Longan (Dimocarpus longan) is commercially grown in many countries including China, India, Taiwan, Thailand and Vietnam (Jiang et al. 2002). In Thailand, longan growing areas cover about 188,574 ha with total production of 1,027,298 t per year. The major production areas in Thailand are located in the northern region consisting of Chiang Mai, Chiang Rai, Lamphun and Phayao Provinces (Office of Agricultural Economics 2017). Longan fruit is non-climacteric fruit and will not continue to ripen once removed from the tree (Drinnan 2004). Consequently, fruit must be harvested when their skin become yellow-brown and their flesh reaches optimal eating quality. Browning of longan fruits are associated with desiccation, heat stress, senescence, chilling injury and pest or pathogen attack (Pan 1994). The most important fruit rot disease of longan caused by fungi including Lasiodiplodia sp., Pestalotiopsis sp. and Xylaria sp. (Chang-ngern et al. 2010). In this study, we conducted morphological and phylogenetic analyses using ITS and EF1-α genes to identify the potential causal agent of longan fruit rot disease.

Longan fruit rot symptoms were observed and collected from a longan orchard during August 2018 until October 2018 at Lamphun Province, Thailand. Longan fruits were incubated in a plastic box for observation of symptom development. The fungal pathogen was isolated from the peel of longan fruit by tissue transplanting technique on potato dextrose agar (PDA) (Barnes 1968) and incubated at room temperature for three days. The fungi were re-isolated by hyphal tip isolation and stored on a PDA slant at 4 °C for further experimentation.

Samples of longan fruit rot were obtained and characterised by observation of the colour of the pericarp. The infected pericarp became brown and developed to a dark-brown and black colour (Fig. 1a). After incubation of longan fruit in a moist chamber, fungal mycelia grew on fruit and formed conidiomata (Fig. 1b). Conidia were released from conidiomata when stored in a moist chamber for three days (Fig. 1c). There were nine fungal pathogens (FRLP1-FRLP9) isolated from infected fruit.

Fig. 1
figure 1

Fruit rot symptoms on longan; a Longan fruit pericarp browning; b Conidiomata formed on pericarp (arrow); c Conidia released from conidiomata (arrow)

Full size image

The pathogenicity test of nine isolates were conducted on postharvest longan fruits according to the method of Than et al. (2008) by placing mycelial discs on both wounded (wounded with sterilised needle pricks) and unwounded longan fruits and kept at room temperature. Longan fruits with PDA discs were used as controls. The spore suspension technique was also used to compare. The symptom development was observed at 3, 5 and 7 days after inoculation.

The results showed that at three days after inoculation by mycelial disc and spore suspension, the fungus isolate FRLP1 caused the most severe fruit rot symptoms using both methods. Symptoms on wounded longan fruits, showed the exocarp and endocarp turning dark brown in colour with greyish mycelial growth around the inoculation site, progressing to cover the whole fruit in five days. During the development of the disease, conidia and conidiomata were observed on inoculated fruits while the control showed no symptoms. The isolate FRLP1 was re-isolated from infected longan fruits, fulfilling Koch’s postulates.

The fungus was cultured on PDA and incubated at room temperature for observation of colony characteristics. Fungal sporulation was induced by transferring fungal discs onto 2% water agar (WA) overlaid with sterilised grass leaf (Imperata cylindrica) as a substrate and incubated at room temperature (Suwanakood et al. 2005). Observation and measurement of conidial characteristics were conducted under a compound microscope with a digital camera (Carl Zeiss, Germany).

The colony of the fungus isolate FRLP1 was initially white with woolly aerial mycelia on PDA (Fig. 2a), then became pale grey in colour after 2 weeks of incubation. Conidiomata were produced on 2% WA overlaid with grass within 10 days (Fig. 2b). Conidiomata were uniloculated, dark brown to black in colour and appeared on the surface of the grass leaf (Fig. 2c). Fungal paraphyses were hyaline, cylindrical shape, aseptate, ends rounded and arising between conidiogenous cells (Fig. 2d). Conidiogenous cells were hyaline, cylindrical, base swollen, holoblastic, proliferating percurrently to form one or two closely spaced annellations. The conidia size was 23.7–28.2 × 12.4–14.9 μm and the shape was ellipsoidal with rounded shape at both apex and base. The immature conidia were hyaline and aseptate when released from conidiomata (Fig. 2e). Mature conidia became dark brown with middle one septate and longitudinal striations formed by melanin deposit on inner surface wall of conidia (Fig. 2f). The morphological features of the fungus isolate FRLP1 were similar to other Lasiodiplodia species. However, Lasiodiplodia species could be distinguished by size and shape of conidia and paraphyses (Abdollahzadeh et al. 2010; Alves et al. 2008; Burgess et al. 2006; Coutinho et al. 2017; Dou et al. 2017; Kwon et al. 2017; Linaldeddu et al. 2015; Machado et al. 2014; Munirah et al. 2017; Pavlic et al. 2004; Phillips et al. 2013; Trakunyingcharoen et al. 2015; Yang et al. 2017). For example the conidial size of Lasiodiplodia theobromae was 19.5–27 × 9.1–15.3 μm with ovoid shape and septate paraphyses compared to L. pseudotheobromae which conidia size were 23.5–32 × 12.7–18 μm, which were larger than L. theobromae, had more ellipsoid and did not taper as strongly towards the base (Table 1).

