Next Article in Journal
Regulation of Sixth Seminal Root Formation by Jasmonate in Triticum aestivum L.
Next Article in Special Issue
Genetic Diversity and Pathogenicity of Botryosphaeriaceae Species Associated with Symptomatic Citrus Plants in Europe
Previous Article in Journal
Effect of Three Nanoparticles (Se, Si and Cu) on the Bioactive Compounds of Bell Pepper Fruits under Saline Stress
Previous Article in Special Issue
Identification of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Brief Report

Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China

1
Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan 430070, China
2
Key Lab of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
Plants 2021, 10(2), 218; https://doi.org/10.3390/plants10020218
Submission received: 5 December 2020 / Revised: 21 January 2021 / Accepted: 21 January 2021 / Published: 23 January 2021
(This article belongs to the Special Issue Citrus Fungal and Oomycete Diseases)

Abstract

:
The Nanfengmiju (Citrus reticulata cv. Nanfengmiju), a high-quality local variety of mandarin, is one of the major fruit crops in Jiangxi Province, China. Citrus melanose and stem-end rot, two common fungal diseases of Nanfengmiju, are both caused by Diaporthe spp. (syn. Phomopsis spp.). Identification of the Diaporthe species is essential for epidemiological studies, quarantine measures, and management of diseases caused by these fungi. Melanose disease was observed on Nanfengmiju fruit in Jiangxi Province of China in 2016. Based on morphological characterization and multi-locus phylogenetic analyses, three out of 39 isolates from diseased samples were identified as D. passifloricola. Since these three isolates did not cause melanose on citrus fruit in the pathogenicity tests, they were presumed to be endophytic fungi present in the diseased tissues. However, our results indicate that D. passifloricola may persist as a symptom-less endophyte in the peel of citrus fruit, yet it may cause stem-end if it invades the stem end during fruit storage. To the best of our knowledge, this is the first report of D. passifloricola as the causal agent of the stem-end rot disease in Citrus reticulata cv. Nanfengmiju.

1. Introduction

As the earliest citrus producer in the world, China has over 4000 years of history of citrus cultivation. The citrus industry of China covers more than 20 provinces [1]. Recently, the cultivation area reached 2.5 million ha, and the production was about 38 million tons [2]. Melanose, one of the most common fungal diseases of citrus worldwide [3,4], generally occurs in many citrus-growing regions of China, such as Chongqing, Fujian, Guangdong, Guangxi, Hunan, Jiangxi, Shaanxi, Shanghai, Zhejiang, and so on [5,6,7]. All commercial citrus varieties are susceptible to melanose. Typical symptoms of melanose disease are small, discrete, sunken spots with a yellowish, reddish-brown to black color. Symptoms begin as tiny pustular lesions, then, pustular lesions disappear and become hardened gummed areas with a sandpaper-like surface [3,8,9]. Diaporthe spp. (syn. Phomopsis) are the causal agents of melanose and can also cause stem-end rots on fruit during the storage period. Since 95% of citrus is consumed as fresh fruit in China, melanose and stem-end rots diseases reduce the economic value of this crop seriously.
At present, Diaporthe citri is the only known causal agent of citrus melanose disease in the world. The species was first found as the causal agent of stem-end rot of citrus fruit in Florida, USA [10]. After that, D. citri was also associated with melanose of citrus fruit, leaves, and shoots and gummosis of perennial branches worldwide [11,12,13,14]. All Citrus species are susceptible to it [4]. In China, D. citri has been isolated in many citrus growing regions, including Guangxi [15], Guangdong [16], Fujian [17], Jiangxi [18], Sichuan [19], Taiwan, Guizhou, Yunnan, Hubei, Jiangsu [20], Zhejiang, and Shanghai [5]. In addition to D. citri, D. citriasiana, and D. citrichinensis have also been found to be pathogens of stem-end rot of citrus fruit in China. D. citriasiana distributes in Shaanxi and Jiangxi Provinces, China. D. citrichinensis is only found in Shaanxi Province, China [5].
The genus Diaporthe, belonging to the Diaporthaceae, Diaporthales, Ascomycota, shows high species diversity. Many species are harmful plant pathogens and exhibit broad host ranges [21,22,23,24,25]. A single species of Diaporthe is commonly associated with different hosts, while a single host may be infected by multiple species of Diaporthe [26,27]. Up to now, over 1020 names “Diaporthe” and around 950 names of the asexual morph “Phomopsis” are recorded in MycoBank lists (accessed July, 2020; http://www.mycobank.org), of which more than 100 Diaporthe or Phomopsis species have been reported in China [5,6,28,29,30,31,32,33]. In the past, morphological characteristics and host associations were the basis of the identification of Diaporthe species. The typical morphological characteristics of Diaporthe spp. are immersed ascomata and erumpent pseudostroma with elongated perithecial necks for the sexual morph [34] and black conidiomata with dimorphic conidia (alpha and beta conidia) for the asexual morph [35]. In some species, there are intermediates between alpha and beta conidia named gamma conidia [36]. However, morphological traits tend to vary in response to changes in environmental conditions, thus they may not be sufficiently reliable for the identification of Diaporthe at the species level [37]. With the development of molecular identification, multi-locus phylogenies combined with morphological characterization have been developed to identify Diaporthe species [21,24,30,37,38]. Nuclear ribosomal internal transcribed spacer regions (ITS), beta-tubulin gene (TUB), translation elongation factor 1-α gene (TEF), histone-3 gene (HIS), and calmodulin gene (CAL) are commonly employed markers to identify Diaporthe species [21,31,37,38].
The Nanfengmiju (Citrus reticulata cv. Nanfengmiju), a high-quality local variety of mandarin, is one of the major fruit crops in Jiangxi Province. The accumulation of dead citrus wood results in the increase of fungal inocula in orchards of Jiangxi. Currently, melanose has become the major fungal disease of Nanfengmiju, immensely reducing the commercial value of citrus production. The identification of Diaporthe spp. is essential for the epidemiology, quarantine measure, and management of citrus melanose and stem-end rot diseases. In this study, morphology, and sequences of five loci (ITS, TUB, TEF, HIS, and CAL) were employed to identify and characterize Diaporthe species on citrus fruit.

2. Results

2.1. Morphological Characterization of D. passifloricola

Thirty-nine isolates (Supplementary Figure S1), were obtained from 10 diseased citrus fruit with typical melanose symptoms. Of these, three isolates preliminarily identified as D. passifloricola with the ITS marker were designated as NFIF-3-11, NFIF-3-19, and NFIF-3-21, and sorted out for further study. All three isolates showed the same culture characteristics on four kinds of media. After three days of incubation, the diameter of colonies on potato dextrose agar (PDA), malt extract agar (MEA), corn meal agar (CMA), and oatmeal agar (OMA) media reached 53–69 mm ( x - = 60), 51–63 mm ( x - = 57), 43–56 mm ( x - = 51), and 44–51 mm ( x - = 49), respectively. The colonies were fluffy with smooth margins. After 30 days of incubation, the surface of colonies on PDA, CMA, and OMA media had a uniform whitish appearance, whereas the colony grown on MEA presented yellowish patches (Figure 1).
Sporulation was induced on PDA and 1/10 PDA medium supplemented with sterilized pine needles (PNA). Conidiomata (pycnidia) were solitary to aggregated, black, sub-globose to globose, up to 200 µm in diameter. Conidial masses were hyaline to creamy, yellowish. Conidial droplets were exuded from central ostioles. Pycnidial walls consisted of 3–6 layers, medium brown (Figure 2). All three isolates produced dimorphic conidia. Alpha (α) conidia were (6.9–) 7.2–8 (–8.2) µm × 3.1–4.1 µm (x = 7.6 × 3.6 µm², n = 30), aseptate, bi-guttulate, hyaline, fusoid, and ellipsoid, smooth, apex subrounded to rounded, base subtruncate to truncate. Beta (β) conidia were (22.3–) 23.7–26.6 (–27.9) µm × 1–2 µm (x = 25.1 × 1.5 µm², n = 30), aseptate, slightly curved to spindle-shaped, smooth, base truncate. Gamma (C) conidia were not observed.

2.2. Pathogenicity Test

In pathogenicity tests, non-wounded Nanfengmiju fruit were used to test the ability of three isolates to cause citrus melanose and stem-end rot diseases. At 15 days after inducing melanose symptom, three isolates of NFIF-3-11, NFIF-3-19, and NFIF-3-21 did not cause any symptoms, while the positive control D. citri strain caused typical reddish-brown to black lesion spots symptoms (Figure 3B). On the contrary, all the fruit inoculated with conidial suspension of isolates NFIF-3-11, NFIF-3-19, and NFIF-3-21, as well as positive control fruit inoculated with D. citri strain showed typical rot symptoms at 7 days after inoculation. No significant symptom was observed on negative control fruit inoculated with sterile water (Figure 3C). Re-isolation was performed following Koch’s postulation method. The strains were re-isolated from the experimentally inoculated fruit with stem-end rot symptoms. The identity of the re-isolated strains was confirmed by amplification and sequencing of ITS, TUB, TEF, HIS, and CAL molecular markers.

