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Article

Unveiling Species Diversity of Plectosphaerellaceae (Sordariomycetes) Fungi Involved in Rhizome and Root Rots of Ginger in Shandong Province, China

by
Qian Zhao
1,
Ao Jia
1,2,
Hongjuan Yang
1,
Jinming Hu
1,
Xuli Gao
1,
Weiqin Zhao
1,
Lifeng Zhou
3,
Miao Zhang
4,
Zhaoxia Li
1 and
Weihua Zhang
1,*
1
Shandong Key Laboratory of Bulk Open-Field Vegetable Breeding, Ministry of Agriculture and Rural Affairs Key Laboratory of Huang Huai Protected Horticulture Engineering, Institute of Vegetables, Shandong Academy of Agricultural Sciences, Jinan 250100, China
2
College of Plant Protection, Shandong Agricultural University, Tai’an 271018, China
3
Lanling Vegetable Industry Development Center, Linyi 277700, China
4
Weifang Agricultural Technology Extension Center, Weifang 261061, China
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(9), 2180; https://doi.org/10.3390/microorganisms13092180
Submission received: 6 August 2025 / Revised: 9 September 2025 / Accepted: 15 September 2025 / Published: 18 September 2025
(This article belongs to the Special Issue Advances in Fungal Plant Pathogens: Diagnosis, Resistance and Control)
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Abstract

Ginger holds significant economic importance in both China and worldwide agriculture. Fungi from the family Plectosphaerellaceae are globally recognized as aggressive plant pathogens. However, the effects of Plectosphaerellaceae species on ginger have been poorly understood. In this research, we identified two novel Musidium species (M. shandongensis sp. nov. and M. zingiberis sp. nov.), one newly recorded species (Gibellulopsis serrae) and one new host record (Plectosphaerella cucumerina) from the rotten rhizomes and roots of ginger in Shandong Province, China, utilizing morphological observations combined with multilocus phylogenetic analysis of the 28S large subunit (LSU), internal transcribed spacer (ITS) region, and translation elongation factor 1-alpha (TEF1-α) gene, along with pathogenicity analyses. Key diagnostic features include M. shandongensis exhibiting abundant mycelium ropes and coils, M. zingiberis showing dark olivaceous colonies, G. serrae producing brown chlamydospores, and P. cucumerina displaying conspicuous guttulae conidia. Comparative analyses with closely related taxa were based on detailed morphological descriptions, illustrations, and phylogenetic analyses. Artificial inoculation of healthy ginger in vitro and in vivo assays caused characteristic symptoms, such as wilt, leaf yellowing, and rhizome necrosis, identical to those observed on naturally infected plants. Our findings broaden current knowledge on the diversity of Plectosphaerellaceae associated with ginger, revealing them as serious threats to ginger cultivation in China.

1. Introduction

Ginger (Zingiber officinale Roscoe), which originates from Southeast Asia, is globally cultivated primarily for its underground rhizomes [1]. It contains a variety of bioactive components that exhibit potent antioxidant, antitumor, and anti-inflammatory effects [2]. Traditionally, it has been widely utilized as a vegetable, spice, and herbal medicine [3,4]. In China, the cultivation area of ginger has reached 63,806 hectares, with a production of 672,914.37 tons, representing approximately 15% of the global output (FAOSTAT 2023). Despite its high economic value, ginger remains very susceptible to multiple devastating diseases, including soft rot, bacterial wilt, rust, anthracnose, and leaf spot [5,6,7]. Notably, rhizome rot has become a serious threat to ginger cultivation, causing substantial yield losses in ginger-growing regions worldwide. According to recent studies, rhizome rot can lead to yield losses ranging from 20 to 100% [8,9,10], and the occurrence area of ginger rhizome rot has been consistently increasing [11]. A wide range of pathogens are associated with this disease, including fungi (Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii), bacteria (Ralstonia solanacearum, Pectobacterium brasiliense, Pseudomonas spp.), oomycetes (Pythium spp.), and plant-parasitic nematodes (Meloidogyne incognita) [12,13,14].
Gibellulopsis, Musidium, and Plectosphaerella belong to Plectosphaerellaceae, Glomerellales in Sordariomycetes (Index Fungorum 2025) [15]. The family Plectosphaerellaceae was established by Zare et al. [16] and currently comprises 24 genera. Species of Plectosphaerellaceae are globally distributed and thrive in various ecological niches. They are primarily soil-borne saprotrophs or parasites of plants, other fungi, insects, and animals [17,18,19,20,21].
Gibellulopsis, originally proposed by Batista & da Silva Maia [18], was revived by Zare et al. [16], with G. piscis as its type species. The genus Gibellulopsis exhibits hyaline vegetative hyphae, verticillium-like conidiophores, and pigmented intercalary or terminal chlamydospores. Currently, seven species are accepted within Gibellulopsis, isolated from both terrestrial and aquatic environments (Index Fungorum 2025) [15]. Most taxa occurred in soil and decaying plants, while some inhabited cloud water, human and animal tissues, water-damaged structures, and packaging materials [22]. The species G. serrae was named by Giraldo López & Crous [22] for a new combination based on Cephalosporium serrae, Verticillium serrae, and Hyalopus serrae as the synonyms. It is generally known as a frequent soil-borne plant pathogen, and for its salt tolerance properties [23]. To date, G. serrae has been reported in only Cuba, Italy, Canada, India, Germany, Israel, and Japan, but has not been recorded in China (USDA Fungal databases 2025) [24].
The monotypic genus Musidium was established by Giraldo López & Crous [22] to accommodate a species formerly known as Acremonium stromaticum, which was described based on isolates obtained from Musa sp. in Honduras [25]. Musidium is characterized by septate hyphae, poorly branched conidiophores, slimy heads conidia, and dark olivaceous stromata. The type species, M. stromaticum, is commonly referred to as a phytopathogen that colonizes the root, rhizosphere, and leaf of Musa sp. [22]. In addition, the species of M. stromaticum has been reported as pathogens causing brown rhizome rot in ginger [26]. To date, M. stromaticum has been reported in only Colombia, Costa Rica, Honduras, Panama, Philippines, Tanzania, England, and Japan (USDA Fungal databases 2025) [24].
The genus Plectosphaerella was proposed by Klebahn [27] on the basis of P. cucumerina as its type species. Previously, it was a member of the Hypocreaceae (Sordariomycetes, Hypocreales) [28]. It was subsequently placed in Sordariaceae (Sordariomycetes, Sordariales) on the basis of centrum development type [29]. Subsequently, Zare et al. [16] introduced the family Plectosphaerellaceae to accommodate the genus. Its asexual morph was known as Plectosporium [30]. Réblová et al. recommended Plectosphaerella rather than Plectosporium as the accepted generic name, given the former’s priority and extensive use in phytopathology [31]. The genus Plectosphaerella typically exhibits simple or sparsely branched conidiophores, phialide-like conidiogenous cells, and mostly cylindrical conidia arranged in slimy heads [16]. According to Index Fungorum (2025) [15], the genus includes 24 accepted species, although P. himantia, P. kunmingensis, and P. melaena are not considered for Plectosphaerella. Species of Plectosphaerella are globally distributed, occupying various ecological niches [17,32], and are commonly mentioned as fungal pathogens on diverse plants, including melon, pumpkin, ranunculus, tomato, pepper, and bamboo [33,34,35,36]. Also, some species act as endophytic fungi [37] or serve as opportunistic pathogens on arthropods [20]. The combination P. cucumerina, proposed by Gams [38], has been acknowledged as a ubiquitous species with a broad environmental tolerance. It is the causative agent of root rot and wilt diseases in 86 plant species from 54 genera around the world [17,22,30,35,36,37,38,39,40]. In addition, it is relevant in the clinical field [41,42] and occurs in soil, dung, and paper [30].
Although Gibellulopsis, Musidium, and Plectosphaerella have been well studied in recent years [17,22,43], knowledge concerning these genera within ginger remains limited. From August 2023 to December 2024, we observed ginger exhibiting symptoms of wilt, leaf yellowing, basal stem browning, rhizome rot, and root necrosis. The symptoms were similar to those caused by fungi of the family Plectosphaerellaceae. This study focused on the identification of new fungal taxa obtained from the rot rhizomes and roots of ginger sampled in Shandong Province, the largest ginger-producing region in China. Through a polyphasic approach combining morphological features, multilocus phylogenetic inference and pathogenicity assessment, we proposed two novel species of Musidium, namely M. shandongensis sp. nov. and M. zingiberis sp. nov., reported G. serrae as a newly recorded species in China, and identified P. cucumerina as a new host record infecting ginger. Detailed morphological descriptions, illustrations, and phylogenetic data of all identified species are provided. To our knowledge, this is the first identification and characterization of Gibellulopsis, Musidium, and Plectosphaerella species associated with ginger in China.

