Lasiodiplodia fici sp. nov., Causing Leaf Spot on Ficus altissima in China

High temperatures and the seasonality in tropical ecosystems favours plant pathogens, which result in many fungal diseases. Among these, diseases caused by Botryosphaeriaceae species are prominent as dieback, canker and leaf spots. In this research, we isolated one leaf-spot-causing Botryosphaeriaceae species from Ficus altissima leaves, which were collected in Guangzhou, Guangdong Province, China. Isolation and identification of the pathogen were based on morphological and molecular aspects. Based on multigene phylogenetic analysis of combined internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1) and beta-tubulin gene (tub2), the fungus associated with leaf spots on F. altissima is described as Lasiodiplodia fici, a novel species. Pathogenicity assays were conducted by inoculating the fungus onto detached shoots and plants under controlled environmental conditions. The results revealed that the L. fici isolates can infect the plant tissues under stress conditions by developing disease symptoms on detached shoots within three days. However, when it was inoculated onto the leaves of the host and grown in natural conditions, the progression of the disease was slow. The putative pathogen was re-isolated, and Koch’s assumptions were satisfied. This is the first report of Lasiodiplodia species causing disease on Ficus altissima. Results from the present study will provide additional knowledge on fungal pathogens associated with forest and ornamental plant species.


Introduction
Fungi are one of the key organisms in forest ecosystems. They live in the ecosystems as decomposers, mutualists and pathogens while contributing to continuous nutrient recycling and affecting biodiversity [1]. However, fungal pathogens associated with forest plants are understudied [2]. Among a wide range of pathogens, opportunistic pathogens are of great concern as they act when the environmental conditions are favourable for disease development [3][4][5]. Botryosphaeriaceae species are well-known opportunistic fungal pathogens causing devastating forest diebacks for a wide range of hosts [6,7]. Diseases caused by Botryosphaeriaceae species have higher significance as most of them infect the perennial parts of the plants [7]. Sydow and Theissen [8] introduced Botryosphaeriaceae in 1918, and the taxonomic status of Botryosphaeriaceae was controversial until it was reintegrated by Schoch et al. in 2006 [9]. However, following both morpho molecular aspects, Botryosphaeriaceae consists of 23 genera and over 100 species that are confirmed with DNA sequence data [7,[10][11][12][13][14].
In 1894, Ellis introduced the Lasiodiplodia [15] with L. tubericola as the type species. Index Fungorum (March 2022) lists 84 Lasiodiplodia epithets. However, there are 71 species accepted with molecular data [7]. Lasiodiplodia is a cosmopolitan genus which has an extensive host and geographical range. Species of Lasiodiplodia are pathogenic on economically important fruit crops [6,16]. The pathogenicity of these species is varied, even within the same host in different countries. Even within a country, a single species can infect several different hosts [4]. As an example, Lasiodiplodia theobromae is one of the major pathogens of grapevine dieback, and the pathogenicity of this species varies in different countries [17][18][19][20][21]. In addition, Lasiodiplodia species have already been reported to cause disease in a broad range of forest plants, including Acacia sp. [12], Eucalyptus [22] and Ficus [23].
The banyan tree (Ficus) is one of the genera of Moraceae and the only genus of the banyan Trib. Ficeae Trec [24]. This species is distributed from the southwest to the east and the south of China, while it is relatively rare in the rest of the country. This tree is a large tree reaching a height of 15-25 m; the bark is dark grey, the chest is about 50 cm in height with a wide crown and old trees often have rusty brown aerial roots [25]. The leaves are thin, tough, elongated and oval, with dark green surfaces and shiny and complete leaf arrangements [25]. The following are common characteristics of banyan trees: they come in pairs or are born with the axils of deciduous branches, they are yellow or reddish when mature, they are oblate, they have three basal bracts, they are broadly ovoid and they are persistent [25,26]. This plant is an important tropical forest tree as well as an ornamental plant ( Figure 1). They are widely used as bonsai and landscaping trees [26]. Therefore, the isolation and characterisation of the diseases associated with this plant have both economic and ecological significance in China. Banyan trees are highly susceptible to diseases and insect pests [27]. African snails, Ficus thrips, banyan grey silk moth, vermillion hair spot moth and banyan tree louse are the pests most widely associated with this host. Banyan trees are susceptible to several fungal diseases, including Anthracnose (Colletotrichum gloeosporiodes), a leaf spot caused by Cercospora spp. and Corynespora cassiicola, grey mould (Botrytis spp.), Phomopsis dieback (Diplodia spp.), Southern blight (Sclerotium rolfsii) and Verticillium wilt (Verticilliumalboatrum) [28,29].
In a sampling survey of banyan trees in Guangzhou City, Guangdong Province, China, a new leaf disease was observed. The objectives of this study were to (i) isolate and characterise the pathogen and (ii) confirmed the pathogenicity of isolated species. As result, a novel Lasiodiplodia species was identified by morphology and phylogenetic analysis based on ITS, tef1 and tub2. Pathogenicity assays were conducted to confirm the pathogenicity, and Koch's postulates were fulfilled. A detailed description and graphical illustration of new Lasiodiplodia species are provided.

