Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest water-soaked brown lesions on Syzygium samarangense in Chiang Rai, Thailand

Abstract Background Syzygium samarangense (Wax apple) is an important tropical fruit tree with high economic and nutrient value and is widely planted in the tropics or subtropics of Asia. Post-harvest water-soaked brown lesions were observed on mature fruits of ornamental wax apples in Chiang Rai Province, Thailand. A fungus with morphological characters, similar to Lasiodiplodia, was consistently isolated from symptomatic fruits. Phylogenetic analyses, based on ITS, LSU, TEF1-a and tub2, revealed that our isolates were closely related to, but phylogenetically distinct from, Lasiodiplodia rubropurpurea. New information Morphological comparisons indicated that pycnidia and conidiogenous cells of our strains were significantly larger than L. rubropurpurea. Comparisons of base-pair differences in the four loci confirmed that the species from wax apple was distinct from L. rubropurpurea and a new species, L. syzygii sp. nov., is introduced to accommodate it. Pathogenicity tests confirmed the newly-introduced species as the pathogen of this post-harvest water-soaked brown lesion disease on wax apples.


Introduction
Wax apple [Syzygium samarangense (Blume) Merrill and Perry] belongs to the Myrtaceae and was naturalised in the Philippines thousands of years ago (Lim 2012, Shen et al. 2012. As a kind of juicy tropical fruit like watermelon with economic importance, it has been commonly and widely cultivated in many Asian countries (Nesa et al. 2014). Every part of S. samarangense also has potential medicinal values (Shen et al. 2012).
Due to the fruit characteristics, such as thin peel and tender pulp with high respiratory intensity, wax apples are prone to damage by pathogens and cannot be stored for a long time (Yang et al. 2009). This causes a significant post-harvest loss. Many studies suggest that wax apple is mainly threatened by fungal diseases. For example, a new fruit rot of wax apple caused by Phytophthora palmivora was reported in southern Taiwan during the rainy periods in 1982 (Lin et al. 1984). Yang et al. (2009) andChe et al. (2015) reported Lasiodiplodia theobromae as the causal agent of black spot disease on harvested wax apple fruits. Pestalotiopsis samarangensis was isolated from the fruit rot in wax apples from markets in Thailand (Maharachchikumbura et al. 2013). Chrysoporthe deuterocubensis caused cankers on wax apple and branches in Taiwan (Fan et al. 2013).
The present study reports a new post-harvest water-soaked brown lesion disease on wax apples caused by Lasiodiplodia sp. in Chiang Rai,Thailand. Morphological and multi-locus phylogenetic analyses revealed that our strain represented a novel species. A pathogenicity test on fruits confirmed the pathogenic relationship between L. syzygii and Syzygium samarangense.

Sample collection, isolation and morphology
Rotten wax apple fruits were occasionally collected from a food market near Mae Fah Luang University in Chiang Rai, Thailand. On the third day after the wax apple fruits were collected, it was observed that there were conidiomata bulges on the surface of the fruit, white hyphae and the fruit turned black, rotted and had cytoplasmic extravasation. Diseased samples were conserved in self-sealing bags and then taken back to the laboratory and photographed. Before isolation, diseased fruits were surface disinfected with 70% ethanol for 30 s, 1% sodium hypochlorite (NaClO) for 1 min and repeatedly twice rinsed in sterile distilled water for 30 s. Pure cultures were obtained by single-conidium isolation following a modified method outlined by Chomnunti et al. (2011) and Maharachchikumbura et al. (2013). The morphology of fungal colonies was recorded following the method of Hu et al. (2007). Fungal mycelium and spores were observed under a light microscope and photographed. The holotype specimen is deposited in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). The ex-type and isotype cultures are deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC) and the Mae Fah Luang University Culture Collection (MFLUCC) in Thailand.

DNA extraction, PCR reaction and sequencing
Fungal cultures were grown on PDA at 28°C. When colonies nearly covered the entire Petri dish (90 mm diam.), fresh mycelia were scraped from the agar surface with sterilised scalpels. Genomic DNA was extracted using a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416) following the manufacturer's protocol. DNA amplification was performed in a 25 μl reaction volume following Liang et al. (2018). Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer regions and intervening 5.8S rRNA region (ITS) and LR0R and LR5 for 28S rRNA (LSU) region (Vilgalys andHester 1990, Rehner andSamuels 1994). Two protein-coding gene fragments, the β-tubulin (tub2) and translation elongation factor 1-alpha (TEF1-a) were amplified with primer pairs BT2A/ BT2B (Glass andDonaldson 1995, O'Donnell andCigelnik 1997) and EF1-688F/ EF1-986R, respectively (Carbone and Kohn 1999, Alves et al. 2008). Purification and sequencing of the PCR amplicons were done by SinoGenoMax, Beijing. The DNA sequences are deposited in the GenBank and their accession numbers are provided in Table 1. The DNA base differences of the four loci amongst our strains and ex-type or representative strains of relative taxa are shown (Table 2). T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   T   Table 1.  T   T   T   T   T   T   T   T   T   T   T   T   T   T   Table 2.
DNA base pair differences between Lasiodiplodia syzygii and L. rubropurpurea in four separate loci. = ex-type T number is 27461. Phylogenetic analyses were constructed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference methods. First, the ambiguous regions were excluded from the alignment and gaps were treated as missing data. The MP analysis was done with PAUP v. 4.0b10 (Swofford 2002), using the heuristic search option with 1,000 random taxa addition and tree bisection and reconnection (TBR) as the branch swapping algorithm. Maxtrees was set to 5000. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each tree generated. The Maximum Likelihood (ML) analysis was performed using IQ-tree (Nguyen et al. 2015, Chernomor et al. 2016. Nucleotide substitution models were selected under the Akaike Information Criterion (AIC) by jModelTest2 (Darriba et al. 2012) on XSEDE in the CIPRES web portal (Miller et al. 2010 . HKY+I was selected as the best model for ITS, for LSU, HKY+I+G, for TEF1-a, HKY+I+G and for β-tubulin, GTR+G was selected as the best model. Six Markov Chain Monte Carlo runs were launched with random starting trees for 1,000,000 generations and sampling every 1,000 generations. The first 25% resulting trees were discarded as burn-in.