2. Materials and Methods

2.1. Samples Collection, Fungal Isolation, and Morphological Observations

Ginger plants exhibiting brown lesions and rot symptoms on rhizomes, roots, and stems were sampled from Anqiu City, Changle County, Laizhou City, Laiwu District, Pingdu City, and Yishui County in Shandong Province, China, from 2023 to 2024. All the sampling sites are major ginger-producing areas whose cultivation exceeds approx. 3335 hectares. The samples were carefully sealed in resealable plastic bags, properly labeled with sampling information, and subsequently transported to the laboratory.
Fungal isolation was performed using the tissue separation method. Symptomatic tissues (~25 mm2) were excised from the margins of the necrotic lesions following surface sterilization with 75% ethanol (30 s) and 5% NaClO (1 min), then rinsed thrice with sterile water. The sterilized tissues were dried on sterile filter paper and then transferred to potato dextrose agar (PDA; composed of 20% potato extract, 2% glucose, and 1.5% agar) Petri dishes, followed by incubation in the dark at 25 °C for 5–7 d. Pure colonies were obtained after sequential passage by hyphal tip transfer on PDA. The obtained isolates were stored on PDA slants for short-term preservation at 4 °C, and in 25% glycerol for long-term preservation at −80 °C. The specimens were deposited in the Fungal Herbarium of the Shandong Academy of Agricultural Sciences (SAAS), and corresponding living cultures were preserved in Qian Zhao’s personal culture collection (QZ).
Colony characteristics and diameters were determined from cultures grown on PDA and oatmeal agar (OA, 3% filtered oat flakes and 1.5% agar) media after incubation for 7 days at 25 °C in the dark and imaged with a digital camera (Canon EOS M100, Tokyo, Japan). Color descriptions were based on Rayner’s method [44]. The micromorphological characteristics, including shapes, colors, and sizes of hyphae, conidiophores, conidiogenous cells, and conidia, were examined and documented with lactic acid as a mountant using a biological microscope (Leica DM 1000, Wetzlar, Germany) equipped with a Leica MC 170HD digital camera. At least 30 random measurements of each structure were recorded using the Nano Measurer v. 1.2 software (Version 1.2, Shanghai, China). Newly described taxa were registered in the Fungal Names database.

2.2. DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA was extracted from actively growing mycelia cultured on PDA using the Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The 28S large subunit (LSU) region, the internal transcribed spacer (ITS) region, and the translation elongation factor 1-alpha (TEF1-α) gene were amplified and sequenced using the corresponding primer pairs ITS4/ITS5 [45], LR0R/LR5 [46,47], and EF1-983F/EF1-2218R [48]. Polymerase chain reaction (PCR) was performed in a 25 μL mixture, comprising 12.5 μL of 2 × Taq PCR Master Mix (Biomed Biotech Co., Ltd., Beijing, China), 1 μL each of forward and reverse primers (10 μM), 1 μL of template DNA, and distilled deionized water to adjust the volume. The PCR parameters included an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 60 s, with a final elongation step at 72 °C for 10 min. The annealing temperature was optimized by performing a gradient PCR, and 50 °C was chosen as it consistently yielded specific, strong amplification products. Amplified products were visualized on 1% agarose electrophoresis gels and sequenced by Tsingke Biotech Co., Ltd. (Beijing, China).