2.3. Phylogenetic Analyses

For preliminary identification, the MegaBlast search was performed for ITS region of three isolates in NCBI’s GenBank nucleotide database. All three isolates (NFIF-3-11, NFIF-3-19, and NFIF-3-21) showed 100% identity to Diaporthe ueckerae (KY565426) and Phomopsis sp. (KX510126, XP677503, KM229696, FJ233186, and GU595054), 99% identity to D. phaseolorum (LC360110), D. longicolla (KF577903), D. ueckerae (KY565424, KY565425), and D. passifloricola (NR_147595).
Multi-locus phylogenetic analyses were carried out based on the sequences of ITS, TUB, TEF, HIS, and CAL. To verify if these five loci were congruent and could be combined together, single locus analysis was also performed for each locus. The results indicated that the topology of single-locus trees was congruent (Supplementary Figures S2–S6). Fifteen new sequences were generated from three isolates in this study. Other published sequences of Diaporthe spp. were downloaded from GenBank database. In total, 2738 characters of 101 strains from 80 Diaporthe spp., including one outgroup species D. citri (CBS 135422), were employed for Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP) analyses to construct phylogenetic tree. The dataset consisted of 611 characters of ITS (1–611), 868 characters of TUB (612–1479), 527 characters of TEF (1480–2006), 581 characters of HIS (2007–2587), and 578 characters of CAL (2588–3165), respectively. MP analyses of combined data generated a single most parsimonious tree (tree length (TL) = 5416, consistency index (CI) = 0.449, retention index (RI) = 0.739, rescaled consistency index (RC) = 0.332, and homoplasy index (HI) = 0.551). Of the 3165 analyzed characters, 1036 characters were parsimony-informative, 431 variable characters were parsimony uninformative, and 1698 characters were constant. Data of each region/loci were shown in Supplementary Table S1. Using the best scoring RA×ML analysis, a final optimization tree with a likelihood value of −30,716.492582 was generated. The matrix data had 1837 distinct alignment patterns in the ML analysis, with 39.30% of gaps and completely undetermined characters. Estimated base frequencies were as follows: A = 0.212443, C = 0.325722, G = 0.238041, T = 0.223795, with substitution rates AC = 1.252910, AG = 4.007552, AT = 1.250610, C = 1.175745, CT = 5.302300, GT = 1.000000. The gamma distribution shape parameter alpha = 0.938818 and the TL = 6.170537. The ML and MP tree of combined data had similar topology to BI tree. The posterior probabilities (PP) values calculated from BI, bootstrap support (BS) values calculated from ML and MP analyses were plotted in Figure 4 and Supplementary Figure S7. The combined loci analyses grouped three isolates (NFIF-3-11, NFIF-3-19, and NFIF-3-21) together with 0.97 of Bayesian posterior probabilities values (BIPP), 99% of Maximum likelihood bootstrap values (MLBS), and 94% of Maximum parsimony bootstrap values (MPBS), respectively. The isolates were classified as D. passifloricola with 1 of BIPP, 75% of MLBS, and 67% of MPBS, and distinct from D. durionigena, D. rosae, D. miriciae, and D. ueckerae. The analysis of polymorphic nucleotides in each locus of D. passifloricola, D. durionigene, and D. rosae also found 11, 4, 4, and 11 polymorphic nucleotides in ITS, TUB, TEF, and CAL, respectively (Supplementary Table S2). While there was no polymorphic nucleotide in HIS sequence of three species.
Materials examined: CHINA, Jiangxi Province, Fuzhou city, Nanfeng district, on fruit of Citrus reticulata cv. Nanfengmiju, August 2016, C. Chaisiri (living culture: CCTCC M 2020452 = NFIF-3-21).

3. Discussion

Diaporthe passifloricola was identified from leaf spots on Passiflora foetida in Malaysia [39]. The colonies of this species on MEA, OA, and PDA are dirty white. Alpha conidia are aseptate, hyaline, smooth, guttulate, fusoid-ellipsoid, tapering towards both ends, apex subobtuse, base subtruncate, (5–) 6–7 (–9) × 2.5 (–3) µm. Gamma conidia are not observed. Beta conidia are spindle shaped, aseptate, smooth, hyaline, apex acutely rounded, base truncate, tapering from lower third towards apex, curved, (20–) 22–25 (–27) × 1.5 (–2) µm. In this study, the colonies of the isolates on PDA were dirty white, which are similar to those of D. passifloricola [39], D. durionigena [40], D. rosae [41], and D. ueckerae [42], while that of D. miriciae is buff [23]. Morphological characteristics of alpha (bi-guttulate) and beta conidia of three isolates are consistent with those of D. passifloricola ex-type strain (CBS 141329) [39]. The sizes of alpha and beta conidia of three isolates are larger than those of D. durionigena [40] and D. rosae [41]. The alpha conidia of D. miriciae are not described of guttulate characterized [23], and the beta conidia of D. ueckerae are not observed in a previous study [42]. Thus, morphological characteristics of the three isolates are the most consistent with those of D. passifloricola. Taking into account that morphological characteristics sometimes vary with environmental conditions, they are not always reliable to identify the isolates to species level in genus of Diaporthe [37]. Thus, further molecular identification is necessary.
The sequence of the ITS region was once used alone to identify Diaporthe species. However, there are many intraspecific variations in ITS locus of certain Diaporthe species. Sometimes the intraspecific variation is even greater than interspecific variation, which makes it difficult to identify Diaporthe species with ITS sequence alone [43,44]. Currently, multi-locus phylogenetic analyses have been applied for the identification of Diaporthe species [37,45]. Thus, although ITS sequences of all three isolates showed 100% similarity with D. ueckerae (KY565426) in this study, it was unreliable, due to many intraspecific variations in ITS regions of Diaporthe species.
The combined use of the five loci (i.e., ITS, TUB, TEF, HIS, and CAL) is shown to be the best way to generate a phylogenetic tree to determine the boundaries of Diaporthe spp. [21,31,33,37,38,45]. After preliminary identification with ITS locus, four species of D. passifloricola, D. rosae, D. ueckerae, and D. miriciae were found to have high identity to the three isolates obtained in this study. Thus, five loci of ITS, TUB, TEF, HIS, and CAL were further employed to perform phylogenetic analysis.
The main molecular traits of D. passifloricola have been described in 2016 [39]. For ITS region, D. passifloricola (KX228292.1) shows 98% (556/567) similarity to D. miriciae (KJ197284.1) and 90% (466/519)–93% (402/430) similarity to five ‘Phomopsis tersa’ (e.g., KF516000.1 and JQ585648.1). For HIS sequence, D. passifloricola (KX228367.1) exhibits 100% identity (380/380) to D. absenteum (KP293559.1) and 99% identity (378/380) to ‘Diaporthe sp. 1 RG-2013’ (KC343687.1). Meanwhile, for TUB sequence, D. passifloricola (MB817057) is 99% similar to ‘Diaporthe sp. 1 RG-2013’ (KC344171.1 (513/517)) and D. miriciae (KJ197264.1 (589/595)). However, the difference among D. passifloricola and other two species D. durionigene and D.rosae, which have the closest genetic distance with D. passifloricola, has not been reported. In this study, polymorphic nucleotides in ITS, TUB, TEF, and CAL sequences of D. passifloricola, D. durionigene, and D. rosae are determined and can distinguish three species well.
The taxonomy of Diaporthe is complex. Many Diaporthe spp. were classified according to different criteria, i.e., host associations, morphological characteristics [26,28,46,47], or sequences of ITS region [22,26,48]. It is suggested that only those type strains, whose identification has been widely recognized, should be accepted as references for the taxonomy of this genus [37,49,50]. Moreover, several isolates included type strains from previous publications are selected for references with phylogenetic analysis in this study. While MegaBlast search was performed for each locus on NCBI, the Diaporthe species showing the highest similarity with the sequencing of each locus of the isolates were not the type strains. Thus, the species identified by us are different from those retrieved by a single locus MegaBlast search on NCBI.
Before this study, 22 Diaporthe spp. associated with citrus were known in the world [5,6,25,37,51,52]. They are either pathogens, endophytes, or saprobes on citrus [6,11,25,52,53,54]. This is the first time that D. passifloricola has been isolated from C. reticulata cv. Nanfengmiju.
In previous studies, 15 Diaporthe spp. have been reported to be associated with citrus in China [5,6]. Of them, three species are pathogens on citrus, i.e., D. citri, D. citriasiana, and D. citrichinensis. D. citri is identified as the causal agents of melanose disease as well as stem-end rot disease. In addition to being a pathogen, D. citri is also found as an endophyte in non-symptomatic twigs and as a saprobe on dead twigs. Two species, D. citriasiana, and D. citrichinensis, can only cause stem-end rot symptom on ponkan fruit (Citrus reticulata) [5]. The other 12 Diaporthe spp. were identified as endophytes or saprobes on citrus [6]. All of these indicate that the symbiotic relationship and ecological function of Diaporthe spp. with citrus plants is complex and variable.
Endophytes are defined as all organisms inhabiting plant organs which, at some time in their lives, can colonize internal plant tissues without causing significant damage to the host [55]. So defined, endophytes may also encompass asymptomatic latent pathogens. Sometimes asymptomatic fungi can cause diseases on their host plants under certain conditions. It’s reported that several Plectosphaerella spp. isolated from symptomless tomatoes and peppers can cause disease symptoms on tomato and pepper, and even basil and parsley when artificially inoculated [56,57]. Epichloë festucae is a well-known endophytic fungus of perennial ryegrass (Lolium perenne). However, a E. festucae noxA mutant is associated with severe stunting of the host as a result of hyphal hyper-branching and increased biomass [58]. Some fungal saprobes and pathogens can be isolated from rice (Oryza sativa) as endophytes [59]. In this study, since D. passifloricola isolates failed to cause melanose on citrus fruit, they are supposed to be the endophytic fungi colonizing diseased tissues with melanose symptoms. However, our results show that this species can induce stem-end rot symptoms on artificially inoculated citrus fruit. Thus, D. passifloricola could be a potential causal agent of stem-end rot disease during transportation and storage.
The disease spots of citrus melanose are formed by host hypersensitive response (HR). When the pathogens penetrate epidermal cells of the citrus, they are arrested and killed at the infection sites by hosts along with the development of melanose symptoms [60,61,62]. As a result, it is difficult to isolate pathogens in old disease spots. The disease spots were not newly formed, which might be the reason why we failed to isolate the pathogen causing melanose symptoms.

