (Mordue, 1974; Mahadevakumar et al., 2016, 2018). Plants (n=60) were inoculated with sclerotial bodies (2 sclerotia/plant) near stem soil interface under green house and covered with polythene bags (at 27 ± 2°C and 80% RH). Non-inoculated plants (n=20) served as controls. The development of foot rot disease was observed eight days after inoculation. A total of 48 plants showed the foot rot symptoms and 12 inoculated plants and control plants remained healthy. The identity of the fungus was confirmed by morphological and cultural characters after re-isolation. C. morifolium is an important flower crop in Karnataka. S. rolfsii is known to be associated with blight and collar rot of Chrysanthemum spp. from Kerala (Beena et al., 2002) but no species (host) identity provided. Therefore, to the best of our knowledge, this is the first report of foot rot disease caused by Athelia rolfsii on C. morifolium in India. Early diagnosis of this disease will help the farmers to adopt suitable management practices to avoid loss.

First report of Lasiodiplodia theobromae associated with panicle blight of grapes (Vitis vinifera L.) in India.

Grape (Vitis vinefera L.) is a popular horticulture crop in Karnataka, India. A fungal pathogen caused panicle blight on panicles with immature fruit and severity increased subsequently in the grape growing regions of Devanahalli and Doddaballapur, Karnataka, between August and September 2019. The disease incidence varied from 15 to 18 percent in around 45 hectares of grape vineyards surveyed. The associated fungal pathogen was isolated on Potato Dextrose Agar (PDA) medium (HiMedia Laboratory, Mumbai, India) amended with Chloramphenicol. A total of 12 fungal isolates were obtained and identified based on morphology. Fungal cultures obtained from all the panicle blight affected samples were fluffy grayish to black with profuse, dense mycelium. Microscopic examinations revealed that the conidia ellipsoidal, two celled and hyaline when young, and developed dark brown pigments at maturity. Mature conidia measured 18.24±2.35 to 26.62±3.11 µm long and 10.32±1.08 to 12.57±1.82 µm width (n=30). The fungal pathogen was identified as a Lasiodiplodia sp. based on colony morphology and microscopic features. A total of three representative isolates L. theobromae (Vv12, Vv15, and Vv19) were selected for molecular identification based on ITS-rDNA, tub2 and EF-1α gene sequences and phylogenetic analysis. Genomic DNA was isolated from 12 day old cultures and ITS-rDNA, tub2 and EF-1α genes were amplified using ITS1/ITS4; Bt2a/Bt2b and EF1-728F/986R primer pairs, respectively (White et al., 1990; Glass and Donaldson, 1995, Carbone and Kohn, 1999). PCR amplicons were sequenced and the sequences were deposited in GenBank with the accession number ITS: MZ855866.1; MZ855867.1; MZ855868.1; tub2: MZ868708.1; MZ868709.1; MZ868710.1 and EF-1α: OM604750; OM604751; OM604752 respectively. The phylogeny was constructed based on combined ITS, EF-1α and the tub2 regions. Maximum Likelihood (ML) analysis was conducted and an ML tree was constructed with the substitution models (branch support was evaluated by 1,000 bootstrap replications). Combined phylogeny confirmed that the sequences shared a common clade with L. theobromae. Based on micro-morphological features and multi-locus sequence phylogeny, the associated fungal pathogen was identified as L. theobromae. There are no reports on the occurrence of L. theobromae causing panicle blight on grapes from India. Further, the pathogens association was confirmed through pathogenicity assay conducted on field harvested healthy bunches of grapes maintained under humid chamber. A total of 10 grape bunches were inoculated with a mycelial disc on the rachis of the panicle and incubated in a moist chamber for 5 days and control sets were inoculated with only agar plugs. The experiments were conducted in three replicates and repeated twice. A total of 21 panicle bunches developed typical rot symptoms 12-days post inoculation. The identity of the pathogen was confirmed based on micromorphology and cultural features after re-isolation (n=5), thus proving the Koch postulates and confirming the association of L. theobromae with panicle blight of grapes. Lasiodiplodia species are known to cause dieback, stem blight, leaf blights and spots on various crop plants. Mathur (1979) mentioned the occurrence of L. theobromae on grapes, however, no further details are available on the part associated, as well as morphological and molecular confirmation of L. theobromae. This is the first report of the L. theobromae causing panicle blight disease of grapes in India. Further, understanding the host range for L. theobromae and its variation will help to draw suitable disease management strategies.

First report of Athelia rolfsii (=Sclerotium rolfsii) causing foot rot disease of chia (Salvia hispanica L.) in India.