2.3. Phylogenetic Analyses

Consensus sequences newly generated in this study were assembled using DNAMAN v. 7.0 (Lynnon Biosoft, San Ramon, CA, USA) and deposited in GenBank. Additional reference sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 10 June 2025). Details for all sequences are provided in Table 1. Individual gene alignments were conducted using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html, accessed on 10 June 2025) [49] with default settings and then manually adjusted in MEGA v. 7 [50]. Phylogenetic analysis was performed based on concatenated LSU, ITS, and TEF1-α loci using Bayesian inference (BI) and maximum likelihood (ML) methods. BI analysis was conducted using MrBayes v. 3.2.1 [51]. The best-fit models of nucleotide substitution for each partition were determined by MrModeltest v. 2.3 [52] under the Akaike Information Criterion (AIC). All characters were assigned equal weights, and alignment gaps were considered as missing data. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 1 million generations, sampling every 1000 generations, and stopping when the mean standard deviation of split frequencies was less than 0.01. The first 25% of sampled trees were removed as burn-in, and the posterior probability (PP) above 0.95 was considered significant [53]. ML analysis was conducted in IQ-TREE v1.6.8 [54], using optimal nucleotide substitution models determined by PartitionFinder v2.0 [55]. The branch support estimation utilized a bootstrapping (BS) method with 5000 replicates [56]. Bootstrap values over 70% were considered significant [49]. The resulting trees were visualized using FigTree v. 1.4.3 [57]. Both the BI and ML trees were rooted with Monilochaetes infuscans CBS 379.77.
Table 1. List of species, collections, and sequences used in the phylogenetic analyses in Figure 1.
Table 1. List of species, collections, and sequences used in the phylogenetic analyses in Figure 1.
SpeciesVoucher/CultureGenBank Accession Number
LSUITSTEF1-α
Acremonium acutatumCBS 140.62OQ055348OQ429437OQ470734
CBS 682.71TNG_056976NR_163811OQ470735
CBS 829.73OQ055350OQ429439OQ470736
A. aeriumCBS 189.70TOQ055352NR_189420OQ470738
CBS 379.70COQ055351OQ429440OQ470737
A. alternatumCBS 407.66TNG_056977NR_144913OQ470739
A. brunneisporumCBS 413.76TNG_243194NR_190249OQ470741
A. chlamydosporiumCBS 414.76TOQ055361NR_189421OQ470748
A. multiramosumCBS 147436TNG_242036NR_189426OQ470770
A. sordidulumCBS 385.73TNG_056992NR_159618OQ470782
Gibellulopsis aquaticaCBS 117131TLR025850LR026720LR026414
G. catenataCBS 113951TLR025851LR026721LR026415
G. fuscaCBS 308.38LR025852LR026722LR026416
CBS 560.65TLR025854LR026724LR026418
CBS 120818LR025856LR026726LR026420
G. nigrescensCBS 470.64LR025860LR026730LR026422
CBS 100829LR025862LR026732LR026423
CBS 120949NTLR025868LR026738LR026429
G. serraeCBS 290.30TLR025872LR026742LR026433
CBS 383.66LR025874LR026744LR026435
CBS 892.70TLR025885LR026755LR026445
CBS 100826LR025886LR026756LR026446
SAAS 311704PV702888PV702874PV701791
SAAS 410805PV702887PV702873PV701790
Monilochaetes infuscansCBS 379.77GU180645LR026764LR026460
Musidium shandongensisSAAS 381414TPV702883PV702869PV701786
SAAS 403027PV702884PV702870PV701787
M. stromaticumCBS 132.74LR025919LR026785LR026479
CBS 133.74LR025920LR026786LR026480
CBS 135.74ALR025922LR026787LR026482
CBS 135.74CLR025923LR026788LR026483
CBS 135.74FLR025925LR026790LR026484
CBS 863.73THQ232143DQ825969LN810533
M. zingiberisSAAS 381402TPV702881PV702867PV701784
SAAS 442806PV702882PV702868PV701785
Plectosphaerella alismatisCBS 113362TLR025932LR026794LR026489
P. citrullaeCBS 131740LR025933LR026795LR026490
CBS 131741TLR025934LR026796LR026491
P. cucumerinaCBS 137.33NTLR025935LR026797LR026492
CBS 137.37TLR025936LR026798LR026493
CBS 101014LR025945LR026807LR026502
CBS 131739NTLR025947LR026809LR026504
SAAS 311708PV702886PV702872PV701789
SAAS 481921PV702885PV702871PV701788
P. delsorboiCBS 116708TLR025948LR026810LR026505
P. melonisCBS 489.96TLR025950LR026812LR026507
CBS 525.93LR025951LR026813LR026508
P. populiCBS 139623TKR476783KR476750LR026527
CBS 139624MH878144KR476751LR026528
P. ramiseptataCBS 131743LR025969LR026831LR026529
CBS 131861TLR025970LR026832LR026530
Verticillium alboatrumCBS 130340ETLR025984LR026847LR026543
V. alfalfaeCBS 130603TLR025988LR026851LR026547
V. dahliaeCBS 127.79BLR025989LR026852LR026548
CBS 179.66LR025992LR026854LR026549
CBS 384.49LR026000LR026861LR026554
V. nonalfalfaeCBS 113707LR026071LR026932LR026587
CBS 130339TLR026074LR026935LR026590
V. nubilumCBS 457.51TLR026076LR026937LR026591
V. tricorpusCBS 126.79LR026078LR026939LR026592
CBS 255.57LR026081LR026942LR026595
V. zaregamsianumCBS 130342TLR026098LR026959LR026610
The ex-type, ex-epitype and ex-neotype are indicated using “T”, “ET” and “NT” after strain numbers, and newly generated sequences are indicated in bold.
Microorganisms 13 02180 g001
Figure 1. Phylogenetic tree of Plectosphaerellaceae generated by Bayesian analysis based on concatenated sequences of the LSU, ITS and TEF1-α loci. The tree was rooted to Monilochaetes infuscans (CBS 379.77). Bayesian posterior probability (PP ≥ 0.90) and RAxML bootstrap support values (ML ≥ 70%) were shown at the nodes (PP/ML BS). Strains marked with “T”, “ET” and “NT” are ex-type, ex-epitype, and ex-neotype, respectively. Newly generated sequences are indicated in red.
Figure 1. Phylogenetic tree of Plectosphaerellaceae generated by Bayesian analysis based on concatenated sequences of the LSU, ITS and TEF1-α loci. The tree was rooted to Monilochaetes infuscans (CBS 379.77). Bayesian posterior probability (PP ≥ 0.90) and RAxML bootstrap support values (ML ≥ 70%) were shown at the nodes (PP/ML BS). Strains marked with “T”, “ET” and “NT” are ex-type, ex-epitype, and ex-neotype, respectively. Newly generated sequences are indicated in red.
Microorganisms 13 02180 g001

2.4. Pathogenicity Test

Pathogenicity tests were performed on all Plectosphaerellaceae species isolated from ginger to fulfill Koch’s postulates, as none of them have been previously documented as causal agents of ginger rhizome rot.
In vitro assay: Rhizomes from ginger cv. Laiwu Dajiang were surface-sterilized in 20% ethanol for 30 s, then placed in Petri dishes containing a single layer of moistened sterile filter paper. For each fungal isolate, 5 mm-diameter mycelial disks were inoculated onto wounds created on the rhizomes using a sterile cork borer. The mycelial disks were obtained from 7-day-old colonies of G. serrae (SAAS 311704), M. shandongensis (SAAS 381414), M. zingiberis (SAAS 381402), and P. cucumerina (SAAS 481921). Control rhizomes were similarly treated but inoculated with sterile PDA disks. Each fungal strain was tested on nine replicate rhizomes. After incubation at 25 ± 2 °C for 7 days, disease severity was assessed using a 0 to 5 scale as follows: 0 = no symptoms; 1 = 1–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; and 5 = 81–100% of the rhizome surface exhibiting necrosis. The disease severity index (DSI) was calculated as: DSI = [Σ(number of rhizomes in each disease class × class score)/(total number of rhizomes × maximum disease score)] × 100.
In vivo assay: Two-month-old seedlings of ginger cv. Laiwu Dajiang were transplanted into 628 mL pots containing sterilized commercial potting substrate. Seven days after transplanting, each plant was inoculated via soil drenching with 5 mL of a conidial suspension (106 conidia mL−1), prepared from 10-day-old cultures of each tested fungal species. Plants treated with sterile water served as negative controls. Each fungal isolate was tested on nine seedlings. The seedlings were maintained in a greenhouse (25 ± 2 °C, 70% relative humidity, natural light) for 14 days. Subsequently, rhizomes were removed from pots, carefully washed, and scored for browning symptoms using the 0 to 5 scale described above. To satisfy Koch’s postulates, the fungi were re-isolated from symptomatic rhizomes.