4. Materials and Methods

4.1. Fungal Isolation

In 2016, 10 citrus fruit of Nanfengmiju with typical symptoms of melanose were collected from a citrus orchard in Fuzhou City of Jiangxi Province (Figure 3A). The discrete and sunken black spots were observed on the fruit surface. Pieces of small sections about 5 mm2 from the margin of the lesion were cut off and soaked in 75% ethanol solution for 1 min. The sections were surface disinfested with 1% sodium hypochlorite solution (NaClO) for 1 min, rinsed three times with sterilized water, dried, and then incubated on PDA plates amended with 100 μg/mL streptomycin and 100 μg/mL ampicillin at 25 °C for 2 to 5 days. Hyphal tips growing from the pieces of the sample were transferred onto fresh PDA plates and incubated at 25 °C for 30 days as previous methods [7]. After sporulation, single-spore-isolation was performed as previously described [63]. All single-spore cultures were stored on half strength PDA slants in Eppendorf tubes at 4 °C, and on dried filter paper discs at −20 °C, respectively. A living culture of D. passifloricola in this study was deposited in China Center for Type Culture Collection (CCTCC), Wuhan, China.

4.2. Morphological Characterization

Sporulation was induced on PDA, MEA, CMA, OMA, and PNA. After inoculation, isolates were incubated at 25 °C with 12 h of light and 12 h of dark for 30 days. Conidia were harvested from the top of mature pycnidia. Pycnidia were picked up from pine needles with sterile toothpicks. The length and width of 30 conidia were measured with a stage micrometer under a Motic BA200 light microscope (Motic China Group Co., Ltd., Xiamen, China). The morphology of conidiomata was observed under OLYMPUS SZX16 stereo microscope (Olympus Corporation, Tokyo, Japan). Images of conidia were captured using a digital camera Nikon Eclipse 80i on a compound light microscope (Nikon Corporation, Tokyo, Japan) imaging system. Images of culture plates were captured using Cannon 600D digital camera (Cannon Inc., Tokyo, Japan). Colony and pycnidia color was investigated with a color chart according to the method of Rayner [64].

4.3. Pathogenicity Test

Pathogenicity tests were carried out on detached Nanfengmiju fruit (Citrus reticulata cv. Nanfengmiju). Non-wounded citrus fruit were washed with tap water, then surface disinfested with 75% of ethanol and rinsed with sterile water. Pycnidia with alpha conidia were induced as mentioned above and diluted to 106 conidia/mL with sterile water. To stimulate melanose symptoms, 300 μL of conidial suspensions was dropped on a piece of cotton, and then placed on the bottom of the fruit as previously described with a slight modification [65]. The inoculated fruit were placed in a plastic chamber with 95% relative humidity, incubated under the condition of 12 h of light and 12 h of dark at 25 °C for 15 days. Since Diaporthe spp. were the causal agents of both melanose and stem-end rot diseases on citrus fruit, their ability to cause stem-end rot symptom was also determined. The stems of citrus fruit were removed carefully, and 10 μl of conidial suspension (106 conidia/mL) of each strain was inoculated onto stem ends as previously described [5]. Then, the inoculated fruit were placed in a plastic chamber with wet towel tissues at the bottom. The chamber was wrapped with plastic film to maintain 95% relative humidity and incubated at 25 °C in the dark for 7 days. In all the pathogenicity tests, the conidial suspension (106 conidia/mL) of D. citri strain NFHF-8-4 [7] and sterile distilled water were used as positive and negative controls, respectively. Symptoms on fruit were observed. Four fruit were inoculated for each strain, and the experiments were repeated at least twice.
To authenticate the causal agent, tissue pieces from the margin of lesions on the experimentally inoculated and diseased fruit were placed on PDA to re-isolate the fungus. Molecular identification of the isolate was performed using the sequence of ITS, TUB, TEF, HIS, and CAL loci as mentioned below.

4.4. DNA Extraction, PCR Amplification, and Sequencing

DNA extraction was performed as previously described [66]. Fragments of ITS, TUB, TEF, HIS, and CAL were amplified by polymerase chain reaction (PCR) using primer pairs ITS1/ITS4 [67], Bt-2a/Bt-2b [68], EF1-728F/EF1-986R [69], CYLH3F/H3-1b [68,70], and CAL-228F/CAL-737R [69], respectively. Twenty-five microliters of PCR reaction included 1 μL genomic DNA (100–500 ng/μL), 1 μL (10 mM) of each primer, 9.5 μL double-distilled water, and 12.5 μL 2× Taq PCR Master Mix (Aidlab Biotechnologies Co., Ltd., Beijing, China). PCR amplification was carried out with an initial denaturation step at 95 °C for 3 min followed by 40 cycles, consisting of a denaturation step at 95 °C for 30 sec, an annealing step for 50 sec, an elongation step at 72 °C for 2 min, and a final step at 72 °C for 5 min. The annealing temperatures were 51 °C for the amplification of partial ITS, 55 °C for the amplification of partial TUB, TEF, and CAL, and 58 °C for the amplification of partial HIS, respectively, as mentioned previously [31]. The size of PCR products was verified by gel electrophoresis in Tris-borate-EDTA (TBE) buffer using 1% agarose gel. Sequencing was carried out at Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., Wuhan, China.

4.5. Phylogenetic Analyses

The preliminary identifications of the isolates obtained in this study were determined using newly generated ITS sequences with all available type-derived sequences listed in previous studies [6,24,25,37,51]. Based on the result of preliminary identification, Diaporthe species with the closest genetic distance to the isolates in this study were selected. Sequences (ITS, TUB, TEF, HIS, and CAL) of them were downloaded from NCBI’s GenBank nucleotide database (www.ncbi.nlm.nih.gov). All sequences used in this study are listed in Table 1, including 15 sequences of three new isolates. The reference isolates were selected from ex-type, ex-epitype, and holotype cultures. Five-locus phylogenetic analyses were conducted to identify isolates to species level according to previous studies [21,30,37]. Sequences of five loci (ITS, TUB, TEF, HIS, and CAL) were assembled. Alignments of assembled sequences were performed with L-INS-i iterative refinement method by MAFFT alignment, a version available online [71], and manual adjustment was conducted where it was necessary by BioEdit v.7.2.5 [72]. ML trees were generated with 1,000 replicates using RA×ML-HPC BlackBox v.8.2.10 [73], which was available on the CIPRES Science Gateway v.3.3 Web Portal [74]. The RAxML software selected general time reversible model of evolution including estimation of invariable sites (GTRGAMMA+I). MP analyses were carried out with 1,000 replicates using Phylogenetic Analyses Using Parsimony (PAUP*) v.4.0b10 [75], with tree bisection and reconnection (TBR) branch-swapping algorithm. All characters were weighted equally, and the alignment gaps were treated as missing characters. Descriptive tree statistics including TL, CI, RI, RC, and HI were calculated for parsimony analyses. MrModeltest v.2.3 [76] was used to perform statistical selection of the best-fit model of nucleotide substitution and the corrected Akaike information criterion (AIC) determined above was incorporated into evolutionary models in the analysis (Supplementary Table S1). BI analysis was performed by using MrBayes v.3.2.2, with Markov Chain Monte Carlo (MCMC) algorithm. Four simultaneous of MCMC chains were run for 20,000,000th generations, and trees were sampled frequency every 100th generations, resulting in a total of 20,000 trees, and started from a random tree topology. The calculation of BI analyses was stopped when the average standard deviation of split frequencies fell below 0.01. The first 10% of trees were discarded as burn-in phase of analysis, and the remaining 180,000 trees were summarized to calculate the PP in the majority rule consensus tree. Phylogenetic analyses and full alignment of datasets were submitted to TreeBASE (www.treebase.org) with the study ID: 27334.

5. Conclusions

Our results indicate that D. passifloricola, may occur as an asymptomatic endophyte in the peel of citrus fruit. If is manages to invade the fruit stalk, however, it may induce typical stem-end rot symptoms during transportation and storage. To the best of our knowledge, this is the first time D. passifloricola has been isolated from Citrus reticulata cv. Nanfengmiju in China and identified as a causal agent of stem-end rot disease in this crop.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/10/2/218/s1, Table S1 nucleotide substitution models, MP and ML alignment properties, Table S2 Polymorphic nucleotides in ITS, TUB, TEF, and CAL sequences of D. passifloricola, D. durionigene, and D. rosae, Figure S1. The prevalence of Diaporthe species on citrus in Jiangxi Province, China based on phylogenetic identification. Numbers (%) indicate the number of obtained isolates of certain species and the percentage among the total 140 isolates [1]. Yellow color indicate 39 isolates of Diaporthe sp. were found in this study, Figure S2. The phylogenetic tree is generated from the analysis of sequences of ITS locus. A, Maximum likelihood and B, Maximum parsimony. Bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with Diaporthella corylina CBS 121124. Ex-type, ex-epitype and ex-isotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given, Figure S3. The phylogenetic tree is generated from the analysis of sequences of TUB locus. A, Maximum likelihood and B, Maximum parsimony. Bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with Diaporthella corylina CBS 121124. Ex-type, ex-epitype and ex-isotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given, Figure S4. The phylogenetic tree is generated from the analysis of sequences of TEF locus. A, Maximum likelihood and B, Maximum parsimony. Bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with Diaporthella corylina CBS 121124. Ex-type, ex-epitype and ex-isotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given, Figure S5. The phylogenetic tree is generated from the analysis of sequences of HIS locus. A, Maximum likelihood and B, Maximum parsimony. Bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with Diaporthella corylina CBS 121124. Ex-type, ex-epitype and ex-isotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given, Figure S6. The phylogenetic tree is generated from the analysis of sequences of CAL locus. A, Maximum likelihood and B, Maximum parsimony. Bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with Diaporthella corylina CBS 121124. Ex-type, ex-epitype and ex-isotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given, Figure S7. The phylogenetic tree is generated from the analysis of the combined sequences of five loci (ITS, TUB, TEF, HIS, and CAL). A, Maximum likelihood and B, Maximum parsimony, bootstrap support values ≥50%, (MLBS/MPBS) are displayed at the nodes. The tree is rooted with D. citri CBS 135422. Ex-type, ex-epitype and holotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given.

