First report of Phytopythium vexans causing gummosis and root rot of Khasi Mandarin (Citrus reticulata Blanco) in north eastern states of India.

Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), ß tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of ß tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.

First Report of Stem Rot Caused by Athelia rolfsii on Curry Leaf Tree (Murraya koenigii) in Tripura, India.

Murraya koenigii is an important medicinal plant of India and commonly known as curry leaf tree grown in tropical and subtropical regions. The leaves of curry tree are used as a herb due to the presence of following important active constituent bismahanine, murrayanine, murrayafoline-A, bi-koeniquinone-A, murrayazolidine etc. (Jain et al. 2017). During mid-July 2019, stem rot disease symptoms were observed on curry leaf trees at the College of Agriculture, Lembucherra, Tripura (India). The disease symptoms consisted of rotting, wilting and blighting with disease incidence ranging from 8 to 10%. Initially, infected plants gradually withered and white mycelia mats appeared on the surface of the lower stem at the soil line. Infected stem samples were collected and surface was sterilized with 0.25% sodium hypochlorite for 1 min, washed thrice with sterilized distilled water and placed in Petri plates containing 2% water agar. After three days of incubation at 26°C, hyphae produced from plant bits were transferred into Petri plates containing potato dextrose agar. Ten isolates were collected from the diseases samples. Pure cultures were obtained as abundant, aerial and white mycelia with round to irregular sclerotia of 0.8 to 1.5 mm in diam. The sclerotia were initially white in color but later turned into brown color. The pathogen was identified as Athelia rolfsii based on morphology (Aycock 1966). To confirm the identification, the genomic DNA was extracted from a mycelia mat of the isolates using ZR fungal/Bacterial DNA miniprep kit (Irvine, CA) and the internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers, ITS1 and ITS4 (White et al. 1990). A 550 bp PCR product was sequenced and showed 99% similarity with Athelia rolfsii isolate (GenBank accession MH854711).The generated sequence was submitted to GenBank (Accession MT535585). After identification of the pathogen a pot experiment was conducted to confirm the pathogenicity. Earthen pots (29 cm. diam.) were filled with sterilized soil and kept in a green house. Ten curry leaf plants (50 days old) were grown from seed in the separate pot were inoculated with 15-day old mycelia mats prepared in potato dextrose broth. The stem of each curry plant were artificially injured with the help of sterile blade and wrapped with moistened sterilized cotton containing the mycelial mat. Five curry leaf plants artificially injured and inoculated with sterilized distilled water were used as control. The Earthen pots with plants were individually covered with plastic bags and kept in the green house at 26°C for approximately 15 days. The inoculated plants started showing symptoms of stem rot six days after inoculation and started drying onward. The symptoms of stem rot on the inoculated plants were similar to those observed in the field. The fungus was re-isolated from the inoculated plants and A. rolfsii identification was confirmed based on morphology. No symptoms were observed on the control plants. The obtained culture was deposited in the Indian Type Culture Collection, Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi, India (ITC-8666). To the best of our knowledge this is the first report of stem rot disease of curry leaf plant caused by A. rolfsii in India and worldwide. Due to medicinal, flavour and aroma properties, it is regularly used in India. Curry leaf plant is regularly used as a medical herb in India and therefore this disease poses a significant risk to production.

Genetic diversity of phytoplasma strains inducing phyllody, flat stem and witches' broom symptoms in Manilkara zapota in India.

During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches' broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25 kb and ~ 1.3 kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by 16S rRNA gene sequence comparison analysis. Virtual RFLP analysis of 16S rRNA gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.

Characterization and genetic diversity of phytoplasmas associated with elephant foot yam (Amorphophallus paeoniifolius) in India.

