Identification of Lasiodiplodia Pseudotheobromae Causing Fruit

plants

Brief Report Identiﬁcation of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China

Jianghua Chen 1,2, Zihang Zhu 1, Yanping Fu 1,2, Jiasen Cheng 1, Jiatao Xie 1 and Yang Lin 1,*

1 Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan 430070, China; [email protected] (J.C.); [email protected] (Z.Z.); [email protected] (Y.F.); [email protected] (J.C.); [email protected] (J.X.) 2 National R & D Center for Citrus Postharvest Technology, Huazhong Agricultural University, Wuhan 430070, China * Correspondence: [email protected]; Tel.: +86-27-87280487

Abstract: Considering the huge economic loss caused by postharvest diseases, the identification and prevention of citrus postharvest diseases is vital to the citrus industry. In 2018, 16 decayed citrus fruit from four citrus varieties—Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)—showing soft rot and sogginess on their surfaces and covered with white mycelia were collected from storage rooms in seven provinces. The pathogens were isolated and the pathogenicity of the isolates was tested. The fungal strains were identified as Lasiodiplodia pseudotheobromae based on their morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin (TUB) gene sequences. The strains could infect wounded citrus fruit and cause decay within two days post inoculation, but could not infect unwounded fruit. To our knowledge, this is the first report of citrus fruit decay caused by L. pseudotheobromae in China. Keywords: citrus; fruit decay; Lasiodiplodia pseudotheobromae

Citation: Chen, J.; Zhu, Z.; Fu, Y.; Cheng, J.; Xie, J.; Lin, Y. Identification of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China. 1. Introduction Plants 2021, 10, 202. https://doi.org/ Citrus is the second-largest fruit crop in China. It is cultivated in over 20 provinces, 10.3390/plants10020202 with an area of approximately 2.5 million ha [1,2]. China has an over 4000-year history of citrus cultivation. There are an abundant and excellent variety of resources in China. Received: 4 December 2020 Fruit decay is a common disease during postharvest transportation and storage. Generally, Accepted: 19 January 2021 the decay rate is 20% to 30%, and sometimes it can reach up to 50%, which causes serious Published: 21 January 2021 postharvest economic losses [3]. Therefore, disease control is vital for the postharvest period of citrus fruit. Publisher’s Note: MDPI stays neutral Common postharvest citrus diseases include green mold, blue mold, sour rot, an- with regard to jurisdictional claims in thracnose, Diplodia stem-end rot, Diaporthe stem-end rot, brown rot, and Alternaria published maps and institutional affil- stem-end rot, etc. In 2018, a survey of postharvest citrus diseases in storage rooms found iations. that the average fruit decay rate exceeded 30% in seven provinces. In addition to the common postharvest diseases, 16 decayed fruit showing soft rot and soggy lesions with dense white hyphae were also observed. In this study, the causal agent of the citrus fruit rot disease was determined via morphological observation, molecular identification, and Copyright: © 2021 by the authors. pathogenicity tests. Licensee MDPI, Basel, Switzerland. This article is an open access article 2. Results distributed under the terms and 2.1. Fungal Isolates conditions of the Creative Commons Attribution (CC BY) license (https:// In 2018, a total of 2402 citrus fruit from four different citrus varieties (Satsuma man- creativecommons.org/licenses/by/ darin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin 4.0/). (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)) were taken

Plants 2021, 10, 202. https://doi.org/10.3390/plants10020202 https://www.mdpi.com/journal/plants Plants 2021, 10, x FOR PEER REVIEW 2 of 9