Author Contributions

Conceptualization, C.C., Y.L. and C.-X.L.; validation, C.C., X.-Y.L., Y.L. and C.-X.L.; formal analysis, C.C. and X.-Y.L.; investigation and resources, C.C., X.-Y.L., W.-X.Y. and Y.L.; data curation, C.C., X.-Y.L., Y.L., W.-X.Y. and C.-X.L.; writing, C.C., C.-X.L. and Y.L.; funding acquisition, Y.L. and C.-X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (number 2017YFD020200103).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Alignment data generated in the current study are available in TreeBASE (accession http://purl.org/phylo/treebase/phylows/study/TB2:S27334). All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.

Acknowledgments

We thank Kevin D. Hyde (Center of Excellence in Fungal Research, Mae Fah Luang University, Thailand) and Jian-Kui Liu (Fungal Research Laboratory, University of Electronic Science and Technology of China, China) for technical assistance and invaluable advice. The authors sincerely thank the reviewers and editor for their contributions to improve the manuscript during the revision process.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Deng, X.X.; Peng, C.J.; Chen, Z.S.; Deng, Z.N.; Xu, J.G.; Li, J. Citrus Varieties in China; China Agriculture Press: Beijing, China, 2008. [Google Scholar]
  2. FAO. Citrus Fruit—Fresh and Processed Statistical Bulletin 2016; Food and Agriculture Organization of the United Nations: Rome, Italy, 2017. [Google Scholar]
  3. Kucharek, T.; Whiteside, J.; Brown, E. Melanose and phomopsis stem-end rot of citrus. In Plant Pathology Fact Sheet; Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida: Florida, FL, USA, 2000; pp. 26–30. [Google Scholar]
  4. Timmer, L.W.; Garnsey, S.M.; Graham, J.H. Scab Diseases, revised edition: 31–32 ed.; American Phytopathological Society Press: St. Paul, MN, USA, 2000; p. 92. [Google Scholar]
  5. Huang, F.; Hou, X.; Dewdney, M.M.; Fu, Y.S.; Chen, G.Q.; Hyde, K.D.; Li, H.Y. Diaporthe species occurring on citrus in China. Fungal Divers. 2013, 61, 237–250. [Google Scholar] [CrossRef]
  6. Huang, F.; Udayanga, D.; Wang, X.H.; Hou, X.; Mei, X.F.; Fu, Y.S.; Hyde, K.D.; Li, H.Y. Endophytic Diaporthe associated with Citrus: A phylogenetic reassessment with seven new species from China. Fungal Biol. 2015, 119, 331–347. [Google Scholar] [CrossRef]
  7. Chaisiri, C.; Liu, X.Y.; Lin, Y.; Li, J.B.; Xiong, B.; Luo, C.X. Phylogenetic analysis and development of molecular tool for detection of Diaporthe citri causing melanose disease of citrus. Plants 2020, 9, 329. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Bach, W.J.; Wolf, F.A. The isolation of the fungus that causes citrus melanose and the pathological anatomy of the host. J. Agric. Res. 1928, 37, 243–252. [Google Scholar]
  9. Gopal, K.; Lakshmi, L.M.; Sarada, G.; Nagalakshmi, T.; Sankar, T.G.; Gopi, V.; Ramana, K.T.V. Citrus melanose (Diaporthe citri Wolf): A review. Int. J. Curr. Microbiol. App. Sci. 2014, 3, 113–124. [Google Scholar]
  10. Fawcett, H.S. The cause of stem-end rot of citrus fruits (Phomopsis citri n. sp.). Phytopathology 1912, 2, 109–113. [Google Scholar]
  11. Mondal, S.N.; Vicent, A.; Reis, R.F.; Timmer, L.W. Saprophytic colonization of citrus twigs by Diaporthe citri and factors affecting pycnidial production and conidial survival. Plant. Dis. 2007, 91, 387–392. [Google Scholar] [CrossRef] [Green Version]
  12. Chen, G.Q.; Jiang, L.Y.; Xu, F.S.; Li, H.Y. In vitro and in vivo screening of fungicides for controlling citrus melanose caused by Diaporthe citri. J. Zhejiang Univ. (Agric. Life Sci.) 2010, 36, 440–444. (In Chinese) [Google Scholar]
  13. Jiang, L.Y.; Xu, F.S.; Huang, Z.D.; Huang, F.; Chen, G.Q.; Li, H.Y. Occurrence and control of citrus melanose caused by Diaporthe citri. Acta Agric. Zhejiangensis 2012, 24, 647–653. (In Chinese) [Google Scholar]
  14. Udayanga, D.; Manamgoda, D.S.; Liu, X.Z.; Chukeatirote, E.; Hyde, K.D. What are the common anthracnose pathogens of tropical fruits? Fungal Divers. 2013, 61, 165–179. [Google Scholar] [CrossRef]
  15. Huang, L. The diseases of Citrus spp. of Guangxi. Guangxi Nong Xun 1943, 4, 27–61. (In Chinese) [Google Scholar]
  16. Guangdong Station of Plant Quarantine. The preliminary investigation results of Citrus spp. insects and diseases in East Guangdong province. 1955; 1–3. (In Chinese) [Google Scholar]
  17. Qiu, W.F. The records of diseases and insects of economic plants in Fujian (1, 2, 3). Xin Nong Ji Kan 1941, 1, 70–75, 161–166, 209–229. (In Chinese) [Google Scholar]
  18. Plant quarantine Station; Department of Agriculture; Jiangxi Academy of Agriculture. The Records of Plant Insects and Diseases in Jiangxi, the Part of Plant Diseases; Jiangxi People’s Publishing House: Nanchang, China, 1960; pp. 1–247. (in Chinese) [Google Scholar]
  19. Li, L. The index of the parasitic fungi of Szechwan, China. Pl. Dis. Rep. Suppl. 1948, 173, 1–38. [Google Scholar]
  20. Chinese Research Institute of Pomelogy and Citrus. The Records of Chinese Fruit Trees’ Diseases and Pests; China Agriculture Press: Beijing, China, 1994. (In Chinese) [Google Scholar]
  21. Guarnaccia, V.; Groenewald, J.Z.; Woodhall, J.; Armengol, J.; Cinelli, T.; Eichmeier, A.; Ezra, D.; Fontaine, F.; Gramaje, D.; Gutierrez-Aguirregabiria, A.; et al. Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe. Persoonia 2018, 40, 135–153. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Santos, J.M.; Phillips, A.J.L. Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Divers. 2009, 34, 111–125. [Google Scholar]
  23. Thompson, S.M.; Tan, Y.P.; Shivas, R.G.; Neate, S.M.; Morin, L.; Bissett, A.; Aitken, E.A.B. Green and brown bridges between weeds and crops reveal novel Diaporthe species in Australia. Persoonia 2015, 35, 39–49. [Google Scholar] [CrossRef] [Green Version]
  24. Udayanga, D.; Castlebury, L.A.; Rossman, A.Y.; Chukeatirote, E.; Hyde, K.D. Insights into the genus Diaporthe: Phylogenetic species delimitation in the D. eres species complex. Fungal Divers. 2014, 67, 203–229. [Google Scholar] [CrossRef] [Green Version]
  25. Guarnaccia, V.; Crous, P.W. Emerging citrus diseases in Europe caused by species of Diaporthe. Ima Fungus 2017, 8, 317–334. [Google Scholar] [CrossRef] [Green Version]
  26. Mostert, L.; Crous, P.W.; Kang, J.C.; Phillips, A.J.L. Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: Morphological, cultural, molecular and pathological characterization. Mycologia 2001, 93, 146–167. [Google Scholar] [CrossRef]
  27. Rehner, S.A.; Uecker, F.A. Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis. Can. J. Bot. 1994, 72, 1666–1674. [Google Scholar] [CrossRef]
  28. Chi, P.K.; Jiang, Z.D.; Xiang, M.M. Flora Fungorum Sinicorum; Science Press: Beijing, China, 2007; Volume 34. (In Chinese) [Google Scholar]
  29. Dissanayake, A.J.; Zhang, W.; Liu, M.; Hyde, K.D.; Zhao, W.S.; Li, X.H.; Yan, J.Y. Diaporthe species associated with peach tree dieback in Hubei, China. Mycosphere 2017, 8, 533–549. [Google Scholar] [CrossRef]
  30. Gao, Y.H.; Liu, F.; Duan, W.J.; Crous, P.W.; Cai, L. Diaporthe is paraphyletic. Ima Fungus 2017, 8, 153–187. [Google Scholar] [CrossRef] [PubMed]
  31. Yang, Q.; Fan, X.L.; Guarnaccia, V.; Tian, C.M. High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described. MycoKeys 2018, 39, 97–149. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Fan, X.L.; Yang, Q.; Bezerra, J.D.P.; Alvarez, L.V.; Tian, C.M. Diaporthe from walnut tree (Juglans regia) in China, with insight of the Diaporthe eres complex. Mycol. Prog. 2018, 17, 841–853. [Google Scholar] [CrossRef]