Sampling and Isolation
During our tropical fungal survey at the South China Botanical Garden in Guangzhou City, Guangdong Province, China, a leaf spot disease was observed in banyan trees. A part of the infected leaf becomes brown and shows chlorosis. Then, the leaf spot becomes wider, and the plant tissue dies. When a leaf is severely damaged, leaf tissue becomes a grey/white transparent film ( Figure 1). In a total of 20 banyan trees, 17 were reported to have these symptoms. From these infected trees, 10 plants were randomly selected, and from each plant, 10 leaves were collected. Almost all the leaves had similar disease symptoms ( Figure 1D,E); thus, the samples were pooled, and 10 leaves were selected for tissue isolation. Tissue isolation resulted in 15 isolates, which were identical and resembled the morphology of Lasiodiplodia species. Thus, three strains (ZHKUCC 21-0125, ZHKUCC 21-0126 and ZHKUCC 21-0127) were selected for further analyses.
Culture characteristics: Colonies on PDA reached 7 cm diameter after five days at 28 • C. The upper view was filamentous, flat and turbid. The aerial hyphae were fluffy, dense and turned grey and black with time. The back became black. Note: Three isolates obtained in this study were phylogenetically closely related to L. iranensis, L. chiangraiensis and L. thailandica. However, isolates from this study formed a distinct linage with 100% ML, 100% MP and 1.0 BYPP support ( Figure 2). Morphologically, these species can be distinguished from the dimensions of their conidia (Table 1), in which Lasiodiplodia fici developed conidia with a higher L/W ratio. In terms of the nucleotide comparison, Lasiodiplodia fici (ZHKUCC 21-0125) and Lasiodiplodia chiangraiensis (MFLUCC 21-0003) differed in one base pair (bp) in ITS, seven in tef1 and two in tub2. Lasiodiplodia fici (ZHKUCC 21-0125) and Lasiodiplodia iranensis (CBS 12471) differed in one base pair (bp) in ITS, 13 in tef1-α, and CBS 124710 did not have tub2 sequence. Lasiodiplodia fici (ZHKUCC 21-0125) and Lasiodiplodia thailandic (CPC 2279) differed in one base pair (bp) in ITS and fifteen in tef1, but tub2 of these two species are identical. Based on these polyphasic approaches, we introduced Lasiodiplodia fici as a new species.

Pathogenicity Assay
In this study, sporulation of Lasiodiplodia fici was difficult to perform, even after trying different media (MEA, WA and PDA) and pine needles. Therefore, the pathogenicity test was carried out by the mycelial plug method. From actively growing cultures on PDA, mycelial plugs 50 mm in diameter and 20 mm in height were used. After 24 h, all wounded inoculated detached shoots had small lesions. Three days later, leaves fell from one infected shoot of the isolate ZHKUCC 21-0127. Then, leaf spots enlarged. Five days later, all infected leaves of three isolates had fallen off, while non-inoculated leaves did not. The disease development and progress of the isolate ZHKUCC 21-0125 were higher than the other two strains. No disease symptoms developed on any of the controls. All non-wounded samples were the same as the control leaves without any disease. Under field conditions, wounded leaves inoculated with mycelial and developed the disease slowly. After seven days of inoculation, ZHKUCC 21-0125 produced the largest lesion ( Figure 4). However, disease establishment and progress were slower on plants inoculated in their natural habitat. All diseased leaf tissues were subjected to re-isolation of the pathogen, and the species was confirmed as Lasiodiplodia fici using sequence data ( Figure 5).

