Colletotrichum Species Causing Anthracnose of Rubber Trees in China

Anthracnose caused by Colletotrichum is one of the most severe diseases of Hevea brasiliensis. However, research on the diversity and geographical distribution of Colletotrichum remains limited in China. In this study, we investigated the phylogenetic diversity of Colletotrichum isolates associated with symptomatic tissues of H.brasiliensis from four provinces of China (Hainan, Guangdong, Guangxi, and Yunnan). Based on multi-locus phylogenetic analyses and phenotypic characteristics, five species were distinguished, including two known species (C. fructicola, C. siamense), one novel species of C. gloeosporioides species complex (C. ledongense), and two novel species of C. acutatum species complex (C. bannanense and C. australisinense). Of these, C. siamense and C. australisinense have been recognized as major causative agents of anthracnose of H. brasiliensis.


Results
Multilocus-based phylogenetic analysis. We collected 62 isolates of Colletotrichum spp. from diseased leaves of Hevea brasiliensis from the main rubber tree growing regions in China, and identified them based on phylogeny and morphological characteristics. Based on the BLAST search results on the NCBI database with the ITS sequences, all Colletotrichum isolates in this study were preliminarily allocated to the following species complexes: 40 isolates belong to the C. gloeosporioides species complex and 22 isolates belong to C. acutatum species complex.
The phylogram in Fig. 1 summarizes the isolates in the C. gloeosporioides species complex. The combined alignment (ITS, TUB2, CAL, ACT, GAPDH, CHS-1, and GS) contained 87 sequences, including C. boninense (CBS 123755) as an outgroup and 3,383 characters including gaps. The gene boundaries in the alignment were: ITS: 1-493, TUB2: 494-1165, CAL: 1166-1817, GAPDH: 1818-2067, ACT: 2068-2312, CHS-1: 2313-2539, GS: 2540-3383. For Bayesian analysis, The results of MrModeltest recommended a GTR + I + G model with inverse gamma distributed rate for ITS and CHS-1, a TN93 + I model for TUB2, a HKY + I model for GAPDH, a GTR + G model with gamma distributed rates for CAL and GS, a K2 + G with gamma distributed rates model for ACT. The Bayesian consensus tree (not shown) confirmed the tree topology of the maximum likelihood tree. Isolates from rubber trees in the C. gloeosporioides complex clustered in three clades ( Fig. 1): ten isolates clustered with the ex-type isolates of C. fructicola, 28 isolates clustered with the ex-type isolates of C. siamense, while two isolates formed a distinct clade (posterior probability = 0.93) most closely related to C. syzygicola and C. cordylinicola. Figure 2 demonstrates the phylogenetic relationships isolates in the C. acutatum species complex. The concatenated alignment (ITS, TUB2, ACT, GAPDH, and CHS-1) contained 58 isolates, with C. orchidophilum (CBS 632.80) as outgroup. The dataset comprised 1, 649 characters including the alignment gaps. The gene boundaries in the alignment were: ITS: 1-478, TUB: 479-961, GAPDH: 962-1197, ACT: 1198-1417, CHS-1: 1418-1649. For Bayesian analysis, The results of MrModeltest recommended a T92 + G model with gamma distributed rate for ITS, a HKY + G model with gamma distributed rates for TUB2, GAPDH, ACT. a TN93 + G model with gamma distributed rate for CHS-1. The maximum likelihood tree confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. Isolates from rubber trees in the C. acutatum complex clustered in two clades (Fig. 2). Nineteen isolates formed a distinct clade (posterior probability = 0.93) most closely related to C. cairnsense. The remaining three isolates also formed a distinct clade (posterior probability = 0.93) most closely related to C. laticiphilum.

Pairwise homoplasy index (PHI) test.
A pairwise homoplasy index test using a 6-gene dataset (ACT, CAL, GAPDH, GS, ITS, and TUB2) was performed to determine the recombination level between C. ledongense and the closely related species C. syzygicola. No significant recombination events were detected between C. ledongense and C. syzygicola (Φw = 1) (Fig. 3).
A 5-locus concatenated dataset (ACT, GAPDH, CHS, ITS, and TUB2) was used to determine the recombination level between C. bannaense and the closely related species C. laticiphilum, as well as between C. australisinense and C. cairnsense. No significant recombination events were detected in the PHI test.
Pathogenicity tests. All tested isolates from symptomatic rubber tree leaves were pathogenic to rubber tree leaves. Leaves of the control plants inoculated with sterile water did not develop any symptoms 7 days post-inoculation. The inoculated Colletotrichum isolates could be re-isolated from the periphery of these lesions, thereby fulfilling Koch's postulates. The pathogenicity tests (Fig. 4) demonstrated that all tested isolates were moderate or highly aggressive on Wenchang11 and 7-33-97 clone. C. siamense in the C. gloeosporioides species complex, and C. australisinense in the C. acutatum species complex were aggressive on all test rubber tree clone. However, C. fructicola was avirulent towards IAN873 clone. C. ledongense was avirulent towards both test rubber clone IAN873 and PR107, but appeared to be the highest aggressiveness on Wenchang11. C. bannaense appeared to be only weak and medium aggressive on IAN873 clone, but avirulent towards PR107. These results indicated that rubber tree Clone Wenchang11 and 7-33-97 are susceptible to Colletotrichum species.
Taxonomy. Based on the multi-locus phylogeny and morphological characteristics, the 62 isolates from H. brasiliensis were identified as five species of Colletotrichum, including three new species and two species newly recorded from Hevea brasiliensis in China (C. siamense and C. fructicola). Etymology. Named after the collection site, Xishuangbanna city, Yunnan province, China.
Etymology. Named after the collection site, Ledong city, Hainan province, China.