Fig. 2
figure 2

Morphological characteristic of the fungus isolate FRLP1; a White and woolly colony and aerial mycelium on PDA; b Conidiomata on grass (arrow); c Conidiomata formed; d paraphyses hyaline, cylindrical and aseptate (arrow); e immature conidia hyaline and aseptate; f mature conidia dark brown colour with one-septate. Bar = 10 μm

Full size image
Table 1 Morphological characteristics of Lasiodiplodia species
Full size table

Moreover, the fungus isolate FRLP1 was confirmed by molecular characterisation using PCR amplification of ITS and EF1-α genes. Genomic DNA was extracted from fungal mycelia according to the method of Myoung et al. (2009). The PCR amplification was performed by EconoTag PLUS GREEN 2X Master Mixed (Lucigen, USA) using ITS gene primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3′) (White et al. 1990) and the EF1-α gene was amplified by using primers EF1-688F (5’-CGGTCACTTGATCTACAAGTGC-3′) and EF1-1251R (5’-CCTCGAAC TCACCAGTACCG-3′) (Cruywagen et al. 2017). The PCR products were analysed by 1.7% agarose gel electrophoresis stained by RedSafe (iNtRON, South Korea).

Nucleotide sequences were directly analysed using fluorescent dye-terminator sequencing on ABI Prism™ 3730xl DNA sequencers (Applied Biosystems, Foster City, CA). All obtained sequences were analysed and aligned using BLAST and MEGA 7 software (Kumar et al. 2016), and then deposited in GenBank. The phylogenetic tree was inferred by using the Maximum Likelihood (ML) method based on the Tamura-Nei model with 1,000 replicates of bootstrap compared to previously reported L. pseudotheobromae, L. theobromae and other members in the family Botryosphaeriaceae (Table 2) which was performed in MEGA 7.

Table 2 Accession code of the sequences used in the phylogenetic analysis
Full size table

The combined ITS and EF1-α dataset of Lasiodiplodia sp. isolate FRLP1 was deposited in Thailand Bioresource Research Center (TBRC) and deposition number was TBRC10378. The combined dataset containing 1,098 characters (563 from ITS accession MK368390 and 535 from EF1-α accession MK376951) was grouped into L. pseudotheobromae clade and separated from L. theobromae clade and other members of the family Botryosphaeriaceae (Fig. 3). The nucleotides of Lasiodiplodia sp. isolate FRLP1 shared 99% identity to the reference L. pseudotheobromae isolate MHGNUF120 (accessions KY404091 and KY404090) (Kwon et al. 2017).

Fig. 3
figure 3

The maximum likelihood tree generated from the combine analysis of ITS and EF1-α dataset. Designated out group taxon is Neofusicoccum luteum. Bootstrap support values from 1,000 replications are shown in the branches

Full size image

From morphological and phylogenetic analysis, the result showed that the fungal isolate FRLP1 could be identified as L. pseudotheobromae, a member of Botryosphaeriaceae, which was commonly found as endophytes and pathogens of various plants in tropical and subtropical regions (Rosado et al. 2016). These fungi are generally regarded as opportunistic pathogens with a latent endophytic stage causing numerous diseases when the host plants are exposed to stress or favorable conditions for disease development (Slippers and Wingfiled 2007). Botryosphaeriaceae were entering the fruit through scars at the stem-end of fruit and fungi colonised and latently remained in the pericarp (Ladanyia 2008). Symptoms of the fruit rot disease cause by L. pseudotheobromae, will develop after harvest especially under high temperature and high humidity conditions (Zhang 2014).

Lasiodiplodia pseudotheobromae was reported for the first time on grapevine in Brazil as a grapevine trunk pathogen (Correia et al. 2013) and mostly found in Africa, Europe and Latin America (Adetunji and Oloke 2013). In the case of Thailand, L. pseudotheobromae has been reported to cause canker, decline, dieback, stem end rot, and fruit rot on a wide range of plants (Farungsang et al. 1992; Trakunyingcharoen et al. 2013). L. pseudotheobromae has been identified as a causal agent of inflorescence blight of longan in Puerto Rico (Serrato-Diaz et al. 2014). To our knowledge, this is the first report of L. pseudotheobromae causing fruit rot disease of postharvest longan in Thailand.