Biological and molecular characterization of seven Diaporthe species associated with kiwifruit shoot blight and leaf spot in China

Copyright: © 2021 Y. Du, X. Wang, Y. Guo, F. Xiao, Y. Peng, N. Hong, G. Wang. This is an open access, peerreviewed article published by Firenze University Press (http://www.fupress. com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


INTRODUCTION
Kiwifruit is known "the king of fruits" due to its rich nutritional content, abundant dietary fibres, balanced nutritional composition of minerals, high vitamin C content, antioxidant properties and other human health-beneficial metabolites, including carotenoids and flavonoids Pan et al., 2020;Wu et al., 2020). The centre of origin of kiwifruit is the mountains and ranges of southwestern China (Yue et al., 2020). Kiwifruit has a short history of domestication, starting from the early 20th century Li et al. 2017aWu et al., 2020. Through decades of domestication and substantial efforts for selection from wild plants, several important horticultural species have been commercially cultivated, including Actinidia chinensis, A. deliciosa, A. eriantha, and A. arguta Song et al., 2020). Actinidia chinensis and A. deliciosa are the major species cultivated in China (Huang, 2009), which had a kiwifruit cultivation area of 240,000 ha in 2018, producing 2.55 MT of fruit, accounting for nearly 55% of the global kiwifruit (FAO, 2018;Guo et al., 2020a).
During the past decades, with the steadily increasing duration of kiwifruit monoculture and the rapid expansion of production, diseases have become prevalent in orchards and nurseries. Branch blight and leaf spot diseases are widespread and prevalent, and these diseases cause serious economic losses in China, and affect development of the kiwifruit industry. Accurate identification of cause of these diseases is important for development of effective biosecurity and trade policies. The most common disease symptoms observed in kiwifruit plantations consist of branch blight, leaf spot, bacterial blossom blight, and fruit rot. These symptoms are related to several fungi (Hawthorne et al., 1982;Pennycook, 1985;Pan et al., 2018) and bacteria (Zhang et al., 2019).
Leaf spot of kiwifruit, caused by Alternaria alternata, Diaporthe spp., Glomerella cingulata, Pestalotiopsis spp. and Phomosis spp., has been previously reported in Korea and New Zealand (Jeong et al., 2008;Hawthorne and Otto, 2012). Didymella bellidis has been reported as the major cause of leaf spot in China (Zou et al., 2019).
The blossom blight of kiwifruit occurs in many countries. Several pathogens have been reported as the causal agents of this disease, including Pseudomonas viridiflava, P. fluorescens, P. syringae, P. fluorescens P. syringae pv. syringae, P. syringae pv. actinidiae, and Botrytis cinerea (Conn and Gubler, 1993;Koh et al., 2001;Shin et al., 2004;Young et al., 2009;Zhang et al., 2019) Fruit rot of kiwifruit can be divided into field rot and postharvest fruit rot. Field rot, caused by Sclerotinia sclerotiorum, affects immature fruits on vines (Pennycook, 1985). More than seven fungi have been reported to be associated with postharvest fruit rots of kiwifruit (Beraha and O'Brien, 1979;Hawthorne et al., 1982;Pennycook, 1985), Diaporthe spp. and Botryosphaeria spp. were reported as the major causes of postharvest fruit rot. In New Zealand, Botrytis cinerea causes storage rot and B. dothidea causes ripening rot. Botryosphaeria dothidea also was the major cause of postharvest fruit rot in Iran (Nazerian et al., 2019). Diaporthe actinidiae has been reported to cause postharvest fruit rot in China, Iran, Korea, and New Zealand (Sommer and Beraha, 1975;Lee et al., 2001;Koh et al., 2005;Mousakhah et al., 2014;Li et al., 2017b). In addition, D. ambigua, D. australafricana, D. novem, and D. rudis have been reported to cause postharvest fruit rot of kiwifruit during cold storage in Chile. Diaporthe ambigua was also isolated from postharvest kiwifruit rots in Greece (Thomidis et al., 2013;. Diaporthe honkongensis has been reported to cause stemend rot in Turkey (Erper et al., 2017). Diaporthe melonis and D. perniciosa have been reported as the major pathogens causing postharvest fruit rots in New Zealand (Beraha and O'Brien, 1979;Hawthorne et al., 1982).