3. Results

3.1. Phylogenetic Analysis

Among the isolates obtained, strains SAAS 311704, SAAS 381402, SAAS 381414, and SAAS 481921 exhibited the characteristic morphological features of Plectosphaerellaceae. BLAST (v. 2.14.0) analyses of LSU, ITS, and TEF1-α sequences (Table 2) further confirmed the taxonomic affiliation of these strains to this family.
To resolve phylogenetic relationships, a concatenated sequence alignment of LSU, ITS, and TEF1-α loci was generated, comprising sixty-two isolates representing six genera: Acremonium (ten sequences), Verticillium (eleven sequences), Musidium (ten sequences), Gibellulopsis (fourteen sequences), Plectosphaerella (sixteen sequences), and Monilochaetes infuscans CBS 379.77 (used as the outgroup). The final dataset contained 2082 characters, including gaps (LSU: 756 bp; ITS: 539 bp; TEF-1α: 787 bp). Of these, 646 characters were variable sites (LSU: 179 bp; ITS: 236 bp; TEF-1α: 231 bp), and 530 characters were parsimony informative (LSU: 149 bp; ITS: 190 bp; TEF-1α: 191 bp). BI analysis was conducted using the best-fit substitution model GTR+I+G across all loci. For ML analysis, the GTR+I+G model was applied to LSU and TEF1-α, while GTR+G was selected for ITS. The topology of multilocus phylogenetic trees obtained from BI and ML inference analyses was congruent; thus, the Bayesian tree is shown (Figure 1). Phylogenetic analyses indicated that the strains of M. zingiberis (SAAS 381402 and SAAS 442806) formed an independent, well-supported branch (1.0 PP, 99% BS) sister to a lineage containing M. stromaticum. In addition, M. shandongensis strains (SAAS 381414 and SAAS 403027) clustered into a separate, strongly supported clade (1.0 PP, 100% BS) closely related to M. zingiberis and M. stromaticum. Moreover, the new G. serrae strains (SAAS 311704 and SAAS 410805) grouped with ex-type strains CBS 290.30 and CBS 892.70 (1.0 PP, 100% BS). The newly obtained strains of P. cucumerina (SAAS 311708 and SAAS 481921) grouped with ex-type strain CBS 137.37 and ex-neotype strains CBS 137.33 and CBS 131739 (1.0 PP, 94% BS). Overall, our isolates represent four phylogenetically distinct lineages, corresponding to two previously known species and two novel species.

3.2. Taxonomy

3.2.1. Gibellulopsis serrae (Maffei) Giraldo López & Crous, Stud Mycol 92: 250 (2018) Figure 2

Fungal Names number: FN 828040.
Materials examined: China, Shandong Province, Qingdao City, Pingdu City, Mingcun Town, Xinan Street, 36°45′36″ N, 119°38′50″ E, 53 m asl, on rhizomes and roots of Zingiber officinale (Zingiberaceae), 7 November 2023, Q. Zhao, H.J. Yang, J.M. Hu, XAJ4; ibid., XAJ16. Yantai City, Laizhou City, Pinglidian Town, 37°17′22″ N, 120°1′31″ E, 27 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 8 October 2024, Q. Zhao, H.J. Yang, A. Jia, PLD5. Yantai City, Laizhou City, Yidao Town, 37°13′58″ N, 120°10′32″ E, 78 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 5 December 2024, Q. Zhao, H.J. Yang, A. Jia, YDZ13.
Mycelium consists of branched, septate, hyaline hyphae with smooth and thin walls, 1.6–2.7 μm in width. Conidiophores arise from either aerial or submerged hyphae, solitary or aggregated, erect or slanted, straight or irregularly curved, unbranched or irregularly branched, bearing 1–2 levels of 1–4 phialides at each node, hyaline, smooth-walled, generally possessing thicker walls than the vegetative hyphae, reaching 45–74 μm in length and 1.8–3.2 μm in basal width. Conidiogenous cells are enteroblastic, monophialidic, terminal or lateral, long-cylindrical to aculeate, hyaline, smooth-walled, with a minute cylindrical collarette and inconspicuous periclinal thickening at the conidiogenous locus, 23–51 μm long and 1.4–2.8 μm wide at the base. Conidia are unicellular, straight, hyaline, thin-walled, displaying morphologies from elongate ellipsoidal to cylindrical with rounded ends, sometimes containing two inconspicuous guttulae, 3.3–5.5 × 1.4–2.6 μm. Chlamydospores are intercalary or terminal, appearing singly or in short chains, subglobose to clavate, thick-walled, gray-brown to dark brown, 5.8–7.9 × 3.1–5.4 μm. Sexual morph was not observed.
Culture characteristics: After 7 days of incubation at 25 °C, colonies on PDA reached 27–29 mm in diameter, exhibiting a flat morphology with moderate, slightly cottony aerial mycelium and white surface appearance. The colony reverse showed an apricot center, gradually fading to creamy white toward the margins. On OA, colonies grew to 24–27 mm in diameter, appearing smooth, felty, membranous with scarce aerial mycelium, hyaline to light gray at the center and pale white at the margin; the reverses were concolorous.
Notes: G. serrae forms a strongly supported clade (1.0 PP, 100% BS) closely related to G. aquatica, G. nigrescens, and G. catenata. Morphologically, G. serrae, G. aquatica, and G. nigrescens produce exclusively 1-celled conidia, while G. catenata may produce both 1- and 2-celled conidia. Conidial dimensions for G. serrae (3.3–5.5 × 1.4–2.6 μm) are shorter than those of G. catenata (4.1–12.9 × 1.5–2.8 μm), and also slightly shorter than those of G. aquatica (3.9–6.1 × 1.6–2.5 μm) and G. nigrescens (4.1–5.6 × 1.6–2.3 μm). Conidiophores further distinguish G. serrae, which bears 1–2 verticillate levels with 1–4 phialides per node, from G. aquatica (1–6 levels, 1–2 phialides) and G. nigrescens (1–4 levels, 1–3 phialides). Additionally, conidiophores of G. serrae (up to 74 μm) are shorter than those of G. aquatica (104 μm) and G. nigrescens (100 μm). Moreover, G. serrae can be distinguished from G. nigrescens by the conidia color (hyaline vs. pale brown with age) and the chlamydospores shape (short chains vs. single). G. serrae has thus far been recorded only from Canada, Cuba, Germany, India, Israel, Italy, and Japan. Consequently, G. serrae is reported for the first time in China.
Microorganisms 13 02180 g002
Figure 2. Gibellulopsis serrae (SAAS 311704). (a,b) Diseased rhizomes of Zingiber officinale. The arrows indicate the disease lesions; (c,d) surface and reverse sides of colony after 7 days on PDA (e,f) and on OA; (g–k) conidiophores; (l,m) conidia; and (n–p) chlamydospores. Scale bars: (g–p) 10 µm.
Figure 2. Gibellulopsis serrae (SAAS 311704). (a,b) Diseased rhizomes of Zingiber officinale. The arrows indicate the disease lesions; (c,d) surface and reverse sides of colony after 7 days on PDA (e,f) and on OA; (g–k) conidiophores; (l,m) conidia; and (n–p) chlamydospores. Scale bars: (g–p) 10 µm.
Microorganisms 13 02180 g002

3.2.2. Musidium shandongensis Q. Zhao & W.H. Zhang, sp. nov. Figure 3

Fungal Names number: FN 572230.
Etymology: shandongensis refers to the collection site, Shandong Province, China.
Materials examined: China, Shandong Province, Jinan City, Laiwu District, Gaozhuang Town, Dongwennan Village, 36°10′44″ N, 117°37′1″ E, 185 m asl, on stems and rhizomes of Zingiber officinale (Zingiberaceae), 14 August 2023, Q. Zhao, H.J. Yang, J.M. Hu, DWN4 (holotype SAAS 381414, ex-type living culture QZ 23829); ibid., DWN23. Weifang City, Xiashan District, Wangjiazhuang Town, Dashuangguotou Village, 36°30′15″ N, 119°22′29″ E, 31 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 30 October 2024, Q. Zhao, H.J. Yang, A. Jia, DSG3, DSG7; ibid., DSG14, DSG16.
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Figure 3. Musidium shandongensis (SAAS 381414). (a–c) Diseased plants, rhizomes and roots of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h–j) conidiophores with conidial heads on mycelial coils; (k–q) conidiophores; (r,s) mycelial coils; (t,u) conidiophores with conidia arranged in slimy heads; (v,w) conidia; and (x) stromatic hyphae. Scale bars: (h–j) 100 µm; and (k–x) 10 µm.
Figure 3. Musidium shandongensis (SAAS 381414). (a–c) Diseased plants, rhizomes and roots of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h–j) conidiophores with conidial heads on mycelial coils; (k–q) conidiophores; (r,s) mycelial coils; (t,u) conidiophores with conidia arranged in slimy heads; (v,w) conidia; and (x) stromatic hyphae. Scale bars: (h–j) 100 µm; and (k–x) 10 µm.
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Mycelium consists of hyaline, branched, septate hyphae with smooth and thin walls, often producing abundant mycelial ropes and coils, 1.5–2.3 μm in width. Conidiophores emerge either individually or in clusters from aerial, substratal, or rope and coiled hyphae. They are straight to flexuous, unbranched or basitonously branched, hyaline, smooth- walled, usually possessing thicker walls than the vegetative hyphae, reaching up to 59.9 μm in length and 3.0 μm in width at the base. Conidiogenous cells are enteroblastic and monophialidic, positioned either terminally or laterally, and exhibit a subulate or acicular shape. They are hyaline, smooth-walled, and feature a short cylindrical collarette along with a prominent periclinal thickening, 16.3–52.3 × 2.0–3.0 μm. Conidia are unicellular, straight, cylindrical to ellipsoidal with rounded ends, hyaline, smooth- and thin-walled, typically produced in slimy heads, 4.0–5.3 × 2.1–3.0 μm. Stromatic hyphae are dark olivaceous, thick-walled, smooth, and formed at the base of plate cultures or along the margins of agar slants. Sexual morph was not observed.
Culture characteristics: On PDA, colonies reached 31–34 mm in diameter after 7 days at 25 °C, presenting as flat, wooly, dirty white, with fimbriate margins, and creamy-white reverses. On OA, colonies grew to 31–36 mm in diameter, with flat, membranous surfaces, scarce aerial mycelium, dirty white centers, pale gray peripheries, filiform margins, and concolorous reverses.
Notes: This type of strain of M. shandongensis formed a distinct lineage sister to the clade harboring M. stromaticum and M. zingiberis (1.0 PP, 100% BS). Morphologically, M. shandongensis can be distinguished from M. stromaticum by the formation of abundant mycelial ropes and coils as well as shorter and wider conidia (4.0–5.3 × 2.1–3.0 vs. 4.2–6.2 × 1.4–2.3 μm). Compared to M. zingiberis, M. shandongensis features more slender conidiophores (59.9 × 3.0 vs. 52.6 × 3.5 μm), narrower conidiogenous cells (16.3–52.3 × 2.0–3.0 vs. 16.5–43.7 × 2.1–3.8 μm), and smaller conidia (4.0–5.3 × 2.1–3.0 vs. 4.1–6.7 × 2.6–3.6 μm). Distinct colony color also differentiates between the two species: M. shandongensis forms dirty intoite and dirty white to pale gray colonies on PDA and OA, whereas M. zingiberis produces dark gray and dark olivaceous to pale gray colonies. The combined phylogenetic and morphological differences support the classification of M. shandongensis as a novel species within Musidium.

3.2.3. Musidium zingiberis Q. Zhao & W.H. Zhang, sp. nov. Figure 4

Fungal Names number: FN 572825.
Etymology: zingiberis is named after the host genus from which it was collected, Zingiber.
Materials examined: China, Shandong Province, Jinan City, Laiwu District, Gaozhuang Town, Dongwennan Village, 36°10′44″ N, 117°37′1″ E, 185 m asl, on stems and rhizomes of Zingiber officinale (Zingiberaceae), 14 August 2023, Q. Zhao, H.J. Yang, J.M. Hu, DWN2 (holotype SAAS 381402, ex-type living culture QZ 23825); ibid., DWN19. Weifang City, Changle County, Yingqiu Town, Lijia Village, 36°35′11″ N, 119°3′15″ E, 56 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 28 April 2024, Q. Zhao, H.J. Yang, J.M. Hu, LJC6; ibid., LJC14. Weifang City, Xiashan District, Wangjiazhuang Town, Dashuangguotou Village, 36°30′15″ N, 119°22′29″ E, 31 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 30 October 2024, Q. Zhao, H.J. Yang, A. Jia, DSG5; ibid., DSG17.
Mycelium consists of branched, septate, hyaline hyphae with smooth and thin walls, frequently forming abundant coils and gracile ropes, 1.6–2.7 μm in width. Conidiophores arise singly or in aggregates from submerged or superficial hyphae, often radiating out from the ropes and coils. They are erect, straight to flexuous at the base, unbranched or mostly with 1–2 lateral branches, hyaline, with smooth cell walls thicker than those of vegetative hyphae, reaching up to 52.6 μm in length and 3.5 μm in width. Conidiogenous cells are enteroblastic, monophialidic, terminal or lateral, subulate or tapering at the top, hyaline, smooth-walled, featuring a distinct periclinal thickening and a cylindrical collarette, 16.5–43.7 × 2.1–3.8 μm. Conidia are aseptate, straight, cylindrical to ellipsoidal with rounded ends, hyaline, thin- and smooth-walled, and typically aggregated in slimy heads, 4.1–6.7 × 2.6–3.6 μm. Stromatic hyphae are dark olivaceous, smooth, branched or unbranched, thick-walled or incrusted, and produced at the base of plate cultures or along the margins of agar slants. Sexual morph was not observed.
Culture characteristics: After 7 days of incubation at 25 °C on PDA, colonies reached 23–27 mm in diameter, appearing flat, felty, dark gray with filiform margins and dark olivaceous reverses. On OA, colonies grew to 25–27 mm in diameter, with flat, membranous surfaces, sparse aerial mycelium, centrally dark olivaceous with pale gray peripheries, margins fimbriate, and reverses concolorous.
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Figure 4. Musidium zingiberis (SAAS 381402). (a–c) Diseased plants and rhizomes of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h–j) conidiophores with conidial heads on mycelial ropes and coils; (k–p) conidiophores; (q) mycelial coils; (r,s) conidiophores with conidia arranged in slimy heads; (t,u) conidia; and (v) stromatic hyphae. Scale bars: (h–j) 100 µm; and (k–v) 10 µm.
Figure 4. Musidium zingiberis (SAAS 381402). (a–c) Diseased plants and rhizomes of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h–j) conidiophores with conidial heads on mycelial ropes and coils; (k–p) conidiophores; (q) mycelial coils; (r,s) conidiophores with conidia arranged in slimy heads; (t,u) conidia; and (v) stromatic hyphae. Scale bars: (h–j) 100 µm; and (k–v) 10 µm.
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Notes: The type culture of M. zingiberis formed a single well-supported clade (1.0 PP, 99% BS) closely related to M. stromaticum. Morphologically, M. zingiberis differs from M. stromaticum in its abundant mycelial coils and gracile ropes, as well as wider conidiophores, conidiogenous cells, and conidia (52.6 × 3.5 vs. 59 × 2.5 μm; 16.5–43.7 × 2.1–3.8 vs. 23–55 × 2–2.5 μm; 4.1–6.7 × 2.6–3.6 vs. 4.2–6.2 × 1.4–2.3 μm). Furthermore, colony color on PDA and OA serves as a key distinguishing feature. M. zingiberis produces colonies ranging from dark gray and dark olivaceous to pale gray, whereas M. stromaticum forms uniformly dirty white colonies. Therefore, the combination of unique morphological traits and multilocus phylogenetic evidence supports the recognition of M. zingiberis as a novel species in Musidium.

3.2.4. Plectosphaerella cucumerina (Lindf.) W. Gams, in Domsch & Gams, Fungi in Agricultural Soils: 160 (1972) Figure 5

Fungal Names number: FN 320609.
Materials examined: China, Shandong Province, Qingdao City, Pingdu City, Mingcun Town, Xinan Street, 36°45′36″ N, 119°38′50″ E, 53 m asl, on rhizomes and roots of Zingiber officinale (Zingiberaceae), 7 November 2023, Q. Zhao, H.J. Yang, J.M. Hu, XAJ8. Linyi City, Yishui County, Yuandongtou Town, Nanqiangyu Village, 35°43′50″ N, 118°22′6″ E, 247 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 19 August 2024, Q. Zhao, H.J. Yang, A. Jia, NQY5, NQY8; ibid., NQY18, NQY21. Weifang City, Anqiu City, Xingan Town, 36°18′39″ N, 119°9′42″ E, 85 m asl, on rhizomes of Zingiber officinale (Zingiberaceae), 29 August 2024, Q. Zhao, H.J. Yang, A. Jia, XAT3.
Mycelium consists of branched, septate, hyaline hyphae with smooth and thin walls, occasionally forming coils, 1.8–3.0 μm in width. Conidiophores are mostly solitary, erect or sinuous, arising from superficial and submerged hyphae, or from hyphal coils. They are typically unbranched, rarely branched, hyaline, and thin-walled, reaching up to 55.4 μm in length and 3.8 μm in width at the base. Conidiogenous cells are enteroblastic, monophialidic, terminal or lateral, discrete, subcylindrical to ampulliform, occasionally sinuous, straight at the apex, gradually tapering to the apex. They are hyaline, smooth-walled, with distinct periclinal thickening and a cylindrical collarette, 11.5–37 μm in length and 1.9–3.6 μm in width at the base. Conidia are acrogenous, unicellular, fusiform or ellipsoidal, hyaline, straight, thin- and smooth-walled, often containing 1–5 conspicuous guttulae, 5.3–8.6 × 2.2–3.5 μm. Neither chlamydospores nor sexual morphs were observed.
Culture characteristics: After 7 days of incubation at 25 °C on PDA, colonies reached 44–46 mm in diameter, appearing flat and repressed, with slimy surfaces and sparse aerial hyphae. The colony center was buff, fading to milky white at the periphery, with a regular margin and concolorous reverse. On OA, colonies grew to 42–46 mm in diameter, presenting as flat with sparse aerial hyphae, creamy white, with a regular margin and concolorous reverse.
Notes: On the basis of multilocus phylogenetic analyses, the isolates of P. cucumerina clustered in a single branch (1.0 PP, 94% BS), including the ex-type P. cucumerina CBS 137.37 and the neotypes of P. cucumerina CBS 131739 and CBS 137.33, which were placed basal to the clade (1.0 PP, 91% BS) containing P. citrullae and P. ramiseptata. The conidia of P. cucumerina are often fusiform or ellipsoidal with up to 5 guttulae, and those of P. citrullae are ellipsoids with up to 2 guttulae. P. ramiseptata is morphologically differentiated from P. cucumerina by its branched and septate conidiogenous cells as well as the production of 1-septate conidia. P. cucumerina was first reported in China from Lycopersicon esculentum [58]. Subsequently, its host range extended to Helianthus annuus, Lagenaria siceraria, Solanum tuberosum, Brassica oleracea, Cucumis sativus, Foeniculum vulgare, Raphanus sativus, Phaseolus vulgaris, and Medicago sativa [40,59,60,61,62,63,64]. The present study represents the first identification of P. cucumerina isolated from ginger.
Microorganisms 13 02180 g005
Figure 5. Plectosphaerella cucumerina (SAAS 481921). (a–c) Diseased rhizomes and roots of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h) mycelial coils; (i) conidiophores radiating out from mycelial coils; (j–m) conidiophores; and (n) conidia. Scale bars: (h–n) 10 µm.
Figure 5. Plectosphaerella cucumerina (SAAS 481921). (a–c) Diseased rhizomes and roots of Zingiber officinale. The arrows indicate the disease lesions; (d,e) surface and reverse sides of colony after 7 days on PDA (f,g) and on OA; (h) mycelial coils; (i) conidiophores radiating out from mycelial coils; (j–m) conidiophores; and (n) conidia. Scale bars: (h–n) 10 µm.
Microorganisms 13 02180 g005

3.3. Pathogenicity Tests

In vitro assay: at 7 days post inoculation (dpi), uninoculated rhizomes remained healthy. Rhizomes inoculated with the four Plectosphaerellaceae spp. developed brown necrotic lesions (Figure 6), with DSI values ranging from 51.1 for M. zingiberis, to 68.9 for M. shandongensis, 75.6 for P. cucumerina, and 97.8 for G. serrae. Furthermore, white aerial mycelia were visible on the surface of the browning rhizomes. These symptoms closely resembled those initially observed on the source rhizomes. In vivo assay: at 14 dpi, control seedlings remained healthy. Seedlings inoculated with the four Plectosphaerellaceae spp. showed symptoms including wilt, leaf yellowing, and rhizome browning and necrosis (Figure 6), with DSI values ranging from 60.0 for M. zingiberis, to 71.1 for M. shandongensis, 77.8 for P. cucumerina, and 100.0 for G. serrae. Specifically, G. serrae infection resulted in bright orange leaf discoloration, defoliation, rhizome rot, and rapid plant death. M. shandongensis and M. zingiberis induced symptoms including leaf twisting-wilting and rhizome brown rot. Ginger seedlings inoculated with P. cucumerina exhibited light leaf yellowing, brown rhizome decay, and yellow-brown necrosis of the basal stem and root. These symptoms were identical to those observed in the field. All of these Plectosphaerellaceae spp. were re-isolated from symptomatic ginger rhizomes and seedlings, thereby establishing the causal link between these isolated fungi and the observed disease symptoms.

4. Discussion

This study investigated the diversity of Plectosphaerellaceae species associated with ginger rhizome and root rots in Shandong Province, China. Based on multi-locus phylogenetic analyses of concatenated LSU, ITS and TEF1-α sequences combined with morphological characteristics and pathogenicity tests, we identified four species: two new species (Musidium shandongensis sp. nov. and M. zingiberis sp. nov.), one newly recorded species (Gibellulopsis serrae) in China, and one new host record (Plectosphaerella cucumerina) on ginger.
The four species introduced in this paper all formed distinct clades in the LSU-ITS-TEF1-α phylogeny, and all were supported by morphological differences that distinguish them. BLAST analyses of the TEF1-α gene yielded identifications that differed from those of the LSU and ITS regions (Table 2), as TEF1-α is known to provide higher resolution for species in Plectosphaerellaceae than do the LSU or ITS loci [17,35]. The relatively high variability of TEF1-α within the Plectosphaerellaceae clade has already been indicated by fine interspecific distinctions [16]. Multi-locus phylogenetic analyses are necessary in delimitation of the various Plectosphaerellaceae species, since no single locus can resolve all known species [22,26,32,37].
Gibellulopsis serrae was proposed as a novel combination to accommodate Cephalosporium serrae, Hyalopus serrae and Verticillium serrae, which formed a well-supported clade distinct from G. nigrescens based on multilocus phylogenetic analysis of LSU, ITS, TEF1-α and RPB2 regions [22]. This species has been previously isolated from diverse hosts, including Abelmoschus esculentus in Cuba, Amaranthus tricolor in Italy, Beta vulgaris in Canada, Musa sp. in India, and Solanum tuberosum in Germany, Israel, and Japan [22]. Furthermore, G. serrae was isolated from soil (Argentina, Israel and New Zealand), granulomas in goldfish (Brazil), human blood (Greece), the human eye (Italy), and wood pulp (Sweden). Interestingly, it has been recognized as a fungicolous species parasitizing Cercospora beticola in Moldavia, Erysiphe sp. in Russia, and Oidium sp. in Odessa [22]. No previous reports of this species associated with any plant host have been published in China. In our study, strains SAAS 311704 and SAAS 410805 clustered with ex-type strains CBS 290.30 and CBS 892.70 (Figure 1), forming a highly supported clade (1.0 PP, 100% BS). We present the first report of G. serrae from China, a new geographical record.
The genus Musidium was introduced for the species originally known as Acremonium stromaticum [22]. Only one species, M. stromaticum, has thus far been accepted in the genus. This species has been documented in Musa sp. mainly in Central America and Europe [22], as well as in Zingiber officinale in India and Japan [25,26]. In the present study, two new Musidium species (M. shandongensis and M. zingiberis) were introduced based on both multilocus phylogenetic analyses (concatenated LSU, ITS, and TEF1-α regions) and distinct morphological features. Giraldo & Crous [22] did not report mycelial ropes and coils in their isolates of M. stromaticum. However, we found that mycelial ropes and coils were common in all the isolates of M. shandongensis and M. zingiberis. Furthermore, both species display wider conidiophores, conidiogenous cells, and conidia compared to those of M. stromaticum.
P. cucumerina, the type species of Plectosphaerella, was previously identified as Venturia cucumerina from Cucumis sativus in Sweden [65]. The genus Plectosphaerella was later established by Klebahn [27] and was also obtained from Cucumis sativus. Plectosphaerella was placed in the family Plectosphaerellaceae based on phylogenetic analysis of the LSU locus [17]. Only three species of sexual morphs have been reported in Plectosphaerellaceae [22,66]. However, the asexual morphs are relatively homogeneous [16], and the main distinguishing characteristics of this genus depend on conidial shape and dimension as well as the occurrence or absence of chlamydospores [17,36]. Species of this genus have diverse nutritional modes and habitat sources, including plant pathogens, plant endophytes, animal pathogens and soil-borne saprobes [17,20,22,30,35,37,67,68]. This morphological simplicity and host variability hinder accurate identification, rendering traditional method insufficient [16,17,30]. While ITS and LSU loci are routinely employed for species delimitation of Plectosphaerella [17,20,69], their resolution is often inadequate due to the limited sequence divergence in this genus [17]. To improve taxonomic clarity, protein-coding genes such as TEF-1α, CaM, TUB2, and RPB2 have been increasingly utilized in recent studies [22,32,37,66,70]. The combination P. cucumerina, introduced by Gams [38], is recognized as a cosmopolitan root-infecting pathogen with a broad host range, including Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Poaceae, and Solanaceae. Compared with these previous studies, we isolated P. cucumerina from decaying rhizomes and roots of ginger. To our knowledge, this is the first documented isolation of P. cucumerina from ginger (Zingiberaceae) worldwide.
The pathogenicity trials revealed that all tested Plectosphaerellaceae spp. were pathogenic, though their virulence levels varied. Detached rhizomes inoculation provided initial evidence of pathogenic potential, which was further supported by symptoms development in intact seedlings inoculation. G. serrae exhibited the highest aggressiveness, triggering severe symptoms including rhizome browning, leaf yellowing, and subsequent plant death. These findings align with Giraldo and Crous [22], who reported similar symptoms on okra, amaranth, beet and potato. M. shandongensis and M. zingiberis caused rhizome browning and leaf curling of the ginger seedlings. Carlucci et al. [17], Raimondo [35] and Li et al. [40] associated symptoms with P. cucumerina such as stunting, root rot, leaf chlorosis, and stem base browning, with this seen as a necrotrophic pathogen in the present investigation. Recent research has demonstrated that Gibellulopsis sp. and P. cucumerina were enriched in diseased ginger crops, serving as potential biomarkers of rhizome rot disease [11]. Ginger diseases are generally categorized into two groups: aerial and soil-borne. Aerial diseases primarily include rust, anthracnose, and leaf spot [6,7]. The typical symptoms of these diseases appear as oval to irregular, chlorotic lesions on the leaves. These distinct lesions are clearly differentiated from the uniform chlorosis of entire leaves caused by soil-borne diseases. The major pathogens responsible for soil-borne diseases in ginger include Fusarium spp., Pythium spp., and Ralstonia solanacearum [5,8]. The four tested Plectosphaerellaceae spp. exhibit symptoms including rhizome and root rot, leaf yellowing, wilting, and subsequent plant death, which is closely similar to disease caused by Fusarium spp. However, Plectosphaerellaceae spp. infection does not induce severe wilting until the rhizome is completely rotten. In contrast, Fusarium spp. infection results in the entire tiller to die, accompanied by rapid wilting and desiccation of the leaves. Additionally, the symptoms caused by Pythium spp. appear on the above-ground part of the rhizome-stem interface as brown lesions that lead to stem rot, leaf chlorosis, and rhizome decay. Moreover, bacteria like Ralstonia solanacearum infection releases a pungent odor while Plectosphaerellaceae spp. infection does not result in any foul odor.