Pathogenicity tests
One isolate of the new Lasiodiplodia species (GUCC 9719.1) was grown on PDA and when the cultures covered the entire surface of the Petri dish, mycelia were scraped off with a sterilised blade. Conidiomata were crushed with a glass rod to prepare a spore suspension of 1× 10 spores/ml. Pathogenicity testing was carried out on five healthy fruits of wax apple bought from the market. Inoculations were carried out in April 2020. The surface of the fruits was wiped with 70% ethanol and allowed to air-dry. Three fruits were slightly wounded by pin-pricking and 3 ml of spores suspension was sprayed on to the wound. The other two wounded fruits were maintained as control and inoculated with 2 ml of sterile deionised water. All inoculated fruits were placed in plastic bags, labelled and a high level of humidity was maintained for seven days by the addition of wet sterile cotton wool in each bag in an illuminated incubator at 28 ± 3°C. Daily observations were made on the development of disease symptoms. When fruits developed the symptoms, they were removed from the bags. Two isolates obtained from the diseased tissue were grown on PDA and then sequenced with primer pairs of the above four DNA markers to confirm the identity. 5
Culture characteristics: Conidia germinate on PDA within 24 hours at room temperature (25-30°C) with germ tubes produced from both ends of the conidia. Colonies with white fluffy mycelium on PDA, after 7 days become olivaceous-grey at the centre, white at the edge, raised, fluffy, dense filamentous.

Etymology
In reference to the host from which the fungus was first isolated. one is shown as Fig. 1. The ML and Bayesian analyses resulted in trees with similar topologies. Strains GUCC 9719.1, GUCC 9719.2, GUCC 9719.3 and GUCC 9719.4 formed an independent well-supported clade sister to Lasiodiplodia rubropurpurea (MP: 100%, ML: 100% and Bayesian posterior probability: 1) Comparison of the DNA base-pair differences between our strains and L. rubropurpurea species in four gene regions (Table  2) confirmed the presence of two species; therefore, a new species is introduced for those isolates from wax apple.

Pathogenicity test on the fruits of wax apple
At the third day after inoculation, water-soaked areas with a few white hyphae began to appear on all inoculated fruits similar to the naturally-infected wax apples ( Fig. 2a and Fig.  3a). The water-soaked symptom of diffusion with abundant hyphae producing mycelium further appeared on inoculated Syzygium samarangense fruits after five days (Fig. 3b). At the 7th day after inoculation, the symptoms spread throughout the fruit (Fig. 3c), together with many white mycelia and more hyphae accompanied by cytoplasmic exosmosis. The control fruits (Fig. 3d) did not show any symptom. The fungi were re-isolated from the lesions of inoculated wax apple fruits and the re-identified (GUCC 9719.3 and GUCC 9719.4) sequencing four gene regions.

Discussion
This study revealed a new species of Lasiodiplodia, L. syzygi from rotting fruits of Syzygium samarangense. Phylogenetic analyses, based on ITS, LSU, TEF1-a and tub2, showed that it is phylogenetically closer to L. rubropurpurea. Comparisons of DNA basepair differences in the four loci, as well as morphological differences, confirmed the novelty of this species. The fungus was proved to be pathogenic and, therefore, it is the causal agent of the post-harvest water-soaked brown lesions on wax apple.
Wax apple (Syzygium samarangense) is known to be affected by many fungal pathogens that often cause economic losses. These include Colletotrichum gloeosporioides (Udayanga et al. 2013) and Lasiodiplodia theobromae which was the causal agent of black spot disease (Che et al. 2015), Pestalotiopsis spp. and Phytophthora spp. The fruit disease of the current study did not show any typical symptoms of black spot caused by L. theobromae. Furthermore, the pink or orange spore masses, typical of anthracnose caused by C. gloeosporioides or epidermal to superficial, acervular conidiomata reported by Maharachchikumbura et al. (2013) for Pestalotiopsis, were not seen in the current study. The fruit rot caused by Phytophthora spp. spread more rapidly (only 2 or 3 days up to a whole fruit) and results in a sour taste on fruits. However, the L. syzygii needed about seven days to completely rot the fruit and did not cause any sour taste in the fruits. Thus, the study reports a new disease on wax apple.
Lasiodiplodia resides in Botryosphaeriaceae, Botryosphaeriales (Hongsanan et al. 2020) and comprises several species known to cause important or potentially important diseases on woody hosts, mostly in the tropics or sub-tropics (Phillips et al. 2019). Very few species of this family appear to be host-specific (Dissanayake et al. 2016). In south-western China and adjoining areas, agriculture and forestry play an important role in the local economy, which might facilitate the spread of this wax apple disease. Thus, research needs to focus on the occurrence of this newly-discovered pathogen in other economically-important plants and in other locations, as well as how to manage it by biological or chemical control approaches. It is also remarkable to find a new disease on such an important commercial fruit indicating that there are numerous new taxa to be discovered in Thailand  and Botryosphaeriaceae ).