During the growing season of 2015 and 2016, leaf yellowing, stunting, and declining symptoms were observed on elephant foot yam in three states of India.The 1.3 kb 16S rDNA fragments were amplified from genomic DNA extracted from all the symptomatic elephant foot yam samples in nested PCR assays using primer pairs, P1/P7 followed by 3F/3R. Pair wise sequence comparison, virtual RFLP and phylogenetic analysis of 16S rRNA gene sequences confirmed association of 'Candidatus Phytoplasma trifolii' (16SrVI-D) and 'Candidatus Phytoplasma oryzae' (16SrXI-B) related strains in Uttar Pradesh, Uttarakhand and Tripura states, respectively. To our knowledge, this is the first report of association of 'Ca. P. trifolii' and 'Ca. P. oryzae' related strains in elephant foot yam in world. In the present study, we also reported Datura stramonium showing witches' broom as a natural weed host for 'Ca. P. trifolii' phytoplasma in Gorakhpur and Kushinagar districts of Uttar Pradesh state, India.

Control of Erysiphe pisi Causing Powdery Mildew of Pea (Pisum sativum) by Cashewnut (Anacardium occidentale) Shell Extract.

The effect of methanolic extract of cashewnut (Anacardium occidentale) shell extract was seen on conidial germination of Erysiphe pisi and powdery mildew development in pea (Pisum sativum). Maximum conidial germination inhibition of E. pisi on glass slides was observed at 300 ppm. Similar effect on floated pea leaves was observed after 48 h at the same concentration. Conidial germination on intact untreated pea leaves was also assessed on II and IV nodal leaves while IV and II nodal leaves were treated with the extract and vice versa. There was tremendous reduction in conidial germination on all the nodal leaves. The disease intensity of pea powdery mildew was significantly reduced by methanolic extract of cashewnut shells. Maximum reduction was observed with 200 ppm where 39% disease intensity was recorded in comparison to 96.53% in the control. The phenolic acid content of pea leaves following treatments with this extract varied and no definite pattern was observed. Out of several phenolic compounds, namely, gallic, ferulic, chlorogenic, and cinnamic acids, only gallic acid was found to be present consistently in all the treatments with varied amounts.

Studies on exudate-depleted sclerotial development in Sclerotium rolfsii and the effect of oxalic acid, sclerotial exudate, and culture filtrate on phenolic acid induction in chickpea (Cicer arietinum).

Exudate depletion from developing sclerotia of Sclerotium rolfsii Sacc. in culture caused reduced size and weight of sclerotia. Germination of exudate-depleted sclerotia was delayed on Cyperus rotundus rhizome meal agar medium when compared with that of control sclerotia. The exudate-depleted sclerotia caused infection in chickpea (Cicer arietinum) plants in a glasshouse. Different temperatures and incubation periods had no effect on the germination ability of the exudate-depleted sclerotia. Oxalic acid, sclerotial exudate, and culture filtrate of S. rolfsii induced the synthesis of phenolic acids, including gallic, ferulic, chlorogenic, and cinnamic acids, as well as salicylic acid, in treated chickpea leaves. Gallic acid content was increased in treated leaves compared with the untreated controls. Maximum induction of gallic acid was seen in both leaves treated with oxalic acid followed by exudate and leaves treated with culture filtrate. Cinnamic and salicylic acids were not induced in exudate-treated leaves. Ethyl acetate fractionation indicated that the sclerotial exudates consisted of gallic, oxalic, ferulic, chlorogenic, and cinnamic acids, whereas the culture filtrate consisted of gallic, oxalic, and cinnamic acids along with many other unidentified compounds.

Plant growth-promoting rhizobacteria-mediated induction of phenolics in pea ( Pisum sativum) after infection with Erysiphe pisi.

Qualitative and quantitative estimation of phenolic compounds was done through high performance liquid chromatography (HPLC) in different parts of pea ( Pisum sativum) after treatment with two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens (strain Pf4) and Pseudomonas aeruginosa (referred to here as Pag) and infection by Erysiphe pisi. The phenolic compounds detected were tannic, gallic, ferulic, and cinnamic acids on the basis of their retention time in HPLC. In all the treated plants, synthesis of phenolic compounds was enhanced. The induction of gallic, ferulic, and cinnamic acids was manyfold more than those in the control. Maximum accumulation of phenolic compounds was observed in plants raised from PGPR-treated seeds and infection with E. pisi. Under pathogenic stress, Pag performed better because a relatively higher amount of phenolics was induced compared with plants treated with Pf4.