Before the advent of molecular biology technology, identification criteria for Diaporthe species were based on the morphological characteristics (e.g., colony appearance in cultures, size and shape of ascomata and conidiomata, sexual state and connections to the asexual state) and host specificity (Rehner and Uecker, 1994;Santos et al., 2011;Gomes et al., 2013;Yang et al., 2018b). Previous studies demonstrated that these characters were generally not sufficient for species level diagnoses, because some species of Diaporthe are not host-restricted and are capable of infecting several taxonomically unrelated host genera (Rehner and Uecker, 1994;Thompson et al., 2011;Elfar et al., 2013;Thompson et al., 2015). Diaporthe helianthi, as the causal agent of stem canker of sunflower, was first reported in the former Yugoslavia. Subsequent studies confirmed Xanthium italicum, X. strumarium, and Arctium lappa as weed hosts of D. helianthi (Thompson et al., 2015). Three other Diaporthe species, D. gulyae, D. kochmanii, and D. kongii, have been identified as the pathogens of sunflower stem canker (Thompson et al., 2011). In addition, character plasticity and cultural variation of Diaporthe hampered species clarification. Application of molecular data has progressed fungal species definition (Hibbett and Taylor, 2013;Yang et al., 2018a). Diaporthe species are being redefined, based on the combination of morphological, cultural and phytopathogenic characteristics, mating types and DNA sequence data Crous, 2017, 2018;Fan et al., 2018). Adoption of multi-locus phylogeny has provided clear resolution of classification and species .
China is an important kiwifruit-growing country and leader in kiwifruit cultivation. However, in recent years, the incidence and systematic identification of the Diaporthe species associated with branch blight of kiwifruit were only assessed in two orchards of Hubei and Anhui provinces. Only 36 strains were obtained and identified as D. tulliensis, D. actinidiae and D. eres, and it is unclear which species is responsible for the disease in different host varieties or species in different provinces (Bai et al., 2017). In addition, leaf spot of kiwifruit caused by Diaporthe species has been rarely reported in China, which makes effective prevention and control of the disease challenging. Therefore, larger scale surveys are needed to give increased understanding of the relative role of Diaporthe spp. in fungal branch blight and leaf spot found on kiwifruit in China.
The present sampled plants with shoot blight and leaf spot symptoms for pathogen isolation. Phylogenetic analyses based on the nuclear ribosomal internal transcribed spacer region (ITS), translation elongation factor 1-alpha (EF1-α) and beta-tubulin (TUB) genes, coupled with morphology of representative strains, were carried out to determine the diversity of pathogens. After pathogenicity determination, species associated with kiwifruit shoot and leaf blight were identified. This study has provided valuable information on pathogen ecology, as a basis for improving management strategies for these economically important diseases.

Sampling and pathogen isolation
Surveys of incidence of shoot blight and leaf spot diseases were conducted in 16 orchards located in nine provinces of China, including Anhui, Chongqing, Henan, Hubei, Fujian, Shandong, Shanxi, Sichuan, and Zhejiang, from October 2017 to May 2019. A total of 106 samples with symptoms of shoot blight and/or leaf spot were collected from Actinidia chinensis ('Cuiyu', 'Donghong', 'Hongyang', 'Huangjin', 'Jinyan', and 'Longzanghong'), and A. deliciosa ('Cuixiang', 'Hayward', 'Jinkui', and 'Xuxiang'). Six pieces (4-5 mm 2 ) of wood or foliage were cut from each of the diseased tissues neighbouring the asymptomatic regions with a sterile scalpel. After surface sterilization in 1% NaOCl for 45 s, the tissues were treated in 75% ethanol for 45 s, rinsed three times in sterile distilled water for 1 min each, and then dried on sterilized filter paper (Fu et al., 2018). Each tissue piece was placed on potato dextrose agar (PDA; 20% diced potato, 2% dextrose, 1.5% agar, and distilled water) plates and incubated at 25°C in the dark for 3-5 d until fungal colony formation (Bai et al., 2015). Colonies with typical characteristic of Diaporthe spp. were sub-cultured onto fresh PDA plates. The obtained isolates were purified using hyphal tip or single spore methods. Mycelium plugs of purified isolates were transferred to PDA tubes, or stored in 25% glycerol at -80°C for subsequent use (Zhai et al., 2014).