  33. Guo, Y.S.; Crous, P.W.; Bai, Q.; Fu, M.; Yang, M.M.; Wang, X.H.; Du, Y.M.; Hong, N.; Xu, W.X.; Wang, G.P. High diversity of Diaporthe species associated with pear shoot canker in China. Persoonia 2020, 45, 132–162. [Google Scholar] [CrossRef]
  34. Wehmeyer, L.E. The genus Diaporthe Nitschke and its segregates. Univ. Mich. Stud. Sci. Ser. 1933, 9, 1–349. [Google Scholar]
  35. Sutton, B.C. The Coelomycetes. Fungi Imperfecti with Pycnidia, Acervuli and Stromata; Commonwealth Mycological Institute: London, UK, 1980. [Google Scholar]
  36. Rosskopf, E.N.; Charudattan, R.; Shabana, Y.M.; Benny, G.L. Phomopsis amaranthicola, a new species from amaranthus sp. Mycologia 2000, 92, 114–122. [Google Scholar] [CrossRef]
  37. Gomes, R.R.; Glienke, C.; Videira, S.I.R.; Lombard, L.; Groenewald, J.Z.; Crous, P.W. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia 2013, 31, 1–41. [Google Scholar] [CrossRef] [Green Version]
  38. Santos, L.; Alves, A.; Alves, R. Evaluating multi-locus phylogenies for species boundaries determination in the genus Diaporthe. PeerJ 2017, 5, 1–26. [Google Scholar] [CrossRef] [Green Version]
  39. Crous, P.W.; Wingfield, M.J.; Richardson, D.M.; Le Roux, J.J.; Strasberg, D.; Edwards, J.; Roets, F.; Hubka, V.; Taylor, P.W.J.; Heykoop, M.; et al. Fungal planet description sheets: 400–468. Persoonia 2016, 36, 316–458. [Google Scholar] [CrossRef] [Green Version]
  40. Crous, P.W.; Wingfield, M.J.; Chooi, Y.H.; Gilchrist, C.L.M.; Lacey, E.; Pitt, J.I.; Roets, F.; Swart, W.J.; Cano-Lira, J.F.; Valenzuela-Lopez, N.; et al. Fungal planet description sheets: 1042–1111. Persoonia 2020, 44, 301–459. [Google Scholar] [CrossRef] [PubMed]
  41. Wanasinghe, D.N.; Phukhamsakda, C.; Hyde, K.D.; Jeewon, R.; Lee, H.B.; Jones, E.B.G.; Tibpromma, S.; Tennakoon, D.S.; Dissanayake, A.J.; Jayasiri, S.C.; et al. Fungal diversity notes 709–839: Taxonomic and phylogenetic contributions to fungal taxa with an emphasis on fungi on Rosaceae. Fungal Divers. 2018, 89, 1–60. [Google Scholar] [CrossRef]
  42. Udayanga, D.; Castlebury, L.A.; Rossman, A.Y.; Chukeatirote, E.; Hyde, K.D. The Diaporthe sojae species complex: Phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops. Fungal Biol. 2015, 119, 383–407. [Google Scholar] [CrossRef] [PubMed]
  43. Farr, D.F.; Castlebury, L.A.; Rossman, A.Y.; Putnam, M.L. A new species of Phomopsis causing twig dieback of Vaccinium vitis-idaea (lingonberry). Mycol. Res. 2002, 106, 745–752. [Google Scholar] [CrossRef]
  44. Santos, J.M.; Correia, V.G.; Phillips, A.J.L.; Spatafora, J.W. Primers for mating-type diagnosis in Diaporthe and Phomopsis: Their use in teleomorph induction in vitro and biological species definition. Fungal Biol. 2010, 114, 255–270. [Google Scholar] [CrossRef] [PubMed]
  45. Udayanga, D.; Liu, X.Z.; Crous, P.W.; McKenzie, E.H.C.; Chukeatirote, E.; Hyde, K.D. A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis). Fungal Divers. 2012, 56, 157–171. [Google Scholar] [CrossRef]
  46. Uecker, F.A. A world list of Phomopsis names with notes on nomenclature, morphology and biology. Mycol. Mem. 1988, 13, 1–231. [Google Scholar]
  47. Brayford, D. Variation in Phomopsis isolates from Ulmus species in the British Isles and Italy. Mycol. Res. 1990, 94, 691–697. [Google Scholar] [CrossRef]
  48. van Niekerk, J.M.; Groenewald, J.Z.; Farr, D.F.; Fourie, P.H.; Halleen, F.; Crous, P.W. Reassessment of Phomopsis species on grapevine. Australas. Plant. Pathol. 2005, 34, 27–39. [Google Scholar] [CrossRef]
  49. Rossman, A.Y.; Adams, G.C.; Cannon, P.F.; Castlebury, L.A.; Crous, P.W.; Gryzenhout, M.; Jaklitsch, W.M.; Mejia, L.C.; Stoykov, D.; Udayanga, D.; et al. Recommendations of generic names in Diaporthales competing for protection or use. Ima Fungus 2015, 6, 145–154. [Google Scholar] [CrossRef] [Green Version]
  50. Dissanayake, A.J.; Phillips, A.J.L.; Hyde, K.D.; Yan, J.Y.; Li, X.H. The current status of species in Diaporthe. Mycosphere 2017, 8, 1106–1156. [Google Scholar] [CrossRef]
  51. Udayanga, D.; Castlebury, L.A.; Rossman, A.Y.; Hyde, K.D. Species limits in Diaporthe: Molecular re-assessment of D. citri, D. cytosporella, D. foeniculina and D. rudis. Persoonia 2014, 32, 83–101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Guarnaccia, V.; Crous, P.W. Species of Diaporthe on Camellia and Citrus in the Azores Islands. Phytopathol. Mediterr. 2018, 57, 307–319. [Google Scholar]
  53. Douanla-Meli, C.; Langer, E.; Mouafo, F.T. Fungal endophyte diversity and community patterns in healthy and yellowing leaves of Citrus limon. Fungal Ecol. 2013, 6, 212–222. [Google Scholar] [CrossRef]
  54. Murali, T.S.; Suryanarayanan, T.S.; Geeta, R. Endophytic Phomopsis species: Host range and implications for diversity estimates. Can. J. Microbiol. 2006, 52, 673–680. [Google Scholar] [CrossRef] [PubMed]
  55. Petrini, O. Fungal endophytes of tree leaves. In Microbial Ecology of Leaves; Andrews, J.H., Hirano, S.S., Eds.; Brock/Springer Series in Contemporary Bioscience; Springer: New York, NY, USA, 1991; pp. 179–197. [Google Scholar]
  56. Raimondo, M.L.; Carlucci, A. Characterization and pathogenicity of Plectosphaerella spp. collected from basil and parsley in Italy. Phytopathol. Mediterr. 2018, 57, 284–295. [Google Scholar]
  57. Raimondo, M.L.; Carlucci, A. Characterisation and pathogenicity assessment of Plectosphaerella species associated with stunting disease on tomato and pepper crops in Italy. Plant. Pathol. 2018, 67, 626–641. [Google Scholar] [CrossRef]
  58. Takemoto, D.; Tanaka, A.; Kayano, Y.; Saikia, S.; Wrenn, R.; Scott, B. Reactive oxygen as a signal in grass-Epichloë symbiosis. In Epichloae, Endophytes of Cool Season Grasses: Implications, Utilization and Biology. Proceedings of the 7th International Symposium on Fungal Endophytes of Grasses; Young, C.A., Aiken, G.E., McCullry, R.L., Strickland, J.R., Schardl, C.L., Eds.; Samuel Roberts Noble Foundation: Lexington, KY, USA, 2012; pp. 109–112. [Google Scholar]
  59. Fisher, P.J.; Petrini, O. Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytol. 1992, 120, 137–143. [Google Scholar] [CrossRef]
  60. Akai, S. Histology of defense in plants. In Plant Pathology; Horsfall, J.G., Dimond, A.E., Eds.; Academic Press: New York, NY, USA, 1959; Volume 1, pp. 435–467. [Google Scholar]
  61. Arimoto, Y.; Homma, Y.; Misato, T. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. Part 2. Infection mode of D. citri to citrus leaf. Ann. Phytopath. Soc. Jpn. 1980, 46, 575–581. [Google Scholar] [CrossRef]
  62. Arimoto, Y.; Homma, Y.; Misato, T. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. Part 3. Leaf against infection of D. citri. Ann. Phytopath. Soc. Jpn. 1982, 48, 559–569. [Google Scholar] [CrossRef]
  63. Yin, L.F.; Chen, S.N.; Chen, G.K.; Schnabel, G.; Du, S.F.; Chen, C.; Li, G.Q.; Luo, C.X. Identification and characterization of three Monilinia species from plum in China. Plant. Dis. 2015, 99, 1775–1783. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Rayner, R.W. A Mycological Colour Chart; Commonwealth Mycological Institute and British Mycological Society: Kew, Surrey, UK, 1970. [Google Scholar]
  65. Kanematsu, S.; Kobayashi, T.; Kudo, A.; Ohtsu, Y. Conidial morphology, pathogenicity and culture characteristics of Phomopsis isolates from peach, Japanese pear and apple in Japan. Jpn J. Phytopathol. 1999, 65, 264–273. [Google Scholar] [CrossRef]
  66. Hu, M.J.; Cox, K.D.; Schnabel, G.; Luo, C.X. Monilinia species causing brown rot of peach in China. PLoS ONE 2011, 6, e24990. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: San Diego, CA, USA, 1990; pp. 315–322. [Google Scholar]
  68. Glass, N.L.; Donaldson, G.C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microb. 1995, 61, 1323–1330. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Carbone, I.; Kohn, L.M. A method for desianing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999, 91, 553–556. [Google Scholar] [CrossRef]