Discussion
Colletotrichum gloeosporioides was considered as the major species responsible for Colletotrichum leaf disease of rubber trees 10,40 , until C. acutarnm was identified from CLD lesions on Hevea brasiliensis using ITS sequence. Jayasinghe et al. reported that the majority of strains examined from Sri Lanka belong to C. acutatum 2 , and Saha et al. reported this species from India as well. Saha et al. revealed that C. acutatum causes the raised spot symptom, while C. gloeosporioides causes both anthracnose and papery lesions on Hevea leaves in India 3 . In the present study, we found that the fungal pathogen responsible for CLD of rubber trees induces four different disease symptoms: the above mentioned raised spots (caused by species of the C. acutatum species complex), anthracnose, papery lesions (caused by species of the C. gloeosporioides species complex), and dark brown shrinking lesions caused by species of the C. acutatum species complex on young leaves. Referring to the CLD symptoms described by Liu et al. on rubber trees and the morphological character of the causal organism 4 , we suggest that C. acutatum (s. l.) was already one of the Colletotrichum species causing CLD on rubber trees at that time in China, and wrongly identified as C. gloeosporioides, although it was first reported in 2008 9 .
Previous studies of Colletotrichum species causing CLD disease of rubber trees used morphological, culture characterizations, and ITS, which restrains identification to species complexes rather than individual species [1][2][3]10 .
Colletotrichum gloeosporioides and C. acutatum were identified as the two main species responsible for CLD on rubber trees. Following the use of multi-gene phylogenetic analysis, the polyphasic method for studying the genus Colletotrichum significantly changed the classification and species concepts. The combinations of different gene regions were recommended and delineated individual species into distinct species complexes with distinct species [16][17][18][19][20][21] . Based on multi-gene phylogenetic analysis, Damm et al. described new species in the C. acutatum species complexes as Colletotrichum laticiphilum based on TUB2, GAPDH, and CHS-1 sequences, and most differentially with TUB2 17 . Using this approach, Hunupolagama et al. revealed that C. laticiphilum, C. acutatum, C. citri, C. nymphaeae, and C. simmondsii belonging to the C. acutatum species complex, were causative agents of CLD on rubber trees in Sri Lanka 27 . The current study represents the first survey of Colletotrichum species associated with anthracnose of rubber trees in China using a multi-locus phylogenetic approach. The most striking finding of this study is the absence of the C. gloeosporioides and C. acutatum which were previously reported to be the main causal agents of rubber tree anthracnose. However, C. siamense, C. fructicola, and C. ledongense, 3 members of the C. gloeosporiodes species complex, were newly associated with rubber tree anthracnose. In addition, two new species of C. acutatum species complex, C. australisinense and C. bannaense were discovered. These newly recorded species may have been previously identified as C. gloeosporioides or C. acutatum. When comparing China with Sri Lanka, species of C. acutatum species complex on rubber trees are completely different. Colletotrichum siamense in the C. gloeosporioides species complex, and C. australisinense in the C. acutatum species complex are the predominant species in China, in contrast to C. simmondsii in the C. acutatum species complex in Sri Lanka. Baroncelli et al. proposed the Colletotrichum acutatum species complex as a suitable model system to study evolution and host specialization in plant pathogens 41 . Colletotrichum bannaense, C. australisinense, and C. laticiphilum were found in the same host, viz., Hevea brasiliensis. Colletotrichum bannaense is most closely related to C. laticiphilum in our phylogenetic analysis. Colletotrichum bannaense shows limited geographical distribution and is only found in the Yunnan province of China. However, C. laticiphilum is mainly distributed in South Asia, Southeast Asia, and South America, (i.e., Sri Lanka, India, and Colombia) and shows a worldwide geographic distribution 17,27 . Colletotrichum australisinense is relatively distantly related to both of the above species in phylogenetic analysis, mainly distributed in Hainan, Guangdong, Guangxi, and Yunnan provinces of China. It is similar to C. laticiphilum in ITS, TUB2, ACT, and CHS-1 sequences, but there are many differences in the GAPDH sequence (15 bp/ 220 bp). The above mentioned three species are suitable for a study of gene family evolution on a fine scale to uncover evolutionary events in the genome which are associated with the evolution of phenotypic characters important for fungal plant pathogens.
Prior to the molecular era, morphological characters, such as color and growth rate of the colonies, presence or absence of setae, and size and shape of conidia and appressoria, formed the basis for studying the taxonomy of Colletotrichum species 7,13 . In this study, we report significant differences in growth temperatures of Colletotrichum species. At 37 °C, C. bannaense displayed no growth. The other four species grew, but growth rate displayed significant differences on PDA after 7 days (C. siamense, 31-45 mm; C. fructicola, 14-21 mm; C. ledongense, 9-11 mm; C. australisinense, 8 mm). We suggest that growth temperatures may be a valuable physiological criterion for the delineation of species in the genus.