DNA extraction, PCR amplification, and sequencing
Colonies were cultivated on PDA plates where the medium was covered with sterile cellophane which was incubated at 25°C in the dark for 5-7 d. Mycelia was scraped and placed into clean tubes. Total genomic DNA was extracted using a modified CTAB method (Freeman et al., 1996). The quality and quantity of DNA were confirmed visually by staining with Gel Red after electrophoresis in 1% agarose gel and visualization under UV light (λ = 302 nm) trans illumination (Udayanga et al., 2012;Gao et al., 2017). The internal transcribed spacer (ITS) region of the nuclear ribosomal genes was amplified using the primer sets ITS1/ITS4 (White et al., 1990), the primers EF1-728F/EF1-986R (Carbone and Kohn, 1999) were used to amplify part of EF1-α, and the primers Bt-2a/Bt-2b (Glass and Donaldson, 1995) were used to amplify part of TUB. For PCR, an aliquot of 50 µL reaction solution contained 5 µL of 10 × Taq buffer II (Mg 2+ Plus) (TaKaRa), 1 µL of dNTP mixture (2.5 mM each), 1 µL of each primer, 0.5 µL of Taq (5U μL -1 ), 2.0 µL of DNA template, and 39.5 µL of ddH 2 O (Zhai et al., 2014). PCR parameters were initiated at 95°C for 5 min, followed by 35 cycles, each of denaturation at 95°C for 30 s, annealing at appropriate temperature for 30 s (56°C for ITS, 51°C for EF1-α, 61°C for TUB), and extension at 72°C for 30 s, and terminated with a final elongation step at 72°C for 10 min (Guo et al., 2020b). The PCR amplicons were purified and sequenced by Sangon Biotech Company, Ltd. The obtained sequences were analyzed on DNAMAN (v. 9.0; Lynnon Biosoft), and deposited in GenBank (Table 1).

Phylogenetic analyses
Novel sequences generated in this study were blasted against the NCBI's GenBank nucleotide database (www http://blast.ncbi.nlm.nih.gov/) to search for closely similar relatives for a taxonomic framework of the studied  (Katoh and Standley, 2013), and were then manually adjusted in MEGA v. 7 (Kumar et al., 2016). An initial maximum likelihood (ML) phylogenetic analysis was conducted based on EF1-α sequences of 284 isolates obtained in this study and 31 reference strains including one outgroup taxon (Diaporthella corylina CBS121124) deposited in GenBank (Table 1) with a GTR+G substitution model by using IQ-tree v.1.6.8, to give an overview backbone phylogenetic tree for the genus Diaporthe (data not shown). A subset of 43 representative isolates was then selected based on the results of the general EF1-α analysis, and was processed through different phylogenetic analyses conducted individually for each locus and multiple sequences analyses using concatenated ITS, EF1-α, and TUB.
Multi-locus phylogenetic analyses were generated using Maximum likelihood (ML) and Bayesian inference (BI). The maximum-likelihood tree was inferred using the edge-linked partition model in IQ-tree (Nguyen et al., 2015;Minh et al., 2020). For the IQ-tree, the best evolutionary model for each partition was determined using ModelFinder (Minh et al., 2020). In the partition model, IQ-TREE can estimate the model parameters separately for every partition. After ModelFinder found the best partition, IQ-TREE immediately starts the tree reconstruction under the best-fit partition model, Branch supports were assessed with ultrafast bootstrap approximation (UFBoot) of 1000 replicates (Hoang et al., 2017). Additionally, Bayesian inference (BI) was performed on the concatenated loci to construct phylogenies using MrBayes v. 3.2.2 (Ronquist et al., 2003) as described by Crous (2006). MrModeltest v. 2.3 (Nylander, 2004) was used to calculate the best-fit models of nucleotide substitution for each data partition with the corrected Akaike information criterion (AIC). Two analyses of four Markov Chain Monte Carlo (MCMC) chains were conducted from random trees with 8 × 10 6 generations. The analyses were sampled every 1000 generations, which were stopped once the average standard deviation of split frequencies was below 0.01. The first 25% of the trees were discarded as the burn-in phase, and the remaining trees were summarized to calculate the posterior probabilities (PP) of each clade being monophyletic. Phylogenetic trees were visualized in Figtree v.1.4.2 (Rambaut, 2014).