Discussion
In this paper, a new Lasiodiplodia species is described from Ficus altissima in China. The fungus was shown to be pathogenic, causing leaf spots similar to the ones from which it was isolated. Correct species identification is important in plant pathology for early detection and development of management strategies [33]. Species delineation in Botryosphaeriaceae genera, such as Lasiodiplodia has been the subject of debate in recent years. Morphology and molecular phylogeny have become important criteria in this genus to resolve species [6]. In the phylogenetic analysis of this study, L. fici clusters together with Lasiodiplodi iranensis, L. chiangraiensis and L. thailandica. Together, these four species develop a distinct clade from other Lasiodiplodia species in the tree. Even though, within this cluster, these species developed independent lineages, their morphology and sequence data are very similar (Table 1). This clustering variation might be a result of the host specificity of species. Lasiodiplodia iranensis was introduced from Salvadora persica from Iran, and both L. chiangraiensis and L. thailandica were recorded from Thailand, while L. thailandica is from Mangifera indica, while L. chiangraiensis from dead wood. The new species from this study was found on Ficus altissima in China. Further studies are required to define the limits of species in Lasiodiplodia and to determine whether the four species in this clade are distinct or if they constitute variation within a single species.
In this study, we observed that L. fici becomes more aggressive and forms lesions within three days when mycelial plugs were inoculated onto the detached shoots. However, when it was inoculated on the host in its natural habitat, disease progression was slower. Thus, our results confirm the opportunistic nature of this fungal group. Isolate ZHKUCC 21-0125 was more aggressive than the other two strains, showing the variations in pathogenicity among the three strains used in this study. This suggests that Lasiodiplodia might have inter-species variation based on pathogenicity as well. Previous studies have shown that several other Botryosphaeriaceae, such as Botryosphaeria [44] and Neofusicoccum [45], as well as Lasiodiplodia [46], have high genetic diversity. However, in this study, we used the mycelial plug method, and the use of the mycelial plug method over spore suspension also leads to false-positive conclusions, in which weak pathogens can be more aggressive when supplied with a large nutrient base in the agar growth medium [4].
In the present study, one of the main problems we faced was the difficulty to sporulate this fungus. We tried different media (PDA, MEA, WA), adding pine needles to the medium as well as adding host tissues to sporulate this fungus. However, it took over three months for out observations of the fruiting bodies to confirm the morphology. This is the main reason why we did not perform pathogenicity using spore suspension, as we could not obtain spores to make 1 × 10 6 spore suspension. In addition to that, we did not observe any type of fruiting bodies on the leaf surface in this study. Up until today, it is debatable how they become pathogenic and what their infection mechanisms are. Lasiodiplodia species do not produce appressoria or any other structures to become pathogenic on the host. Thus, most studies related to Lasiodiplodia and most of the Botryosphaeriaceae taxa showed similar observations as we did [3,4,16,20]. These species are opportunistic fungal pathogens; when the host becomes stressed due to environmental conditions, they are supposed to develop the disease. Therefore, it is necessary to have natural connections to the host tissues, such as stromata or physical injury. This is the main reason for our observations in the pathogenicity essay.
China has a rich and diverse vegetation cover; thus, identifying different fungal species associated with plants is of great significance for the ecological control of plants. Moraceae represents many ecological and economically important plants, including Ficus spp. and Mulberries [47]. However, there is a limited number of studies on microfungi associated with these hosts, especially with forest plants such as Ficus. Therefore, the new species described here will provide new insight into the disease occurrence in forest plants as well as Moraceae hosts. The new species record of Lasiodiplodia in Botryosphaeriaceae can enrich domestic resources and provide a certain database which is of great significance for enriching fungal species in China.

Samples Collection and Isolation
Samples were collected from the South China Botanical Garden in Guangzhou City, Guangdong Province (E 113 • 21 56 N 23 • 11 24 ). The sample was brought into the laboratory, and the pathogen was isolated using tissue dissociation [33,48,49]. The infected tissue was thoroughly cleaned with sterile water. Then, the samples were cut with sterilized scissors into small pieces (5 mm 2 ) that included both infected and healthy tissues. After that, the leaf cuttings were washed with 75% ethanol for 30 s, 10% NaOCl for 30 s and followed by three times with sterilised water. After that, cuttings were dried on sterilized filter paper, transferred to a PDA plate and incubated for 5-7 days at 25 • C. When the mycelia appeared in the tissue samples, the purification was carried out by hyphal tip isolation. Purification was preformed 3-5 times to obtain pure cultures. All pure cultures obtained in this study and Herbarium materials (as dry cultures) were deposited in the Culture Collection and the Herbarium of Zhongkai University of Agricultural Engineering (ZHKUCC), Guangzhou City, Guangdong, China.