  70. Crous, P.W.; Groenewald, J.Z.; Risède, J.M.; Simoneau, P.; Hywel-Jones, N.L. Calonectria species and their Cylindrocladium anamorphs: Species with clavate vesicles. Stud. Mycol. 2004, 50, 415–430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Katoh, K.; Rozewicki, J.; Yamada, K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2017, bbx108, 1–7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Hall, A.T. BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. Nucleic Acids Res. 1999, 41, 95–98. [Google Scholar]
  73. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  74. Miller, M.A.; Pfeiffer, W.; Schwartz, T. The CIPRES science gateway: A community resource for phylogenetic analyses. In TeraGrid Conference: Extreme Digital Discovery; San Diego Supercomputer Center: San Diego, CA, USA, 2011; Volume 41, pp. 1–8. [Google Scholar]
  75. Swofford, D.L. PAUP* Phylogenetic Analysis Using Parsimony, (*and Other Methods). Version 4.0 b10; Sinauer Associates: Sunderland, MA, USA, 2003. [Google Scholar]
  76. Nylander, J.A.A. MrModeltest v.2. Program Distributed by the Author; Evolitionary Biology Centre, Uppsala Univeristy: Uppsala, Sweden, 2004. [Google Scholar]
  77. Crous, P.W.; Wingfield, M.J.; Schumacher, R.K.; Summerell, B.A.; Giraldo, A.; Gené, J.; Guarro, J.; Wanasinghe, D.N.; Hyde, K.D.; Camporesi, E.; et al. Fungal planet description sheets: 281–319. Persoonia 2014, 33, 212–289. [Google Scholar] [CrossRef]
  78. Dissanayake, A.J.; Camporesi, E.; Hyde, K.D.; Zhang, W.; Yan, J.Y.; Li, X.H. Molecular phylogenetic analysis reveals seven new Diaporthe species from Italy. Mycosphere 2017, 8, 853–877. [Google Scholar] [CrossRef]
  79. Gao, Y.H.; Liu, F.; Cai, L. Unravelling Diaporthe species associated with Camellia. Syst. Biodivers. 2016, 14, 102–117. [Google Scholar] [CrossRef]
  80. Tan, Y.P.; Edwards, J.; Grice, K.R.E.; Shivas, R.G. Molecular phylogenetic analysis reveals six new species of Diaporthe from Australia. Fungal Divers. 2013, 61, 251–260. [Google Scholar] [CrossRef]
  81. Chang, C.Q.; Xi, P.G.; Xiang, M.M.; Jiang, Z.D.; Chi, P.K. New species of Phomopsis on woody plants in Hunan province. Mycosystema 2005, 24, 145–154. [Google Scholar]
  82. Mapook, A.; Hyde, K.D.; McKenzie, E.H.C.; Jones, E.B.G.; Bhat, D.J.; Jeewon, R.; Stadler, M.; Samarakoon, M.C.; Malaithing, M.; Tanunchai, B.; et al. Taxonomic and phylogenetic contributions to fungi associated with the invasive weed Chromolaena odorata (siam weed). Fungal Divers. 2020, 101, 1–175. [Google Scholar] [CrossRef]
  83. Souza, A.R.C.; Baldoni, D.B.; Lima, J.; Porto, V.; Marcuz, C.; Machado, C.; Ferraz, R.C.; Kuhn, R.C.; Jacques, R.J.S.; Guedes, J.V.C.; et al. Selection, isolation, and identification of fungi for bioherbicide production. Braz. J. Microbiol. 2017, 48, 101–108. [Google Scholar] [CrossRef] [Green Version]
  84. Crous, P.W.; Carnegie, A.J.; Wingfield, M.J.; Sharma, R.; Mughini, G.; Noordeloos, M.E.; Santini, A.; Shouche, Y.S.; Bezerra, J.D.P.; Dima, B.; et al. Fungal planet description sheets: 868–950. Persoonia 2019, 42, 291–473. [Google Scholar] [CrossRef]
  85. Chang, C.Q.; Cheng, Y.H.; Xiang, M.M.; Jiang, Z.D.; Chi, P.K. New species of Phomopsis in woody plants in Fujian provice. Mycosystema 2005, 24, 6–11. [Google Scholar]
  86. Thompson, S.M.; Tan, Y.P.; Young, A.J.; Neate, S.M.; Aitken, E.A.B.; Shivas, R.G. Stem cankers on sunflower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe (Phomopsis) species. Persoonia 2011, 27, 80–89. [Google Scholar] [CrossRef] [Green Version]
  87. Dissanayake, A.J.; Chen, Y.Y.; Liu, J.K. Unravelling Diaporthe species associated with woody hosts from Karst Formations (Guizhou) in China. J. Fungi 2020, 6, 251. [Google Scholar] [CrossRef]
  88. Manawasighe, I.S.; Dissanayake, A.J.; Li, X.H.; Liu, M.; Wanasinghe, D.N.; Xu, J.P.; Zhao, W.S.; Zhang, W.; Zhou, Y.Y.; Hyde, K.D.; et al. High genetic diversity and species complexity of Diaporthe associated with grapevine dieback in China. Front. Microbiol. 2019, 10, 1936. [Google Scholar] [CrossRef] [PubMed]
  89. Crous, P.W.; Summerell, B.A.; Swart, L.; Denman, S.; Taylor, J.E.; Bezuidenhout, C.M.; Palm, M.E.; Marincowitz, S.; Groenewald, J.Z. Fungal pathogens of Proteaceae. Persoonia 2011, 27, 20–45. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  90. Santos, L.; Phillips, A.J.L.; Crous, P.W.; Alves, A. Diaporthe species on Rosaceae with descriptions of D. pyracanthae sp. nov. and D. malorum sp. nov. Mycosphere 2017, 8, 485–511. [Google Scholar] [CrossRef]
  91. Milagres, C.A.; Belisário, R.; Silva, M.A.; Lisboa, D.O.; Pinho, D.B.; Furtado, G.Q. A novel species of Diaporthe causing leaf spot in Pachira glabra. Trop. Plant. Pathol. 2018, 43, 460–467. [Google Scholar] [CrossRef]
  92. Crous, P.W.; Summerell, B.A.; Shivas, R.G.; Burgess, T.I.; Decock, C.A.; Dreyer, L.L.; Granke, L.L.; Guest, D.I.; Hardy, G.E.S.J.; Hausbeck, M.K.; et al. Fungal planet description sheets: 107–127. Persoonia 2012, 28, 138–182. [Google Scholar] [CrossRef]
  93. Marin-Felix, Y.; Hernández-Restrepo, M.; Wingfield, M.J.; Akulov, A.; Carnegie, A.J.; Cheewangkoon, R.; Gramaje, D.; Groenewald, J.Z.; Guarnaccia, V.; Halleen, F.; et al. Genera of phytopathogenic fungi: GOPHY 2. Stud. Mycol. 2019, 92, 47–133. [Google Scholar] [CrossRef]
  94. Gao, Y.H.; Su, Y.Y.; Sun, W.; Cai, L. Diaporthe species occurring on Lithocarpus glabra in China, with descriptions of five new species. Fungal Biol. 2015, 119, 295–309. [Google Scholar] [CrossRef]
  95. Yang, Q.; Du, Z.; Tian, C.M. Phylogeny and morphology reveal two new species of Diaporthe from traditional chinese medicine in Northeast China. Phytotaxa 2018, 336, 159–170. [Google Scholar] [CrossRef] [Green Version]
  96. Feng, X.X.; Chen, J.J.; Wang, G.R.; Cao, T.T.; Zheng, Y.L.; Zhang, C.L. Diaporthe sinensis, a new fungus from Amaranthus sp. in China. Phytotaxa 2019, 425, 259–268. [Google Scholar] [CrossRef]
  97. Sommer, N.F.; Beraha, L. Diaporthe actinidiae, a new species causing stem-end rot of chinese gooseberry fruits. Mycologia 1975, 67, 650–653. [Google Scholar] [CrossRef]
  98. Hyde, K.D.; Chaiwan, N.; Norphanphoun, C.; Boonmee, S.; Camporesi, E.; Chethana, K.W.T.; Dayarathne, M.C.; de Silva, N.I.; Dissanayake, A.J.; Ekanayaka, A.H.; et al. Mycosphere notes 169–224. Mycosphere 2018, 9, 271–430. [Google Scholar] [CrossRef]
  99. Doilom, M.; Dissanayake, A.J.; Wanasinghe, D.N.; Boonmee, S.; Liu, J.K.; Bhat, D.J.; Taylor, J.E.; Bahkali, A.H.; McKenzie, E.H.C.; Hyde, K.D. Microfungi on Tectona grandis (teak) in Northern Thailand. Fungal Divers. 2016, 82, 107–182. [Google Scholar] [CrossRef]
  100. Liu, J.K.; Hyde, K.D.; Jones, E.B.G.; Ariyawansa, H.A.; Bhat, D.J.; Boonmee, S.; Maharachchikumbura, S.S.N.; McKenzie, E.H.C.; Phookamsak, R.; Phukhamsakda, C.; et al. Fungal diversity notes 1–110: Taxonomic and phylogenetic contributions to fungal species. Fungal Divers. 2015, 72, 1–197. [Google Scholar] [CrossRef]
  101. Crous, P.W.; Wingfield, M.J.; Roux, J.J.L.; Richardson, D.M.; Strasberg, D.; Shivas, R.G.; Alvarado, P.; Edwards, J.; Moreno, G.; Sharma, R.; et al. Fungal planet description sheets: 371–399. Persoonia 2015, 35, 264–327. [Google Scholar] [CrossRef] [PubMed]
  102. Noriler, S.A.; Savi, D.C.; Ponomareva, L.V.; Rodrigues, R.; Rohr, J.; Thorson, J.S.; Glienke, C.; Shaaban, K.A. Vochysiamides a and b: Two new bioactive carboxamides produced by the new species Diaporthe vochysiae. Fitoterapia 2019, 138, 104273. [Google Scholar] [CrossRef] [PubMed]