Morphological and growth rate analyses
Based on the results of the phylogenetic analyses, 17 representative isolates (including D. alangii: CQ155, FJHJB57; D. compactum: CQ178, SC67; D. eres: HN10, HB25, SX24, HB24, CQ3; D. hongkongensis: CQ51; D. sojae: CQ14, CQ78, CQ16; D. tectonae: CQ58, SC83; D. unshiuensis: CQ7, CQ9) were selected for morphological observations and growth rate assessments. Three-day-old mycelium plugs (5 mm diam.) were taken from the margins of actively growing cultures and transferred onto the centres of 9 cm diam. Petri dishes containing potato dextrose agar (PDA) or 2% tap water agar supplemented with sterile fennel stems. Cultures were incubated at 25°C with a 14 h/10 h fluorescent light/dark cycle (Guo et al., 2020b). Colony diameters were measured daily for 3 d to calculate mycelium growth rates (mm d -1 ). For each representative isolate, these measurements were made in triplicate. Colony shape, density, and pigment production on PDA were noted after seven days. Generation of ascomata and conidiomata on PDA or fennel stems were examined periodically. Shape, colour, and size of asci were observed using light microscopy (Nikon Eclipse 90i or Olympus BX63), and 50 asci, ascospores, conidiophores, and conidia were measured. Leaves of Actinidia chinensis 'Cuiyu', wounded or unwounded, were inoculated with mycelium plugs of isolates in eight replicates, to assess the pathogenicity of representative isolates selected from the seven Diaporthe species. Fresh and healthy leaves were washed under running tap water followed by surface sterilization with 25% ethanol, drying with sterile tissue paper and then air-drying (Hawthorne and Otto, 2012;Mousakhah et al., 2014;Fu et al., 2018). For the wound inoculation method, the mycelium plugs (agar disks) were placed midway on each side of each leaf midrib after wounding three times by pinpricking with a sterilized needle (insect pin, 0.5 mm diam.). For the non-wound inoculation method, mycelium plugs were placed directly on the unwounded leaves. Inoculations with sterile agar plugs were used as negative controls. The experiment was conducted twice. The inoculated leaves were put into a plastic container covered with plastic film and incubated at 25 ± 1°C with a 12 h/12 h light/dark photoperiod. Symptoms and lesion lengths were recorded at 7 d after inoculation, and re-isolations were made from lesion margins to fulfil Koch's postulates.

Pathogenicity and host range
Pathogenicity was also determined on excised segments of 1-year-old woody shoots of A. chinensis ' Cuiyu,' A. chinensis 'Jinyan', A. chinensis 'Hongyang', and A. chinensis 'Huangjin', in five replicates. Green shoots (5 -10 mm diam.) were pruned from healthy kiwifruit vines and cut into 10 cm long segments, rinsed with tap water, surface disinfected with 75% ethanol, and then air-dried. Wounding and non-wounding inoculation methods were used. For the wounding treatment, a superficial wound (5 mm diam.) was made on each shoot segment by removing the cortex with a disinfected 5 mm diam. hole punch (Bai et al., 2015;Sessa et al., 2017). Agar plugs (5 mm diam.) from fungus cultures were inserted into the wounds, and the inoculated parts were sealed with Parafilm to maintain humidity (Mostert et al., 2001). For the non-wound inoculation method, the agar plugs were placed on the surface of unwounded shoots directly and the inoculated parts were sealed with Parafilm to maintain humidity. All inoculated shoots were kept in plastic containers covered with plastic film and maintained in the laboratory at 25°C. The experiment was conducted twice. Lesion lengths were measured at 10 d after inoculation, and pieces were excised from the xylem or phloem tissues under canker lesions neighbouring asymptomatic regions and were cultured to fulfil Koch's postulates.