DNA Extraction, Amplification and Sequencing
Total genomic DNA was extracted from the sterile 7-day-old pure cultures using MagPure Plant AS Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) following the manufacturer's protocols. Three gene regions, ITS, tef1 and tub2, were used for PCR amplification of the isolated strains. The PCR mixtures consisted of 1 µL genomic DNA, 1× NH4 reaction buffer Guangzhou Meiji Biotechnology Co., Ltd., Guangzhou, China), 2 mM MgCl2, 40 µM of each dNTP, 0.2 µM of each primer and 0.5 U Taq DNA polymerase (Guangzhou Meiji Biotechnology Co., Ltd., China), in a total volume of 12.5 µL. Each gene region ITS, tef1 and tub2 was amplified and sequenced following White et al. [12], Carbone et al. [50] and Glass et al. [51], respectively. The PCR was performed in a C1000 TouchTM thermal cycler (Guangzhou Hongtu instrument Co., Ltd., Guangzhou, China), and the positive implications were observed after 1% agarose gel electrophoresis under ultraviolet light using GelDoc XR + (BIO-RAD, Hercules, CA, USA). All positive amplicons were sequenced by Tianyi (Guangzhou, China) Co., Ltd. Sequence data generated in this study were deposited in NCBI Genbank (Supplementary Table S1).
Maximum parsimony analysis was performed using PAUP (phylogenetic analysis using parsimony) v.4.0b10 [54]. Here, the heuristic search was performed with tree bisectionreconnection (TBR) branch swapping and 1000 random sequence additions. In the alignment, ambiguous regions were excluded. Then, gaps were treated as missing data. Tree stability was evaluated by 1000 bootstrap replications. The most parsimonious trees were saved after branches of zero length collapsed. Tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI) were calculated.
The best model of evolution was determined by MrModeltest v. 2.2 for each gene. Maximum likelihood analyses via RAxML v. 8.2.12 [55] were accomplished using RAxML-HPC2 on XSEDE v. 8.2.8 [55,56] using the CIPRES Science Gateway platform [57]. GTR + I + G evolution model was used with 1000 non-parametric bootstrapping iterations. MrBayes v.3.0b4 was [55] used for the Bayesian analyses. The Markov Chain Monte Carlo sampling (BMCMC) analysis was conducted with four simultaneous Markov chains. The MCMC heated chain was set with a "temperature" value and run for 1,000,000 generations. Trees were sampled at every 100th generation. From the 10,000 trees obtained, the first 2000, representing the burn-in phase, were discarded. To calculate posterior probabilities, the remaining 8000 trees were used. New data were submitted to the Faces of Fungi database [58] and Index Fungorum (http://www.indexfungorum.org (accessed on 20 March 2022). New species were described following Jayawardena et al. [59] and Manawasinghe et al. [60].

Pathogenicity Assay
The pathogenicity of the three isolates obtained in this study (ZHKUCC 21-0125, ZKUCC 21-0126 and ZKUCC 21-0127) was tested on healthy Ficus leaves. The inoculum was mycelial plugs. We did not use conidial suspension inoculation since our isolates did not produce enough conidia to make a 1 × 10 6 spore suspension. We used detached shoots and plants grown in natural habitats. Healthy leaves with the same developmental stages were selected. The leaves were surface sterilized with 75% alcohol and wounded by pricking with a sterilized needle. Another set of unwounded leaves was inoculated. Mycelial plugs 5 mm diameter were applied to both wounded and non-wounded leaves and covered with parafilm. Non-colonised PDA plugs were used in the control. There were three replicates for each isolate and control. Once the disease was developed, the pathogen was re-isolated to confirm Koch's postulates.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens11080840/s1, Table S1: GenBank accession numbers of the isolates included in the phylogenetic analysis.

Data Availability Statement:
The sequence data generated in this study are deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank). All accession numbers are given in Supplementary Table S1. of electronic Specimen Bank of plant diseases). Alan J. L. Phillips acknowledges the support from UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT, Portugal (to BioISI). Ishara S. Manawasinghe would like to acknowledge the support from Zhongkai University of Agriculture and Engineering, Guangzhou, China (KA210319288).