Materials and Methods
Collection and isolates. Diseased leaves of rubber trees (Hevea brasiliensis) showing different symptoms (anthracnose, papery lesions, raised spot and shrinking lesions) were collected from 18 fields of four provinces in China (Hainan, Guangdong, Yunnan, and Guangxi). Plant pathogenic fungi were isolated from leaf spots using both single spore and tissue isolation methods. Single spore isolation following the protocol of Wang et al. was adopted for collections with visible foliar sporulation 42 . The procedure described by Niu et al. was used for tissue isolation and single spore cultures 36 . These pure cultures were stored in sterilized water in Eppendorf tubes at 4 °C and stock cultures were stored in PDA slants at 4 °C in the dark. The ex-type living cultures of new species from this study were deposited in the China General Microbiological Culture Collection centre (CGMCC).

Morphological analysis.
Agar plugs (5 mm diameter) were taken from the periphery of actively growing 5-days-old cultures and transferred to the center of 9 cm Petri dishes containing potato dextrose agar (PDA) and synthetic nutrient-poor agar medium (SNA). Cultures were incubated at 25 °C and 37 °C in the dark. The shapes and sizes of 30 conidia, conidiophores, and appressoria were recorded on PDA at 25 °C after 7 days using methods SCIEnTIfIC REPORTS | (2018) 8:10435 | DOI:10.1038/s41598-018-28166-7 described by Liu et al. 23 . SNA was used for observation of the newly described species. Interspecific difference of colony growth was observed at 37 °C. DNA extraction, PCR amplification, and sequencing. Fungal isolates were grown on PDA for 7 days. Mycelia were collected in a sterile centrifuge tube and stored at −80 °C for DNA extraction. Genomic DNA was extracted from all isolates using a modified CTAB protocol as described in Stewart et al. 43 . In the multi-locus analysis of C. gloeosporioides species complex, seven loci, including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), beta-tubulin (TUB2), calmodulin (CAL), a intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), chitin synthase 1 (CHS-1), and glutamine synthetase (GS) gene, were amplified and sequenced using the primer pairs ITS1 + ITS4 44 , T1 45 + Bt-2b 46 , CL1C + CL2C 21 , GDF1 + GDR1 47 , ACT-512F + ACT-783R 48 , CHS-79F + CHS-354R 48 , and GSF1 49 + GSR2 21 , respectively. Five loci (ACT, GAPDH, ITS, TUB2, and CHS-1) were used for the multi-locus analysis of Colletotrichum acutatum species complex.
Phylogenetic analysis. Sequences of ex-type and authentic strains from several contemporary phylogenetic studies 17,21,23,24,28,29,31,32,34,35,[50][51][52] were obtained NCBI GenBank. The dataset was assembled and manually adjusted using MEGA v. 6.0. All gaps were treated as missing data. Nucleotide substitution models were generated using MrModeltest v. 3.6 53 . A maximum likelihood phylogenetic analysis of the dataset was performed with raxmlGUI1.5 54 . Bayesian Inference analyses were performed using MrBayes v. 3.2 55  Genealogical concordance phylogenetic species recognition analysis. Phylogenetically related but ambiguous species were analyzed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. 58 . The PHI test was performed in SplitsTree4 59,60 in order to determine the recombination level within phylogenetically closely related species using a 6-locus concatenated dataset (ACT, CAL, GAPDH, GS, ITS, and TUB2) in C. gloeosporioides species complex, and using a 5-locus concatenated dataset (ACT, GAPDH, CHS, ITS, and TUB2) in the C. acutatum species complex. If the pairwise homoplasy index results were below a 0.05 threshold (Фw < 0.05), it was indicative for significant recombination present in the dataset. The relationship between closely related species was visualized by constructing a splits graph.
Pathogenicity. All strains were selected for pathogenicity testing: Healthy and non-wounded copper brown leaves, collected from the clone Wenchang11, 7-33-97, IAN873, and PR107, were washed with tap water and disinfected in 1% sodium hypochlorite for 3 min. Disinfected leaves were washed three times with sterilized water and then dried on the bench top. Fungal conidia were harvested by flooding 4-day-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 1 × 10 6 conidia/ml. Then, 20 μL conidial suspensions were dropped onto intact, detached copper brown rubber tree leaves. Leaves inoculated with sterile water were used as controls. Inoculated leaves placed on moist tissue paper were maintained in a humidified chamber, incubated at 28 °C, and monitored daily for lesion development. Finally, conidia of each strain were collected from diseased leaves and cultured on a new PDA plate. These were then checked for morphological characteristics to confirm Koch's postulates.