Detached fruits of A. deliciosa 'Hayward' were inoculated with seven representative isolates in quadruplicate to determine isolate pathogenicity. The inoculations were conducted using wounded and non-wounded methods as previously described (Diaz et al., 2017;Li et al., 2017a). Healthy fruits were surface-sterilized with 75 c / o ethanol prior to inoculation, washed three times with sterile water, and were air-dried (Zhou et al., 2015;Erper et al., 2017). For the wounded treatment, each mycelium plug was placed on the fruit after wounding once by pinpricking with a sterilized needle (5 mm deep) (Luongo et al., 2011). For the non-wounded method, the mycelium plug was directly placed on the surface of unwounded fruits. Inoculation with sterile agar plugs was used as controls. Inoculation points were individually wrapped with sterilized moist cotton plugs (Zhai et al., 2014;Bai et al., 2015). Fruits were placed in a sealed plastic container at 25°C with a 12 h/12 h light/dark photoperiod. The tests were repeated twice. Seven days post inoculation, symptoms on the fruit were recorded and the lengths of lesions were measured. Recovery isolations were made from the flesh at the margins of developed lesions.
The host range of the seven Diaporthe species was determined on detached shoots of five Rosaceae fruit tree species, including Malus pumila 'Hong Fushi', Prunus salicina 'Dahongpao', Prunus armeniaca 'Helanxiangxing', Pyrus pyrifolia 'Cuiguan' and Prunus persica 'Youtao'. Shoots of these plants were wound-inoculated (as described above). Five shoots of each host were used for each inoculation treatment

Statistical analyses
Data from repeated tests and among treatments in each test were analyzed using SPSS Statistics 21.0 (Win-Wrap® Basic; http://www.winwrap.com) by one-way analysis of variance, and means were compared using Tukey's test at a significance level of P = 0.05.

Sampling and pathogen isolation
Investigations and analyses of the occurrence and sample collection of kiwifruit branch blight and leaf spot were conducted from October 2017 to May 2019. One hundred and six samples were collected from the surveyed orchards and nurseries for fungus isolations; 80 of the samples were diseased branches and 26 were infected leaves. In total, 284 Diaporthe isolates showed typical Diaporthe spp. cultural characteristics (Table  2), including fluffy, flattened, white, creamy, sulphur or grayish aerial mycelium, with solitary or aggregated, globose, dark pycnidia and the presence of alpha and/ or beta conidia (Sessa et al., 2017;Guo et al., 2020b). Among them, 81 isolates were obtained from diseased leaf samples, and were identified as six species of Diaporthe (D. alangii, D. compactum, D. eres, D. hongkongensis, D. sojae, and D. unshiuensis). In total, 203 isolates were derived from infected shoots, and were identified as seven species of Diaporthe (D. alangii, D. compactum, D. eres, D. hongkongensis, D. sojae, D. unshiuensis and D. tectonae). The kiwifruit species and varieties from which these isolates were obtained included A. chinensis 'Cuiyu', 'Donghong', 'Hongyang', 'Huangjin', 'Jinyan', 'Longzanghong', and A. deliciosa 'Cuixiang', 'Hayward', 'Jinkui', and 'Xuxiang'. Branch blight symptoms were commonly observed at the incision or pruned positions, with reddish-black fusiform or irregular necrotic lesions ( Figure 1c); the lesions would gradually expand along each incision (Figure 1e). Under dry climate conditions, the infected branches turned brown and cracked, with internal discolourations (Figure 1d). Whole branches were withered (Figure 1f). The diseased leaves developed silvery gray or bronze spots, which were sporadically distributed on the leaves (Figure 1a). In the later stage of disease development, the spots were expanded to the edges of the leaves, and the leaves withered and curled at their margins. Scattered pycnidia were observed on the diseased leaves (Figure 1b).