Salvia hispanica L. (Lamiaceae) commonly called 'chia' is an important food crop that has gained significance in recent times globally due to its nutritive value. During a field survey (Mysore district, Karnataka, October, 2021), chia fields were found associated with a characteristic foot rot disease. Further, the presence of mycelial structures along with sclerotial bodies was recorded near the stem-soil interface on the infected plants. The disease incidence ranged 15-21% in an area of approximately 15 hectares of chia fields. The symptoms initially appeared as tan lesions near the stem soil interface and the lesions were colonized by the fast growing mycelium. As the disease progressed, the plants toppled due to death of the stem-root interface region. Infected plants from KM Halli (12º20'90"N; 76º37'68"E) and DMG Halli (12º28'50"N; 76º51'66"E) (n=30) were sampled and associated fungal pathogen isolated on potato dextrose agar (PDA; HiMedia Lab, Mumbai). Fungal mycelia developing from the infected tissues were inoculated on to fresh PDA plates to obtained pure cultures for further identification. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.52.9 mm) were observed after 1012 days of incubation at room temperature. Sclerotia were white at first and turned brown with age. The average number of sclerotia produced per plate ranged from 150 to >280 (n = 10). To further to confirm the identity of the isolates, three representative isolates (SrSh1, SrSh5 and SrSh10) was subjected to molecular identification based on ITS-rDNA sequences. Briefly, genomic DNA was isolated from 12 day old cultures using the CTAB method and ITS-rDNA was amplified using ITS1-ITS4 primers (White et al., 1990). An expected amplicon of >650 bp was obtained and later sequenced from both the directions. The consensus sequences were analysed through nBLAST search which revealed that 100% (643/643 bp) sequence similarity with reference sequences of Athelia rolfsii (S. rolfsii) from GenBank database (KY640622 and AB075298). A phylogenetic tree obtained by the neighbor-joining method using MEGAX shared a common clade with the reference sequences retrieved and computed, thus confirming the identification based on sequence analysis and molecular phylogeny. The representative sequence of A. rolfsii isolates SrSh1, SrSh4 and SrSh7 isolates deposited in GenBank with Accession no OM021878-OM021880. Based on etiology, morphological, cultural and molecular data the pathogen was identified as Athelia rolfsii (Curzi) Tu & Kimbrough (Syn: Sclerotium rolfsii Sacc.) (Mordue, 1974; Mahadevakumar et al., 2016, 2018). Pathogenicity tests were conducted by inoculating the sclerotial bodies near stem soil interface of chia plants grown under green house (at 28 ± 2°C and 70% relative humidity). Briefly, a total of 60 healthy plants were inoculated with sclerotia and covered with polythene bags for 2 days and removed later. Plants (n=20) inoculated without any sclerotia were treated as controls. The development of characteristic foot rot disease was observed after 6-8 days post inoculation. A total of 38 plants showed the foot rot symptoms while control plants remained healthy. The identity of the fungus was confirmed by morphology and molecular sequence analysis after re-isolation. Chia is an important food crop and in recent times has been regarded as super food. Although S. rolfsii is known to be associated with many crops, this is the first report in chia. Therefore, to the best of our knowledge, this is the first report of foot rot disease caused by Sclerotium rolfsii on chia in India. Early diagnosis of this disease will help the farmers to adopt suitable management practices to avoid loss.

First report of Athelia rolfsii (=Sclerotium rolfsii) associated with southern blight disease of Macrotyloma uniflorum in India.

Horse gram (Macrotyloma uniflorum (Lam.) Verdc., Fabaceae) is an important legume crop cultivated widely in the arid and semiarid regions. During a survey carried out in the Mysore district (Karnataka, India, October 2021), horse gram plants showed the symptoms of southern blight disease. Disease incidence ranged from 20-27% in the approximate 52 hectares of horse gram fields evaluated. The symptoms initiated as tan lesions and the developing mycelial threads colonized the infected root-stem interface, causing girdling; lesions on leaves enlarged and developed into distinct spots. Infected parts (leaves & stem) (n=30) were collected in poly bags and incubated in a moist chamber overnight, followed by surface sterilization of small segments of stem, leaf with 2% NaOCl, rinsed with sterile water (SW), and placed onto the potato dextrose agar (PDA, HiMedia Lab, Mumbai) supplemented with chloramphenicol (40 mg/L). The plates were incubated at room temperature (28 ± 2°C) for 5-7 days. The fungal colonies developed from the diseased segments were sub-cultured through hyphal tipping to fresh PDA plates and pure cultures were obtained. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.52.9 mm) were observed after 1012 days of incubation. Sclerotia were white in the beginning and turned to brown later. The average number of sclerotia produced per plate ranged from 112 to 320 (n = 20). To determine the identity of the isolated fungal pathogen, ITS-rDNA was amplified and sequenced using ITS1/ITS4 (White et al. 1990) primers. The amplified PCR product was purified and sequenced directly. The ITS sequences (OM037658 & OM037659) shared 100% (630/643bp) sequence similarity to Athelia rolfsii (KY640622.1, AB075298). The phylogenetic tree (Neighbour-Joining method) constructed based on ITS-rDNA region confirmed that the sequences shared a common clade with reference sequence of A. rolfsii. Thus the identity was confirmed based on micromorphology and phylogenetic analysis. Pathogenicity tests were conducted on a total of 20 plants (5-6 weeks old) in greenhouse conditions (at 28 ± 2°C and 70% relative humidity) by inoculating with sclerotia from 15 days old cultures on stem and leaves and 14 plants were found infected after 5 days of post-inoculation, while uninoculated control plants remained healthy. Similarly, detached leaf assay (Mahadevakumar et al., 2018) was performed under in vitro conditions at 28 ± 2°C in a moist chamber and 28 out of 30 leaves showed the leaf spot symptoms after 3-5 days of inoculation. Uninoculated control leaves remained healthy. The identity of the fungus was confirmed by morphology and molecular analysis after re-isolation. The occurrence as a pathogen on horse gram has not been previously reported elsewhere. This is the first report of southern blight disease caused by A. rolfsii on horse gram from India. Early diagnosis of this leaf spot disease will help the farmers to adopt suitable management practices to avoid loss in production.