Figure 1. The cultural characteristics of Diaporthe passifloricola (NFIF-3-21) on different media. The isolate was incubated at 25 °C in the dark. (A,E), PDA medium, (B,F), MEA medium, (C,G), CMA medium, (D,H), OMA medium. Note: AD, Colonies after 3 days incubation, EH, Colonies after 30 days incubation.
Figure 1. The cultural characteristics of Diaporthe passifloricola (NFIF-3-21) on different media. The isolate was incubated at 25 °C in the dark. (A,E), PDA medium, (B,F), MEA medium, (C,G), CMA medium, (D,H), OMA medium. Note: AD, Colonies after 3 days incubation, EH, Colonies after 30 days incubation.
Plants 10 00218 g001
Figure 2. Asexual reproduction of Diaporthe passifloricola (NFIF-3-21). (A,B), conidiomata on PNA after 30 days incubation, (CF), conidiomata on PDA after 30 days incubation, (G), alpha (α) conidia, (H), beta (β) conidia. Scale bars: AB, 500 µm; CF, 200 µm; GH, 10 µm.
Figure 2. Asexual reproduction of Diaporthe passifloricola (NFIF-3-21). (A,B), conidiomata on PNA after 30 days incubation, (CF), conidiomata on PDA after 30 days incubation, (G), alpha (α) conidia, (H), beta (β) conidia. Scale bars: AB, 500 µm; CF, 200 µm; GH, 10 µm.
Plants 10 00218 g002
Figure 3. (A) Nanfengmiju fruit from Jiangxi Province showing symptoms of melanose. (B) pathogenicity stimulating melanose symptoms on mandarin fruit. For each strain, 300 μL of conidial suspensions is dropped on a piece of cotton, and then placed on the bottom of the fruit. The inoculated fruit are placed in a plastic chamber maintain 95% relative humidity, incubated at 25 °C 12 h of light and 12 h of dark for 15 days. (C) pathogenicity stimulating stem-end rot symptoms on stem-end of mandarin fruit. The stems of citrus fruit are removed carefully, and 10 μL of conidial suspension of each strain is dropped there and incubated at 25 °C in the dark for 7 days. Note: B and C, from left to right are sterile water, conidial suspensions of D. citri (isolate NFHF-8-4) and conidia suspensions of D. passifloricola (isolate NFIF-3-21), respectively.
Figure 3. (A) Nanfengmiju fruit from Jiangxi Province showing symptoms of melanose. (B) pathogenicity stimulating melanose symptoms on mandarin fruit. For each strain, 300 μL of conidial suspensions is dropped on a piece of cotton, and then placed on the bottom of the fruit. The inoculated fruit are placed in a plastic chamber maintain 95% relative humidity, incubated at 25 °C 12 h of light and 12 h of dark for 15 days. (C) pathogenicity stimulating stem-end rot symptoms on stem-end of mandarin fruit. The stems of citrus fruit are removed carefully, and 10 μL of conidial suspension of each strain is dropped there and incubated at 25 °C in the dark for 7 days. Note: B and C, from left to right are sterile water, conidial suspensions of D. citri (isolate NFHF-8-4) and conidia suspensions of D. passifloricola (isolate NFIF-3-21), respectively.
Plants 10 00218 g003
Figure 4. Bayesian inference phylogenetic tree is generated from the analysis of the combined sequences of five loci (ITS, TUB, TEF, HIS, and CAL). Posterior probabilities support values ≥0.7 and Bootstrap support values ≥50%, Bayesian posterior probabilities values (BIPP)/ Maximum likelihood bootstrap values (MLBS)/ Maximum parsimony bootstrap values (MPBS) are displayed at the nodes. The tree is rooted with D. citri CBS 135422. Ex-type, ex-epitype, and holotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given.
Figure 4. Bayesian inference phylogenetic tree is generated from the analysis of the combined sequences of five loci (ITS, TUB, TEF, HIS, and CAL). Posterior probabilities support values ≥0.7 and Bootstrap support values ≥50%, Bayesian posterior probabilities values (BIPP)/ Maximum likelihood bootstrap values (MLBS)/ Maximum parsimony bootstrap values (MPBS) are displayed at the nodes. The tree is rooted with D. citri CBS 135422. Ex-type, ex-epitype, and holotype cultures are indicated in bold. The codes of isolates used for phylogenetic tree are given.
Plants 10 00218 g004
Table 1. GenBank accession numbers of isolates used in this study.
Table 1. GenBank accession numbers of isolates used in this study.
Diaporthe Species Culture No.Host SpeciesOriginGenBank No. Reference(s)
ITSTUBTEFHISCAL
D. acaciarumCBS 138862Acacia tortilisTanzaniaKP004460KP004509KP004504[77]
D. acericolaMFLUCC 17-0956Acer negundoItalyKY964224KY964074KY964180KY964137[78]
D. alangiiCFCC 52556Alangium kurziiChinaMH121491MH121573MH121533MH121451MH121415[31]
D. amaranthophilaATCC 74226Amaranthus sp.USAAF079776[36]
D. ambiguaCBS 114015Pyrus communisSouth AfricaKC343010 KC343978KC343736 KC343494KC343252 [37]
D. angelicaeCBS 111592Heracleum sphondyliumAustriaKC343027 KC343995KC343753 KC343511 KC343269[37]
D. apiculatumCGMCC3.17533Camellia sinensisChinaKP267896KP293476 KP267970 [79]
D. arctiiCBS 136.25Arctium sp.UnknownKC343031 KC343999KC343757 KC343515 KC343273 [37]
D. batatasCBS 122.21Ipomoea batatasUSA KC343040 KC344008 KC343766 KC343524KC343282 [37]
D. beilharziaeVPRI 16602Indigofera australisAustraliaJX862529 KF170921JX862535 [80]
D. caryaeCFCC 52563Carya illinoensisChinaMH121498MH121580MH121540MH121458MH121422[31]
D. chimonanthiSCHM 3614Chimonanthus praecoxChinaAY622993[81]
D. chromolaenaeMFLUCC 17-1422Chromolaena odorataThailandMT214362[82]
D. cichoriiMFLUCC 17-1023Cichorium intybusItalyKY964220KY964104KY964176KY964133[78]
D. citriCBS 135422Citrus sp.USAKC843311KC843187KC843071MF418281KC843157[25,51]
D. compactaCGMCC3.17536Camellia sinensisChinaKP267854 KP293434 KP267928 KP293508[79]
D. convolvuliCBS 124654Convolvulus arvensisTurkey KC343054 KC344022KC343780 KC343538KC343296 [37]
D. cucurbitaeDAOM 42078Cucumis sativusCanada KM453210 KP118848 KM453211 KM453212[42]
D. cuppateaCBS 117499Aspalathus linearisSouth AfricaKC343057 KC344025KC343783 KC343541KC343299[37]
D. diacheniiPH10-1UnknownLithuaniaKR870866[83]
D. durionigenaVTCC 930005Durio zibethinusVietnamMN453530MT276159MT276157[40]
D. durionigenaKCSR1906.7Durio zibethinusVietnamMN453531MT276160MT276158[40]
D. endophyticaCBS 133811Schinus terebinthifoliusBrazil KC343065 KC344033 KC343791 KC343549KC343307 [37]
D. fructicolaMAFF 246408Passiflora edulis x P. edulis f. flavicarpaJapanLC342734LC342736LC342735LC342737LC342738[84]
D. fructicolaMAFF 246409Passiflora edulis x P. edulis f. flavicarpaJapanLC342739LC342741LC342740LC342742LC342743[84]
D. ganjaeCBS 180.91Cannabis sativaUSAKC343112 KC344080KC343838 KC343596 KC343354 [37]
D. glabraeSCHM 3622Bougainvillea glabraChinaAY601918[85]
D. goulteriBRIP 55657aHelianthus annuusAustraliaKJ197289 KJ197270KJ197252 [23]
D. gulyaeBRIP 54025Helianthus annuusAustraliaJF431299 KJ197271JN645803 [23,86]
D. guttulataCGMCC3.20100UnknownChinaMT385950MT424705MT424685MW022491MW022470[87]
D. helianthiCBS 592.81Helianthus annuusSerbia KC343115 KC344083KC343841 KC343599 KC343357 [37]
D. hordeiCBS 481.92Hordeum vulgareNorway KC343120 KC344088KC343846 KC343604 KC343362 [37]
D. hubeiensisJZB320123Vertis viniferaChinaMK335809MK500147MK523570MK500235[88]
D. infecundaCBS 133812Schinus terebinthifoliusBrazil KC343126 KC344094KC343852 KC343610 KC343368 [37]
D. infertilisCBS 230.52Citrus sinensisSurinameKC343052KC344020KC343778KC343536KC343294[37]
D. kongiiBRIP 54031Helianthus annuusAustraliaJF431301 KJ197272JN645797 [23,86]
D. leucospermiCBS 111980Leucospermum sp.AustraliaJN712460[89]
D. longicollaATCC 60325Glycine maxUSAKJ590728 KJ610883 KJ590767 KJ659188KJ612124 [42]
D. longicollaCBS 127267Glycine maxCroatiaKC343199KC344167KC343925KC343683KC343441[42]
D. longicollaCBS 116023Glycine maxUSAKC343198KC344166KC343924KC343682KC343440[42]
D. longisporaCBS 194.36Ribes sp.Canada KC343135 KC344103KC343861 KC343619 KC343377 [37]