2. Results 2.1. Fungal Isolates Plants 2021, 10, 202 In 2018, a total of 2402 citrus fruit from four different citrus varieties (Satsuma2 of 8 mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)) were fromtaken storage from storage houses houses (15 ◦C) (15 in seven°C) in provincesseven provinces (Guangxi, (Guangxi, Hubei, Zhejiang,Hubei, Zhejiang, Hunan, Hunan, Jiangxi, Fujian,Jiangxi, andFujian, Guangdong) and Guangdong) and investigated. and investigated. Except for Except fruit with for fruit typical with green typical mold green and bluemold mold and symptoms,blue mold 402symptoms, diseased 402 fruit diseased samples fruit were samples collected were for fungal collected isolation. for fungal From theisolation. 402 decayed From fruit,the 402 450 decayed fungal strains fruit, 450 were fungal isolated. strains Based were on theisolated. morphological Based on char- the acteristicsmorphological and internalcharacteristics transcribed and internal spacer regionstranscribed (ITS) spacer sequences, regions most (ITS) of themsequences, were identiﬁedmost of them as Geotrichum were identifiedspp., Colletotrichumas Geotrichum spp., DiplodiaColletotrichumspp., Alternariaspp., Diplodiaspp., spp., etc. Al- In addition,ternaria spp., 16 strains etc. In belonging addition, to 16 the strains genus belongingLasiodiplodia towere the genus also isolated Lasiodiplodia from 16 were diseased also fruitsisolated with from soft, 16 water-soaked diseased fruits lesions with soft, (Figure water-soaked1a, Table1). lesions (Figure 1a, Table 1).

Figure 1. Symptoms of of the the diseased diseased fruit fruit and and colony colony morphology morphology on on potato potato dextrose dextrose agar agar (PDA). (PDA). ((a)) SymptomsSymptoms of the diseased citrus citrus fruit. fruit. ( (bb)) Colony Colony morphology morphology of of the the fungal fungal strains strains JX.1, JX.1, GD.2, GD.2, and HN.3 on PDA incubated at at 25 25 °C◦C in in the the dark dark for for 2 2 days; days; (c (c) )Colony Colony morphology morphology of of the the fungal fungal strains JX.1, GD.2,GD.2, andand HN.3HN.3 onon PDAPDA incubatedincubated atat 3535 ◦°CC in the dark for 40 days.

Table 1. Samples used in this study.

No. of Rotten Fruit Except for Lasiodiplodia Total No. of Citrus Varieties Location Those with Green Incidence (%) Strain Investigated Fruit and Blue Mold Symptoms Guangxi, Guilin 1 27 225 0.44 Hunan, Huaihua 5 76 320 1.56 Citrus unshiu Hubei, Wuhan 1 69 258 0.39 Hubei, Yicang 1 18 344 0.29 Zhejiang, Taizhou 2 20 259 0.77 Citrus reticulata Fujiang, Quanzhou 1 7 92 1.09 Blanco cv. Ponkan Citrus reticulata cv. Jiangxi, Nanfeng 3 111 566 0.53 nanfengmiju Citrus reticulata Guangdong, 2 74 338 0.59 Blanco Quangzhou

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2.2. Molecular Characterization Based on the multiple gene analyses, 16 strains were identiﬁed as Lasiodiplodia pseudotheobromae. A MegaBlast analysis showed that they exhibited 100% identity to the type Plants 2021, 10, x FOR PEERstrain REVIEW of L. pseudotheobromae (CBS 116459). Since the 16 strains were of the 4 of 9 same species, three of2.2. them Molecular from Characterization different citrus varieties and producing areas—JX.1, GD.2, and HN.3— were randomlyBased selectedon the multiple for further gene analyses, study (Table16 strains S1). were In total,identified 1727 as charactersLasiodiplodia of 62 isolates from 30pseudotheobromaeLasiodiplodia. Aspecies MegaBlast and analysis one outgroup showed that species they exhibited (Diplodia 100% seriataidentity) to were the used to construct Maximumtype strain of likelihood L. pseudotheobromae (ML) and(CBS Maximum116459). Since Parsimony the 16 strains (MP) were phylogeneticof the same trees. The species, three of them from different citrus varieties and producing areas—JX.1, GD.2, combinedand HN.3—were data included randomly 525 charactersselected for further of ITS study (1–525), (Table 550S1). In characters total, 1727 characters of translation elongation factorof 62 1- isolatesα gene from (TEF 30) Lasiodiplodia (530–1079), species and 643and one characters outgroup of species beta-tubulin (Diplodia seriata (TUB) ) (1085–1727). Based onwere the used MP to construct analysis, Maximum a single likelihood most parsimonious (ML) and Maximum tree Parsimony was constructed (MP) phy- (tree length (TL) = 460,logenetic consistency trees. The combined index (CI) data = included 0.654, retention525 characters index of ITS (RI) (1–525), = 0.854, 550 characters rescaled consistency of translation elongation factor 1-α gene (TEF) (530–1079), and 643 characters of be- index (RC)ta-tubulin = 0.559, (TUB) homoplasy(1085–1727). Based index on the (HI) MP =analysis, 0.346). a single Of all most the parsimonious characters, tree 175 characters were parsimonywas constructed informative, (tree length (TL) 63 variable= 460, consistency characters index (CI) were = 0.654, parsimony retention index uninformative, (RI) and 1,489 characters= 0.854, rescaled were consistency consistent. index (RC) The = topology 0.559, homoplasy of the index ML (HI) tree = 0.346). was similarOf all the to that of the MP tree.characters, The isolates 175 characters were groupedwere parsimony with informative,L. pseudotheobromae 63 variable charactersbut were were distinguished par- from simony uninformative, and 1,489 characters were consistent. The topology of the ML tree L. sterculiaewas similar, L. vitis to that, and of theL. MP mediterranea tree. The isolates, the were species grouped with with the L. pseudotheobromae closest genetic but distance to L. pseudotheobromaewere distinguished(Figure from2). L. sterculiae, L. vitis, and L. mediterranea, the species with the closest genetic distance to L. pseudotheobromae (Figure 2).

74/99 CERC 1961 Lasiodiplodia americana 83/92 CERC 1962 L. americana 99/100 85/94 BL 184 L. exigua CBS 137785 L. exigua 58/64 CBS 124925 L. mahajangana 68/96 CBS 124927 L. mahajangana CMM 4015 L. brasiliense 66/69 CPC 22800 L. brasiliense 69/93 CBS 128313 L. viticola 100/100 UCD 260MO L. viticola CBS 124.13 L. theobromae 98/99 CBS 164.96 L. theobromae 71/79 86/100 CBS 124704 L. gilanensis 86/98 CBS 124705 L. gilanensis 63/82 CBS 128311 L. missouriana 97/100 CBS 128312 L. missouriana CBS 120832 L. plurivora 95/97 CBS 121103 L. plurivora -/55 79/83 CBS 124710 L. iraniensis CBS 124711 L. iraniensis 100/100 CBS 176_26 L. jatrophicola 91/100 CMM 3610 L. jatrophicola 98/98 CBS138653 L. thailandica 92/96 CBS 138760 L. thailandica 98/100 CBS 124706 L. citricola 52/51 87/88 CBS 124707 L. citricola CBS 456.78 L. parva CBS 494.78 L. parva 73/81 CMM 2275 L. euphorbicola 88/94 CMM 3609 L. euphorbicola BOT 29 L. egyptiacae CBS 130992 L. egyptiacae CBS 124708 L. hormozganensis 96/100 CBS 124709 L. hormozganensis 70/85 CMM 3833 L. macrospora -/66 98/100 CMM 3872 L. subglobosa 84/90 CMM 4046 L. subglobosa 80/81 60/74 CMW 4564 L. gravistriata 93/93 CMW 4565 L. gravistriata L. mediterranea 78/98 CBS 137783 CBS 137784 L. mediterranea CBS 124060 L. vitis CBS 116459 60/60 CBS 374.54 79/93 58/53 JX.1 L. pseudotheobromae GD.2 97/99 HN.3 CBS 342.78 L. sterculiae 76/85 CBS 447.62 L. sterculiae CBS 122519 L. margaritacea 96/96 CBS 122065 L. margaritacea CBS 110492 L. crassispora 100/100 CBS 118741 L. crassispora CBS 121770 L. pyriformis 87/92 CBS 121771 L. pyriformis 99/100 68/69 CBS 118740 L. rubropurpurea WAC 12536 L. rubropurpurea 63/69 100/100 CBS 118739 L. venezuelensis WAC 12540 L. venezuelensis 100/100 CBS 116355 L. gonubiensis CBS 115812 L. gonubiensis CBS 112555 Diplodia seriata

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Figure 2. The phylogenetic tree generated from the analysis of the combined sequences of three loci, internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin gene (TUB). Bootstrap support values ≥ 50% (Maximum likelihood bootstrap value (MLBS)/ Maximum parsimony bootstrap value (MPBS) are displayed at the nodes. The tree is rooted with Diplodia seriata (CBS 112555). Ex-type, ex-epitype, and ex-isotype cultures are indicated in bold. Isolates obtained in this study are indicated in italics. The codes of the isolates used for the phylogenetic tree are given. Plants 2021, 10, x FOR PEER REVIEW 5 of 9

Figure 2. The phylogenetic tree generated from the analysis of the combined sequences of three loci, internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin gene (TUB). Bootstrap support values ≥50% (Maximum likelihood bootstrap value (MLBS)/ Maximum parsimony bootstrap value (MPBS) are displayed at the nodes. The tree is rooted with Diplodia seriata (CBS 112555). Ex-type, ex-epitype, and ex-isotype cultures are indicated in bold. Isolates obtained in this study are indicated in italics. The codes of the isolates used for the phylogenetic tree are given.

2.3. Morphological Characteristics Plants 2021, 10, 202 At first, the colonies were white with sparse, fluffy aerial mycelia. After one4 of 8week, they became a pale gray with dark pigment (Figure 1b). Pycnidia were dark brown to black, solitary, globose, and intraepidermal (Figure 3a,b). The pseudparaphyses were hyaline,2.3. Morphological cylindrical-shaped, Characteristics aseptate, sometimes branched, end-rounded, and arising amongAt the ﬁrst, conidiogenous the colonies were cells white (Figure with 3c). sparse, Conidiogenous ﬂuffy aerial mycelia.cells were After hyaline, one week, smooth, cylindrical,they became slightly a pale grayswollen with at dark the pigment base, and (Figure holoblastic1b). Pycnidia (Figure were dark3d). brownConidia to black,were ellipsoidal,solitary, apex globose, and base-rounded, and intraepidermal widest (Figure at the3 a,b).middle, The and pseudparaphyses thick-walled. were Immature hyaline, conidia werecylindrical-shaped, colorless, hyaline, aseptate, and sometimesaseptate, while branched, mature end-rounded, conidia andwere arising dark amongbrown the (Figure 3e,f),conidiogenous one-septate cells with (Figure longitudinal3c). Conidiogenous striations (Figure cells were 3g,h). hyaline, The coni smooth,dial dimensions cylindrical, were slightly swollen at the base, and holoblastic (Figure3d). Conidia were ellipsoidal, apex and (22.5−) 27.8 (−32) µm × (14.5−) 17 (−20) µm (x̅ = 27.8 × 17 µm, n = 50). base-rounded, widest at the middle, and thick-walled. Immature conidia were colorless, hyaline,The strain and aseptate, JX.1 could while grow mature between conidia were10 an darkd 35 brown °C. The (Figure optimal3e,f), one-septategrowth temperature with waslongitudinal 30 °C (Figure striations S1). Meanwhile, (Figure3g,h). all The three conidial of the dimensions strains JX.1, were GD.2, (22.5 − and) 27.8 HN.3 (−32) producedµm a reddish× (14.5− pigment) 17 (−20) atµ m35 (°Cx = (Figure 27.8 × 17 1c).µm, n = 50).

FigureFigure 3. Morphology 3. Morphology of pycnidia, of pycnidia, conidiogenous conidiogenous cells, cells, and thethe conidia conidia of of the the strain strain JX.1. JX.1. (a) Pycnidia(a) Pycnidia induced induced on an on an autoclavedautoclaved citrus citrus twig. twig. (b) (Pycnidiab) Pycnidia induced induced on on a a citrus citrus leaf.leaf. ((cc)) Conidiogenous Conidiogenous layer layer with with paraphyses. paraphyses. (d) Concurrently (d) Concurrently proliferatingproliferating conidiogenous conidiogenous cells. cells. (e) (Hyalinee) Hyaline and and aseptate aseptate conidia. conidia. (f (f)) Septate Septate and dark-walleddark-walled conidia. conidia. (g (,gh,)h Mature) Mature co- nidiumconidium at two atfocal two planes focal planes to show to show the thelongitudinal longitudinal striations. striations. Bars Bars = = 10 10 µm.µm.

◦ 2.4. PathogenicityThe strain JX.1Assay could grow between 10 and 35 C. The optimal growth temperature was 30 ◦C (Figure S1). Meanwhile, all three of the strains JX.1, GD.2, and HN.3 produced a reddishIn the pigment pathogenicity at 35 ◦C assay, (Figure 1maturec). fruits of the Satsuma mandarin were used to test the virulence of the isolates. Firstly, discoloration and necrosis appeared on the fruit surface.2.4. Pathogenicity Then, the Assay lesions enlarged rapidly, became soft and rotten, and were covered with sparseIn the white pathogenicity mycelia. assay, The maturelesions fruits spread of theto Satsumathe whole mandarin fruit within were used3 to 4 to days test after the virulence of the isolates. Firstly, discoloration and necrosis appeared on the fruit surface. Then, the lesions enlarged rapidly, became soft and rotten, and were covered with sparse white mycelia. The lesions spread to the whole fruit within 3 to 4 days after the occurrence of necrosis. Finally, the fruit became soggy. The onset of lesions emerged on wounded Satsuma mandarin fruit within two days of inoculation (dpi) (Figure4). No symptoms of Plants 2021, 10, x FOR PEER REVIEW 6 of 9 Plants 2021, 10, 202 5 of 8 the occurrence of necrosis. Finally, the fruit became soggy. The onset of lesions emerged on wounded Satsuma mandarin fruit within two days of inoculation (dpi) (Figure 4). No symptomsdecay were of observeddecay were on the observed unwounded on fruit.the unwounded The recovered fruit. isolates The showed recovered identical isolates showedmorphological identical traits morphological to the original traits ones. to the original ones.

Figure 4. Pathogenicity of strains JX.1, GD.2, and HN.3 on Satsuma mandarin (Citrus unshiu) fruit. The inoculated fruit Figurewere 4. Pathogenicity placed in a plastic of chamberstrains JX.1, maintained GD.2, and near HN.3 95% relative on Satsuma humidity, mandarin incubated (Citrus at 25 ◦ Cunshiu with) 12 fruit. h of lightThe andinoculated 12 h of fruit were darkplaced for in 4 days.a plastic chamber maintained near 95% relative humidity, incubated at 25 °C with 12 h of light and 12 h of dark for 4 days. 3. Discussion 3. Discussion In this study, 16 L. pseudotheobromae strains were isolated from rotten postharvest citrusIn this fruit study, which were16 L. collected pseudotheobromae from storage strains rooms were in seven isolated provinces from of rotten China. postharvest This is citrusthe firstfruit report which that wereL. pseudotheobromae collected from causesstorage fruit rooms rot on in citrus seven in provinces China. of China. This is the firstThe report conidiaomata that L. pseudotheobromae of the isolates formed causes on fruit citrus rot twigs, on citrus producing in China. slowly maturing conidiaThe conidiaomata with thick walls of the and isolates longitudinal formed striations, on citrus which twigs, are producing typical morphological slowly maturing conidiafeatures with of thethick genus wallsLasiodiplodia and longitudinal[4]. Furthermore, striations, the pseudparaphyseswhich are typical were morphological mostly aseptate. The colonies can grow at 10 ◦C and produce a dark pink pigment at 35 ◦C on features of the genus Lasiodiplodia [4]. Furthermore, the pseudparaphyses were mostly potato dextrose agar (PDA). These morphological characteristics are consistent with those aseptate.of L. pseudotheobromae The colonies [can5,6]. grow Combined at 10 with °C and the resultsproduce of the a dark phylogenetic pink pigment analysis, at these 35 °C on potatoisolates dextrose were identified agar (PDA). as L. pseudotheobromae These morphological. characteristics are consistent with those ofBased L. pseudotheobromae on its morphological [5,6]. and Combined molecular with characteristics, the resultsL. pseudotheobromaeof the phylogenetichas been analysis, thesedistinguished isolates were from identifiedL. theobromae as asL. apseudotheobromae cryptic species [5].. It is a common pathogen of tropical andBased subtropical on its plants morphological and has a wide and host molecular range, including characteristics,Acacia confusa L. pseudotheobromae, Albizia falcataria, has beenEucalyptus distinguishedsp., Mangifera from L. sylvaticaand theobromae, Paulowniaas a cryptic fortune species, and [5].Vitis It is vinifera a common, etc. [ 5pathogen,7–18]. of tropicalL. pseudotheobromae and subtropicalwas plants also isolated and has from a wide the leaf host and range, twig including of Magnolia Acacia candolii confusaas an, Albi- endophyte and a saprobe [19]. It has been reported to be associated with citrus stem-end zia falcataria, Eucalyptus sp., Mangifera sylvaticaand, Paulownia fortune, and Vitis vinifera, etc. rot disease in Bangladesh and the postharvest fruit rot of lemon in Turkey [20,21]. [5,7–18].It L. is difficultpseudotheobromae to evaluate was the also harm isolated of L. pseudotheobromae from the leaf andto citrus twig fruit of Magnolia during the candolii as growthan endophyte and storage and period. a saprobe Although [19]. theIt has fungus been can reported artificially to infectbe associated citrus twigs with and citrus stem-endleaves and rot produce disease pycnidia, in Bangladesh whether itand can the infect postharvest citrus in the fruit field isrot still of unknown. lemon in Due Turkey [20,21].to the wide host range of L. pseudotheobromae, the primary inocula of this disease might alsoIt comeis difficult from other to evaluate plants in the the harm surrounding of L. pseudotheobromae area. At the same time,to citrus since fruit the fungus during the growthhas also and been storage found period. to be an Although endophyte the in severalfungus tropical can artificially and subtropical infect treescitrus [19 twigs,22], and leaveswhether and it produce could endogenously pycnidia, growwhether in citrus it can trees in shouldfect citrus also bein investigated.the field is Follow-upstill unknown. research is needed to clarify the primary inocula and the inoculum source of the disease. Due to the wide host range of L. pseudotheobromae, the primary inocula of this disease might also come from other plants in the surrounding area. At the same time, since the fungus has also been found to be an endophyte in several tropical and subtropical trees [19,22], whether it could endogenously grow in citrus trees should also be investigated. Follow-up research is needed to clarify the primary inocula and the inoculum source of the disease.

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4. Materials and Methods 4.1. Fungal Isolates In 2018, disregarding fruit with typical green mold and blue mold symptoms, 402 diseased fruit samples of 4 different citrus varieties (Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco) were collected from storage houses (15 ◦C) in seven provinces (Guangxi, Hubei, Zhejiang, Hunan, Jiangxi, Fujian, and Guangdong). Fungal isolation was performed as previously described [23]. Single-spore isolation was carried out for all the strains after conidiation.

4.2. Molecular Characterization Genomic DNA was extracted using the CTAB method [24]. Fragments of ITS regions, TEF gene (TEF), and TUB gene were amplified by polymerase chain reaction (PCR) with primer pairs ITS1/ITS4, EF1-688F/EF1-1251R, and Bt2a/Bt2b, respectively [5,25,26]. The annealing temperatures were 54 ◦C for the partial ITS and 55 ◦C for the partial TEF and TUB, as mentioned previously. The size of the PCR products was verified by gel electrophoresis in Tris-borate-EDTA (TBE) buffer using 1% agarose gel. The PCR products were purified using E.Z.N.A.® Gel Extraction Kits (Omega Bio-tek, Norcross, GA, USA) and then cloned into the vector pMD19-T (TaKaRa, Dalian, China). Sequencing was carried out at Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. For each amplicon, three clones were selected for sequencing and assembly to avoid point mutations introduced by PCR and sequencing. The forward and reverse sequences were assembled by BioEdit v.7.2.5 [27]; the sequences of the three loci were deposited in GenBank (www.ncbi.nlm.nih.gov).

4.3. Phylogenetic Analysis Based on a preliminary identiﬁcation by MegaBlast search for ITS regions, all the available type strains with the closest genetic distance were selected for phylogenetic analysis (Table S1). The sequences of the ITS, TEF, and TUB loci were aligned by the MAFFT online service of the European Bioinformatics Institute [28] with L-INS-I iterative reﬁnement method, and manual adjustment was performed when necessary by BioEdit v.7.2.5. Based on the combined dataset of ITS, TEF, and TUB, the ML tree was constructed with 1000 replicates of bootstrap using RAxML-HPC BlackBox v.8.2.10 [29] on the CIPRES Science Gateway v.3.3 Web Portal (https://www.phylo.org). The MP trees were generated with 1000 replicates using Phylogenetic Analyses Using Parsimony (PAUP*) v.4.0b10 [30]. The TL, cCI, RI, RC, and HI were calculated for parsimony and the bootstrap analyses.

4.4. Morphological Characterization To observe the colony morphology, all the strains were incubated on PDA at 25 ◦C in the dark. Meanwhile, to determine their optimal growth temperatures, the strains were incubated at 10, 15, 20, 25, 30, or 35 ◦C in the dark. After conidiation on autoclaved twig and leaf of a Satsuma mandarin at 25 ◦C with diffused daylight for 7 days, the morphology of the conidiogenous cells, paraphyses, and conidia were observed. The dimensions of 50 conidia were also measured under a microscope.

4.5. Pathogenicity Assay To fulﬁll the Koch’s postulates, the pathogenicity of the strains was tested on the detached fruit of Satsuma mandarin. In brief, healthy, mature citrus fruit were surface- sterilized with 75% ethanol and then rinsed with sterile water. After being air dried, the fruit were wounded by an aseptic puncture. Mycelial plugs (6 mm in diameter) cut off from the margin of the activated colonies were transferred onto the wounded or unwounded citrus fruit. Plugs of PDA media were used as controls. The fruit were then incubated at 25 ◦C and 95% relative humidity with 12 h of light and 12 h of dark for 4 days. For each strain, 8 wounded and 8 unwounded fruit were inoculated, and the experiments Plants 2021, 10, 202 7 of 8

were independently repeated twice. To authenticate the pathogens, the causal agents were reisolated.

Supplementary Materials: The following are available online at https://www.mdpi.com/2223-774 7/10/2/202/s1. Figure S1. Effects of ambient temperature on the mycelial growth of strain JX.1. The letters indicate a significant difference (p < 0.05). Table S1. GenBank accession numbers of isolates used in this study. Author Contributions: Conceptualization, J.C. (Jianghua Chen); validation, J.C. (Jianghua Chen) and Z.Z.; formal analysis, J.C. (Jianghua Chen), Y.F., and Y.L.; funding acquisition, Y.F.; project administration, Y.F.; supervision, Y.F., J.C. (Jiasen Cheng), J.X., and Y.L.; writing—original draft, J.C. (Jianghua Chen), Y.F., and Y.L.; writing—review and editing, J.C. (Jianghua Chen), Y.F., and Y.L. All the authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the earmarked fund for China Agriculture Research System, grant number CARS-26. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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