Phylogenetic analyses of isolated fungi
The 43 representative isolates were subjected to multilocus phylogenetic analyses with concatenated ITS, EF1-α, and TUB sequences together with 31 reference isolates from previously described species, including the outgroup sequence of Diaporthe corylina (culture CBS 121124). A total of 1557 characters (ITS: 1-541, EF1-α: 542-935, TUB: 936-1557) were included in the phylogenetic analyses. For the Bayesian analyses, the following priors were set in MrBayes for the different data partitions: the SYM+I+G model with invgamma-distributed rates was implemented for ITS; The GTR+G model with gamma-distributed rates was implemented for EF1-α; and the HKY +G model with propinv-distributed rates was implemented for TUB. For the IQ-tree inference, the SYM+I+G model was selected for ITS, GTR+I+G for EF1-α, and HYK+I+G for TUB. Bayesian posterior probability (PP ≥ 0.5) and Maximum likelihood bootstrap values (ML ≥50) were shown at the dendrogram nodes (Figures 2 and 3).
The multi-locus phylogenetic results showed that 43 representative isolates were assigned to seven species (Figure 2). Five isolates grouped with the type strain and other reference sequences of D. alangii (Bayesian posterior probability = 0.52, Maximum likelihood bootstrap value 89). Four isolates clustered together with the type strain and other reference strains of D. compactum with high support (1.00, 99). Ten isolates clustered together with the ex-type strain and other reference strains of D. eres with high support (1.00, 99). Five isolates grouped with the ex-type strain and other reference strains of D. hongkongensis with strong support (1.00, 100). Seven isolates clustered together with the type strain and other reference strains of D. sojae with strong support (1.00, 100). Three isolates grouped with the reference strains of D. tectonae with high support (0.89, 99). Nine isolates were identified as D. unshiuensis, forming a highly supported subclade (1.00, 100).
The individual alignments and trees of the three single loci used in the analyses were also compared with respect to their performance in species recognition. Each gene used differentiated D. compactum, D. eres, D. hongkongensis, D. sojae, and D. unshiuensis. Moreover, ITS and EF1-α gathered D. tectonae and D. alangii into one clade (data not shown). The phylogenetic analysis of TUB sequences showed that similar clades statuses compared with the phylogenetic analyses based on the concatenated ITS, EF1-α, and TUB sequences (Figure 3). The phylogenetic analysis of TUB sequences was conducted with a HKY+I+F substitution model using IQ-tree v.1.6.8, and 436 characters were included in the phylogenetic analysis.

Pathogenicity tests
Two typical symptoms developed on detached leaves of A. chinensis, which were observed at the wound sites at 7 d post inoculation. One symptom consisted of reddish-brown, round or suborbicular, small, slowly expan-sion lesions, while the other consisted of lesions with black necrotic centres surrounded by round or suborbicular brown halos (Figure 5a). The first of these symptoms developed after inoculations with D. compactum, D. eres, D. sojae or D. unshiuensis, while the second was induced by inoculations with D. tectonae, D. alangii or D. hongkongensis. Lesion diameters caused by the different fungi differed significantly. Diaporthe tectonae, or D. alangii caused large lesions (mean diam. = 10-22 mm) on all the inoculated leaves, those caused by D. hongkongensis or D. eres were smaller (5-8 mm), while those caused by isolates of D. sojae, D. compactum, or D. unshiuensis were smaller still (2-4 mm) (Figure 6a). Unwounded leaves inoculated with Diaporthe spp. isolates remained symptomless. In parallel, no lesions were observed on the leaves that were wound and non-wound inoculated with PDA discs as controls. Koch's postulates were fulfilled by re-isolating each Diaporthe sp. isolate only from symptomatic leaves.