D. lusitanicaeCBS 123212Foeniculum vulgarePortugal KC343136 KC344104KC343862 KC343620KC343378 [37]
D. malorumCBS 142383Malus domesticaPortugalKY435638KY435668KY435627KY435648KY435658[90]
D. manihotiaCBS 505.76Manihot utilissimaRwanda KC343138 KC344106KC343864 KC343622 KC343380 [37]
D. masireviciiBRIP 57892aHelianthus annuusAustraliaKJ197277 KJ197257KJ197239 [23]
D. megalosporaCBS 143.27Sambucus canadensisUnknownKC343140 KC344108KC343866 KC343624 KC343382 [37]
D. melonisCBS 507.78Cucumis meloUSAKC343142 KC344110 KC343868 KC343626KC343384 [37]
D. michelinaSCHM 3603Michelia albaChinaAY620820[30]
D. middletoniiBRIP 54884eRapistrum rugostrumAustraliaKJ197286 KJ197266KJ197248[23]
D. minusculataCGMCC3.20098UnknownChinaMT385957MT424712MT424692MW022499MW022475[87]
D. miriciaeBRIP 54736jHelianthus annuusAustraliaKJ197282 KJ197262KJ197244 [23]
D. miriciaeBRIP 55662cGlycine maxAustraliaKJ197283KJ197263KJ197245[23]
D. miriciaeBRIP 56918aVigna radiataAustraliaKJ197284KJ197264KJ197246[23]
D. neoarctiiCBS 109490Ambrosia trifidaUSAKC343145 KC344113KC343871 KC343629 KC343387 [37]
D. novemCBS 127270Glycine maxCroatia KC343156 KC344124KC343882 KC343640 KC343398 [37]
D. ovalisporaCGMCC3.17256Citrus limonChinaKJ490628 KJ490449 KJ490507 KJ490570[6]
D. pachiraeCOAD2074Pachira glabraBrazilMG559537MG559541MG559539MG559535[91]
D. passifloraeCBS 132527Passiflora edulisSouth AmericaJX069860KY435674KY435633KY435654KY435664[92]
D. passifloricolaCBS 141329Passiflora foetidaMalaysiaKX228292KX228387KX228367[39]
D. passifloricolaNFIF-3-11Citrus reticulata cv. NanfengmijuChinaMG786598MG925398MG925401MK238998MK238995This study
D. passifloricolaNFIF-3-19Citrus reticulata cv. NanfengmijuChinaMG786599MG925399MG925402MK238999MK238996This study
D. passifloricolaNFIF-3-21Citrus reticulata cv. NanfengmijuChinaMG786600MG925400MG925403MK239000MK238997This study
D. phaseolorumCBS 139281Phaseolus vulgarisUSAKJ590738 KJ610893 KJ590739 KJ659220KJ612135 [42]
D. pyracanthaeCBS 142384Pyracantha coccineaPortugalKY435635KY435666KY435625KY435645KY435656[90]
D. racemosaeCBS 143770Euclea racemosaSouth AfricaMG600223MG600227MG600225MG600221MG600219[93]
D. rosaeMFLUCC 17-2658Rosa sp.ThailandMG828894MG843878MG829273[41]
D. rosaeMFLUCC 18-0354Magnolia champacaThailandMG906792MG968951MG968953[94]
D. rosaeMFLUCC 17-2574Senna siameaThailandMG906793MG968952MG968954[94]
D. sackstoniiBRIP 54669bHelianthus annuusAustraliaKJ197287 KJ197267KJ197249 [23]
D. salicicolaVPRI 32789Salix purpureaAustraliaJX862531 KF170923JX862537 [80]
D. sambucusiiCFCC 51986Sambucus williamsiiChinaKY852495KY852511KY852507KY852503KY852499[95]
D. schiniCBS 133181Schinus terebinthifoliusBrazil KC343191 KC344159KC343917 KC343675 KC343433 [37]
D. schoeniMFLUCC 17-2930Schoenus nigricansItalyKY964226KY964109KY964182KY964139[78]
D. sclerotioidesCBS 296.67Cucumis sativusNetherlands KC343193 KC344161KC343919 KC343677 KC343435 [37]
D. serafiniaeBRIP 55665aHelianthus annuusAustraliaKJ197274 KJ197254KJ197236 [23]
D. sinensisCGMCC3.19521Amaranthus sp.ChinaMK637451MK660447MK660449MK660451[96]
D. sojaeCBS 139282Glycine maxUSAKJ590719KJ610875KJ590762KJ659208KJ612116[42]
D. sojae (D. actinidiae)ICMP13683Actinidia deliciosaNew ZealandKC145886KC145941[97]
D. sojae (D. camptothecae)SCHM 3611Camptotheca acuminateChinaAY622996[81]
D. sojae (D. kochmanii)BRIP 54033Helianthus annuusAustraliaJF431295 JN645809[42,86]
D. sojae (D. melonis var. brevistylospora)MAFF 410444Cucumis meloJapanKJ590714 KJ610870 KJ590757 KJ659203KJ612111 [42]
D. stewartiiCBS 193.36Cosmos bipinnatusUnknownFJ889448 JX275421GQ250324JX197415[44,45]
D. subellipicolaKUMCC 17-0153UnknownChinaMG746632MG746634MG746633[98]
D. subordinariaCBS 101711Plantago lanceolataNew Zealand KC343213 KC344181KC343939 KC343697 KC343455 [37]
D. tecomaeCBS 100547Tabebuia sp.Brazil KC343215 KC344183KC343941 KC343699 KC343457 [37]
D. tectonaeMFLUCC 12-0777Tectona grandisThailandKU712430KU743977KU749359KU749345[99]
D. tectonendophyticaMFLUCC 13-0471Tectona grandisThailandKU712439KU743986KU749367KU749354[99]
D. terebinthifoliiCBS 133180Schinus terebinthifoliusBrazil KC343216 KC344184KC343942 KC343700 KC343458[37]
D. thunbergiicolaMFLUCC 12-0033Thunbergia laurifoliaThailandKP715097KP715098[100]
D. tulliensisBRIP 62248aTheobroma cacaoAustraliaKR936130KR936132KR936133[101]
D. ueckeraeCBS 139283Cucumis meloUSAKJ590726KJ610881KJ590747KJ659215KJ612122[42]
D. ueckeraeFAU659Cucumis meloUSAKJ590724KJ610879KJ590745KJ659213KJ612120[42]
D. ueckeraeFAU658Cucumis meloUSAKJ590725KJ610880KJ590746KJ659214KJ612119[42]
D. ueckeraeFAU660Cucumis meloUSAKJ590723KJ610878KJ590744KJ659212KJ612121[42]
D. unshiuensisCGMCC3.17569Citrus unshiuChinaKJ490587KJ490408 KJ490466 KJ490529[6]
D. unshiuensisZJUD51Fortunella margarita (Lour.) SwingleChinaKJ490586KJ490407KJ490465KJ490528[6]
D. unshiuensisZJUD50Fortunella margarita (Lour.) SwingleChinaKJ490585KJ490406KJ490464KJ490527[6]
D. vexansCBS 127.14Solanum melongenaUSA KC343229 KC344197KC343955 KC343713 KC343471 [37]
D. vitimegasporaSTE-U2675Vitis viniferaTaiwanAF230749[26]
D. vochysiaeLGMF1583Vochysia divergensBrazilMG976391MK007527MK007526MK033323MK007528[102]
Diaporthe sp. 1CBS 119639Man, abscessGermanyKC343202KC344170KC343928KC343686KC343444[37]
Diaporthella corylinaCBS 121124Corylus sp.ChinaKC343004KC343972KC343730KC343488KC343246[37]
a ATCC: American Type Culture Collection, Manassas, Virginia, USA; BRIP: Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Center, Beijing, China; CGMCC: China General Microbiological Culture Collection, Beijing, China; COAD: Coleção Octávio Almeida Drummond, Universidade Ferderal de Viçosa, Viçosa, Brazil; DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; FAU: Isolates in culture collection of Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, Maryland, USA; ICMP: International Collection of Micro-organisms from Plants, Landcare Research, Auckland, New Zealand; JZB: Culture collection of Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China; KCSR, VTCC: Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology (IMBT), Vietnam National University, Hanoi, Vietnam; HUMCC: Kunming Institute of Botany Culture Collection, Yunnan, China; LGMF: Culture collection of Laboratory of Genetics of Microorganisms, Federal University of Parana, Curitiba, Brazil; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; SCHM: Mycological Herbarium of South China Agricultural University, Guangzhou, China; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; VPRI: Victorian Plant Pathogen Herbarium, Bundoora, Australia; ZJUD: Diaporthe species culture collection at the Institute of Biotechnology, Zhejiang University, Hangzhou, China; Ex-type, ex-epitype, and holotype cultures are indicated in bold. Isolates obtained in this study are indicated in italics. b ITS: Nuclear ribosomal internal transcribed spacer regions; TUB: Beta-tubulin gene; TEF: Translation elongation factor 1-α gene; HIS: Histone-3 gene; and CAL: Calmodulin gene. Sequences generated in this study are indicated in italics.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Chaisiri, C.; Liu, X.-Y.; Yin, W.-X.; Luo, C.-X.; Lin, Y. Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China. Plants 2021, 10, 218. https://doi.org/10.3390/plants10020218

AMA Style

Chaisiri C, Liu X-Y, Yin W-X, Luo C-X, Lin Y. Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China. Plants. 2021; 10(2):218. https://doi.org/10.3390/plants10020218

Chicago/Turabian Style

Chaisiri, Chingchai, Xiang-Yu Liu, Wei-Xiao Yin, Chao-Xi Luo, and Yang Lin. 2021. "Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China" Plants 10, no. 2: 218. https://doi.org/10.3390/plants10020218

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop