Mango is one of the most economically important fruit crops in the Philippines, providing a livelihood to about 2.5 million local farmers and contributing an estimated Php 41.6 billion to the Philippine national economy primarily through exportation (Fernandez 2011). While the Philippines possesses the ideal climatic conditions for mango production, its production remained stagnant, with a meager increase in production in the past decade. Fresh mangoes face difficulty penetrating the markets of countries with strict sanitary and phytosanitary requirements primarily due to the prevalence of postharvest diseases (Fernandez-Stark 2017). It is estimated that postharvest losses of the mango industry in the Philippines are estimated at 2 to 33% (Mopera 2016). The marketability and quality of mangoes are severely reduced upon the formation of unsightly postharvest disease symptoms. Stem-end and fruit rot are considered the second most damaging mango disease after anthracnose. It occurs only in ripe fruits, wherein the formation of brown to black lesions typically starts at the stem end. The lesions progress with the affected skin remaining firm and the flesh actively necrosing (Prakash and Misra 2001). Diverse fungal species can cause fruit rot, including Dothiorella dominicana, Lasiodiplodia theobromae, Phomopsis mangiferae, and Alternaria alternata. However, L. theobromae is known to be the most common and globally found fruit rot and stem-end rot-causing pathogen (Galsurker et al. 2018).

A survey of mango (Mangifera indica var. Carabao) postharvest diseases in Southern Luzon, Philippines, led to the isolation of a fungal pathogen causing a characteristic fruit rot - water-soaked brown lesions with white mycelia protruding from the wounded area in the late stage of the infection (Fig. 1A). The pathogen was isolated using the protocol of Nelson (2008) and was grown in PDA medium at 30oC under dark conditions. It was observed to produce very thin thread-like white mycelia that turned white to dark grey with age (Fig. 1B). The immature conidia were ovate, hyaline, and aseptate. The morphological characteristics, such as conidial shape and mycelial colour (Fig. 1B), were similar to the description of Neofusicoccum mangiferae by Slippers et al. (2005). In 2012, it was first reported to cause mango fruit rot in Taiwan (Ni et al. 2012) while in 2014, it was first reported to be affecting mango rachises and inflorescences in Puerto Rico (Serrato-Diaz 2014), and in 2015, it was first reported as the causative agent of grapevine dieback in 2015 in China (Dissanayake et al. 2015). Representative cultures were deposited in the Philippine National Collection of Microorganisms (PNCM) of the National Institute of Molecular Biology and Biotechnology (BIOTECH) under the accession ID: BIOTECH 3448.

Fig. 1
figure 1

(A) Infected mango fruit from which isolations were made, (B) 10-day-old culture of Neofusicoccum mangiferae BIOTECH 3448, left plan view, right reverse view

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The identity of BIOTECH 3448 was confirmed by sequencing ITS at 62oC annealing temperature (White et al. 1990). ITS sequencing yielded a 552 nucleotide sequence (deposited in GenBank under accession ID: MK911891). The sequence was compared to the NCBI (https://www.ncbi.nlm.nih.gov/) database using BLAST (Basic Local Alignment Search Tool). The sequence was found to have 99.81% similarity to the reference sequence of N. mangiferae strain CMW 7797 (NR_152945). To further characterize the sequence, representative ITS sequences of other Neofusicoccum genus members were retrieved from the GenBank database and included in a phylogenetic analysis. All sequences were retrieved from the database on February 17, 2023. It was compared with those of other type strains, including N. hellenicum, N. parvum, and N. ningerense. Phylogenetic analysis was conducted using the Maximum Likelihood model of Molecular Evolutionary Genetics Analysis Version 7.0 (MEGA7) (Kumar 2016), accompanied by bootstrap analysis with 1,000 replications. The fungal pathogen (BIOTECH 3448) clustered together with the reference N. mangiferae species forming a clade supported by a 90% bootstrap value, further confirming the identity of the fungal pathogen as N. mangiferae (Fig. 2). In addition, to further corroborate the initial results, the partial alpha subunit of the translation elongation factor 1 (TEF1) was amplified using the primers EF1-728 F/986R (Carbone and Kohn 1999). It was revealed that it is 99.63% similar to the TEF1 sequence of N. mangiferae type strain CMW 7797 (DQ093220).

Fig. 2
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Phylogenetic tree based on the partial internal transcribed spacer (ITS) of BIOTECH 3448 and other member species of Neofusicoccum genus. Values at the nodes indicate bootstrap values. Colletotrichum gloeosporioides served as the outgroup

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As species of Neofusicoccum are generally considered opportunistic, pathogenicity testing was conducted using the protocol of Sun et al. (2008). Mango fruits purchased from the local market were sorted based on size and absence of physical injuries or infections. Only mango fruits of similar size and with no early signs of disease or injuries were used for the pathogenicity testing. Fruits were disinfected using 2% sodium hypochlorite for 5 min, washed twice with sterile distilled water, then air dried under aseptic conditions. Next, six wounds (3 mm deep and 3 mm wide) per fruit were created using a flame-sterilized scalpel. An isolate of BIOTECH 3448 grown in PDA for one week was scraped, and approximately one loopful of mycelia was used in inoculating each wound. A negative control set-up was made by inoculating sterile distilled water, respectively. All samples were placed in a sterile autoclavable plastic containing moistened cotton and incubated at room temperature for 9–15 days in a 1-h light/dark cycle. The pathogenicity testing was conducted twice with two replicates each. Results indicate identical symptoms compared to the symptoms observed in the field. However, nine days after inoculation, inoculated mangoes were covered with black lesions with mycelia emerging from the origin of inoculation (Fig. 3). The pathogen was successfully reisolated after pathogenicity testing, including identity verification using ITS sequencing, which satisfied Koch’s postulates. There has been no report of the presence of N. mangiferae in the Philippines. This is the first report of the presence of N. mangiferae infecting mango fruits in the Philippines.

Fig. 3
figure 3

Pathogenicity test of mango fruit (Mangifera indica var. carabao) inoculated with Neofusicoccum mangiferae and uninoculated mango fruit

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