In the pathogenicity tests conducted on branches of four kiwifruit varieties, all the Diaporthe species were pathogenic to wounded branches. The symptoms induced by representative isolates were similar, as fusiform necrotic lesions and internal discolouration observed at the wound sites (Figure 5b).  (15-25 mm), and those from the remaining isolates were shorter still (3-10 mm) (Figure 6e). Unwounded shoots of kiwifruit inoculated with Diaporthe spp. isolates remained symptomless, and no lesions developed on the shoots that were wound and non-wound inoculated with PDA discs. Each respective Diaporthe species was reisolated from inoculated symptomatic shoots, fulfilling Koch's postulates for these pathogens. Fruits were susceptible to the representative isolates selected from each species, and all tested species caused rots on wounded fruits. Typical symptoms were some sarcocarp tissues swollen with internal softening around the inoculation wounds at early stages, transparent drops streaming from the inoculation punctures, epidermis peeling, and brownish, damp and rotted flesh (Figure 7a). Fruits inoculated with D. alangii (CQ155), D. eres (HN10), D. sojae (CQ14), D. tectonae (CQ58), or D. hongkongensis (CQ21) had larger lesions (mean diam. = 30-42 mm) than those inoculated with D. unshiuensis (CQ7) or D. compactum (CQ178), (13-17 mm) ( Figure  7b). All the non-wounded fruits inoculated with Diaporthe spp. isolates remained symptomless. The negative controls of wounded and unwounded fruits did not produce lesions. Each respective Diaporthe species was re-isolated from the symptomatic fruits, fulfilling Koch's postulates for these pathogens.
In the host range tests, at 10 d post-inoculation, the seven Diaporthe species all caused canker symptoms on detached shoots of the five different fruit crop plants. After removing phloem tissues, maroon and fusiform necrotic lesions emerged in the underlying wood below, and these extended along the inoculated branches. In most cases, the affected shoots of pear and apple showed swollen and the bark cracking at the margins, with dark-brown to reddish cankers and abundant gummosis were observed at the inoculation sites on the branch of plum and apricot. The symptoms produced on peach shoots were black depressed cankers. The Diaporthe isolates caused different degrees of lesioning on detached branches of the different fruit tree species. Diaporthe tectonae or D. alangii isolates caused large lesions (mean length = 18-39 mm), and D. compactum, D. eres, D. hongkongensis, D. sojae, or D. unshiuensis caused shorter lesions (5-15 mm) on Pyrus pyrifolia 'Cuiguan' (Figure 8a). Lesion lengths on Prunus salicina 'Dahongpao' caused by the seven Diaporthe species were of length 5-20 mm, except that two isolates caused larger lesions, with those from D. sojae isolate (CQ14) being 30 mm, and those from D. alangii isolate (SC74) being 53 mm (Figure 8b). The lesion lengths on Malus pumila 'Hong Fushi' caused by the seven Diaporthe species were mostly 5-15 mm in length, except for those from one isolate of D. alangii (SC74) which were longer (70 mm) (Figure 8c). The lesion lengths on Prunus persica 'Youtao' caused by D. alangii isolate CQ155, D. compactum isolate CQ178, D. eres, D. hongkongensis, D. sojae, or D. unshiuensis were 5-10 mm long, and those from the remaining isolates were larger (15-25 mm) (Figure 8d). Diaporthe alangii caused large lesions (60 mm) on Prunus aremeniaca 'Helanxiangxing', followed by D. hongkongensis,D. eres,D. sojae,D. tectonae isolate CQ58,, and the remaining isolates caused short lesions (5-10 mm) (Figure 8e). No lesions were induced in the branches inoculated with non-colonized PDA plugs.

DISCUSSION
Diaporthe spp. previously reported on kiwifruit have been associated with fruit stem-end rots (Sommer and Beraha, 1975;Hawthorne et al., 1982;Lee et al., 2001;Koh et al., 2005;Luongo et al., 2011;Thomidis et al., 2019), and with shoot blight and leaf spots. In the present study, a large-scale investigation of Diaporthe species associated with kiwifruit infections was conducted in nine major cultivation provinces of China. Multilocus phylogenetic analyses and morphological characterization of isolated fungi were employed to evaluate the diversity of Diaporthe species associated with shoot blight and leaf spot of kiwifruit, and pathogenicity tests was performed to fulfill Koch's postulates for representative Diaporthe isolates. This study has shown that seven Diaporthe species, including D. unshiuensis, D. eres, D. sojae, D. hongkongensis, D. compactum, D. alangii, and D. tectonae, were the causal organisms of shoot blight and leaf spot diseases of kiwifruit. As well, D. unshiuensis, D. sojae, D. compactum, D. alangii, and D. tectonae are here first reported as causes of kiwifruit shoot blight and leaf spot. The study was comprehensive, investigating samples from 16 orchards located in nine major kiwifruit production areas in China, and used phylogenetic analyses and morphology to characterize a large number of fungus isolates.
