First report of Berkeleyomyces basicola causing mango root rot and decline in India.

Mango wilt has been a serious constraint in mango (Mangifera indica L.) production in several countries including India (Shukla et al. 2018). Although, several fungal pathogens have been reported associated with the disease, species of Ceratocystis, Verticillium and Lasiodiplodia have been found predominantly responsible for the wilt (Shukla et al. 2018). A twenty-seven-year old mango tree cv. Dashehari at Rehmankhera, Lucknow, Uttar Pradesh, India suffered sudden wilt (Fig. 1A) during February 2020. Though, symptoms were similar to Ceratocystis wilt, no gummosis was observed on trunk or branches which occurred in the majority of Ceratocystis fimbriata infected trees. The infected roots of the wilted tree exhibited dark brown to black discoloration in woody portions (Fig. 1B). Severely affected roots were completely rotten. Similar symptoms of root infection were observed in an additional 16 declining trees within an orchard of 120 trees total (Fig. 2). The infected hard wood samples from live roots of 16 declining and one wilted trees were utilized for isolation by placing stem tissue of discolored and normal colored tissue on surface sterilized fresh carrot discs placed in a moisture chamber (Fig. 1C) for 10 days. Out of 17 tree samples, isolates of Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong, M.J. Wingf. (Nel et al. 2018) obtained from 1 wilted and 9 declining trees were transferred to and maintained in pure culture on potato dextrose agar. Isolates were grown for 7 to 10 days at 23±1 °C temperature in the dark. The isolates were characterized by a greyish black compact mycelial colony (Fig. 1D). Two types of spores, endoconidia (phialospores) and chlamydospores (aleuriospores or amylospores) were observed under microscope. The endoconidia were hyaline, cylindrical in shape with 10 to 42 × 3 to 6 μm (n=50) in size (Fig. 1E). Chains of dark colored chlamydospores (3 to 7 spores in chain) of 24 to 52 × 10 to 12 μm (n=50) size were apparent (Fig. 1E&F). Molecular identification of the fungus isolated from the wilted tree was established by amplifying the ITS1-5.8 rDNA-ITS2 region of fungal genomic DNA and the set of ITS primers (ITS 1 and ITS4) (White et al. 1990) followed by sequencing. The sequence has been submitted to the NCBI database vide accession number MT786402. The present isolate (MT786402) shared >99 percent nucleotide similarity with other B. basicola isolates. The phylogenetic tree was constructed using the ITS1-5.8 rDNA-ITS2 sequences of other B. basicola isolates and other Thielaviopsis spp., C. fimbriata, Chalaropsis thielavioides through neighbor joining method using MEGAX software (Fig. 3) (Kumar et al. 2018). The present isolate formed a distinct cluster along with other B. basicola isolates in a separate clade. Koch's postulate was performed under a transparent polycarbonate sheet roof net house at 14.4 and 42.2 °C minimum and maximum temperatures, respectively. A 100 ml macerated culture suspension consisting of 1000 chlamydospores and endoconidia per ml suspension was inoculated in the rhizosphere of mango seedlings planted in sterilized soil filled in earthen pots, using ten replicates for inoculated and uninoculated plants. Symptoms of necrotic root tissue were observed 90 days after inoculation and were consistent with those observed in the field. The same fungus was re-isolated from infected roots and identity was confirmed. All control plants remained symptom-free and B. basicola was not isolated from the roots. Thus, we conclude that B. basicola is capable of causing root rot disease of mango. To the best of our knowledge this is the first report of B. basicola causing mango root rot and decline across the globe, hitherto unreported. The extent of the root necrosis symptoms associated with mature mango trees demonstrates the potential virulence of B. basicola, although its pathogenicity risk on healthy mature trees is still unknown. However, the possibility of severe losses to the mango industry in world number one mango producer country, India cannot be ruled out, if found widespread.