DNA sequence data are essential for resolving taxonomic questions, redefining species boundaries, and accurate species nomenclature . Phylogenetic analyses individually based on ITS, EF1-α, and TUB sequence data differentiated D. compactum, D. eres, D. hongkongensis, D. sojae, and D. unshiuensis. However, ITS and EF1-α gathered D. tectonae and D. alangii into one clade. Several studies have used three to five concatenated genes simultaneously to separate species within Diaporthe (Santos et al., 2011;Gomes et al., 2013;Gao et al., 2015;Diaz et al., 2017). As ITS and the EF1-α gene have limitations for distinguishing D. alangii and D. tectonae, the concatenated ITS, EF1-α, and TUB phylogenetic analysis was successively employed to discriminate these two fungi. The results showed that the two species clustered into two clades with high bootstrap (1.00/99). These two fungi also differed morphologically, with D. tectonae having shorter alpha conidia than D. alangii.
Since Diaporthe spp. have endophytic, saprobic or pathogenic lifestyles, pathogenicity to kiwifruit was assessed by inoculating leaves, shoots, and fruit of different kiwifruit species, using wound and non-wound inoculation methods. Wound inoculations showed that all the species were pathogenic and caused leaf spot and shoot blight of this host. The different fungi also showed significantly different virulence, with D. alangii and D. tectone as the most aggressive species, followed by D. compactum, D. eres, D. hongkongensis, D. unshiuensis, and D. sojae. It is significant, however, that the inoculations of these seven species on unwounded leaves, branches, and fruits did not cause disease symptoms. These species can be endophytes and opportunist pathogens occurring in a wide range of hosts and later as saprobes on dead host tissues.
Host affiliation has been for species delimitation in Diaporthe, but this has proved uninformative because many Diaporthe spp. have been recorded on a wide range of hosts (Santos and Phillips, 2009;Udayanga et al., 2012;Gao et al., 2015;Guarnaccia et al., 2020). For example, D. lithocarpus was confirmed as the cause of diseases on five plant hosts belonging to different families, and Lithocarpus glabra was shown to host seven different species of Diaporthe (Gao et al., 2014). In the present study, the seven Diaporthe species isolated from kiwifruit were not host-specific. Diaporthe tectonae was first reported to cause branch and twig dieback on Tectonae grandis in Northern Thailand (Doilom et al., 2016), and the present study has showed that D. tectonae could induce shoot blight of kiwifruit in China. Diaporthe alangii was originally isolated from dieback branches of Alangium in China (Yang et al., 2018b). The present study confirmed D. tectonae as the cause of leaf spot and shoot blight of kiwifruit. Diaporthe eres, D. sojae, and D. hongkongensis are pathogens causing shoot canker of grapevine and pear (Dissanayake et al., 2014;Guo et al., 2020b), as well as kiwifruit shoot blight and leaf spot (present study). Diaporthe unshiuensis was reported from the fruit of Citrus unshiu with unidentified symptoms and non-symptomatic branches and twigs of Fortunella margarita (Huang et al., 2015), and the present study showed weak aggressiveness of this species to kiwifruit. Several Diaporthe spp. have been recently recognized as causal agents of diseases of Rosaceae fruit crop plants, including peach, pear, and apple (Bai et al., 2015;Sessa et al., 2017;Tian et al., 2018;  al., 2020b). However, kiwifruit is often in mixed plantings with apple, apricot, pear, peach, and plum in many kiwifruit production areas in China. Results of host range and virulence assessments described here have shown the seven Diaporthe species were pathogenic, not only to kiwifruit, but also to most other Rosaceae fruit crop hosts. This indicates that these pathogens have the potential to infect these alternative hosts, potentially providing pathogen inoculum across these hosts.
In conclusion, identification of these pathogens provides valuable new information to assist understanding of leaf spot and branch blight of kiwifruit. The study has also shown the Diaporthe species responsible for these diseases, which will assist the design of potential disease prevention and management strategies for these economically important diseases.