5. Conclusions

This study discovered and described two new species, one newly recorded species, and one new host record, causing rhizome and root rots of ginger in Shandong Province, China. These new findings significantly enrich our understanding of the biodiversity of the Plectosphaerellaceae family and provide valuable insights into the impact of diseases caused by these newly discovered pathogens on ginger production. Cultivation systems, such as continuous cropping in Shandong Province, may promote the development of ginger rhizome rot diseases. The infection processes and pathogenic mechanisms of the Plectosphaerellaceae species in causing ginger diseases remain largely unknown. Accurate identification of the causal agents and understanding their diversity through morphological, molecular, and pathogenic analyses are essential for effective disease management and the formulation of control strategies. Ongoing research into the comprehensive characterization of the Plectosphaerellaceae species causing this disease, including key virulence determinants and pathogenic mechanisms, will be crucial for developing targeted control measures and safeguarding ginger production in China and worldwide.

Author Contributions

Conceptualization, W.Z. (Weihua Zhang); methodology, W.Z. (Weihua Zhang) and Q.Z.; software, Q.Z.; formal analysis, X.G., L.Z., M.Z. and Z.L.; investigation, Q.Z., A.J., H.Y. and J.H.; resources, Q.Z., A.J. and W.Z. (Weiqin Zhao); data curation, Q.Z.; writing—original draft preparation, Q.Z.; writing—review and editing, W.Z. (Weihua Zhang); supervision, W.Z. (Weihua Zhang); project administration, W.Z. (Weihua Zhang); funding acquisition, W.Z. (Weihua Zhang). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research & Development Program of China (2023YFD1401200), Postdoctoral Innovation Program of Shandong Province (SDCX-ZG-202400139) and Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2024A10).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original sequence data presented in the study are openly available in [NCBI] at [https://www.ncbi.nlm.nih.gov/].

Acknowledgments

The authors thank the Shandong Key Laboratory for Green Prevention and Control of Agricultural Pests for providing the funds. The authors are grateful to Chunhua Shang and Jinan Academy of Agricultural Sciences for collecting diseased ginger samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microorganisms 13 02180 g006
Figure 6. Pathogenicity assays on ginger rhizomes and seedlings for the Plectosphaerellaceae species. Disease symptoms on the rhizomes and seedlings caused by (a–c) Gibellulopsis serrae (SAAS 311704), (d–f) Musidium shandongensis (SAAS 381414), (g–i) Musidium zingiberis (SAAS 381402), and (j–l) Plectosphaerella cucumerina (SAAS 481921). The arrows indicate the disease lesions. (a,d,g,j) upper: the rhizomes were inoculated with sterile PDA disks as a negative control; lower: the rhizomes were inoculated with mycelial disks of the indicated fungal isolates. (b,e,h,k) left: the rhizomes were inoculated with conidial suspension of the indicated fungal isolates; right: the rhizomes were inoculated with sterile water as a negative control. (c,f,i,l) left: the seedlings were inoculated with conidial suspension of the indicated fungal isolates; right: the seedlings were inoculated with sterile water as a negative control.
Figure 6. Pathogenicity assays on ginger rhizomes and seedlings for the Plectosphaerellaceae species. Disease symptoms on the rhizomes and seedlings caused by (a–c) Gibellulopsis serrae (SAAS 311704), (d–f) Musidium shandongensis (SAAS 381414), (g–i) Musidium zingiberis (SAAS 381402), and (j–l) Plectosphaerella cucumerina (SAAS 481921). The arrows indicate the disease lesions. (a,d,g,j) upper: the rhizomes were inoculated with sterile PDA disks as a negative control; lower: the rhizomes were inoculated with mycelial disks of the indicated fungal isolates. (b,e,h,k) left: the rhizomes were inoculated with conidial suspension of the indicated fungal isolates; right: the rhizomes were inoculated with sterile water as a negative control. (c,f,i,l) left: the seedlings were inoculated with conidial suspension of the indicated fungal isolates; right: the seedlings were inoculated with sterile water as a negative control.
Microorganisms 13 02180 g006
Table 2. BLAST results for our strains SAAS 311704, SAAS 381402, SAAS 381414 and SAAS 481921.
Table 2. BLAST results for our strains SAAS 311704, SAAS 381402, SAAS 381414 and SAAS 481921.
StrainMolecular MarkerClosest SpeciesIdentity (%)GenBank Accession NumberIdentities
SAAS 311704LSUGibellulopsis nigrescens CBS 179.40100.00%MH867573.1851/851 (no gaps)
ITSGibellulopsis nigrescens 74_ITS4100.00%OP498056.1556/556 (no gaps)
TEF1-αGibellulopsis serrae MFLUCC:23-030899.75%PP866301.1802/804 (no gaps)
SAAS 381402LSUMusidium stromaticum S20-1100.00%LC743850.1818/818 (no gaps)
ITSMusidium stromaticum CBS 863.7398.77%MH860814.1561/568 (2 gaps)
TEF1-αAcremonium stromaticum CBS 863.73 98.90%LN810533.1899/909 (no gaps)
SAAS 381414LSUMusidium stromaticum S20-199.50%LC743850.1795/799 (no gaps)
ITSMusidium stromaticum CBS 863.7398.03%MH860814.1546/557 (no gaps)
TEF1-αAcremonium stromaticum CBS 863.73 97.84%LN810533.1904/924 (no gaps)
SAAS 481921LSUPlectosphaerella cucumerina CAES PC01100.00%MK143394.1862/862 (no gaps)
ITSPlectosphaerella cucumerina FL08-0027100.00%AB469880.1555/555 (no gaps)
TEF1-αPlectosphaerella cucumerina SKH23026100.00%PV593119.1923/923 (no gaps)
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Zhao, Q.; Jia, A.; Yang, H.; Hu, J.; Gao, X.; Zhao, W.; Zhou, L.; Zhang, M.; Li, Z.; Zhang, W. Unveiling Species Diversity of Plectosphaerellaceae (Sordariomycetes) Fungi Involved in Rhizome and Root Rots of Ginger in Shandong Province, China. Microorganisms 2025, 13, 2180. https://doi.org/10.3390/microorganisms13092180

AMA Style

Zhao Q, Jia A, Yang H, Hu J, Gao X, Zhao W, Zhou L, Zhang M, Li Z, Zhang W. Unveiling Species Diversity of Plectosphaerellaceae (Sordariomycetes) Fungi Involved in Rhizome and Root Rots of Ginger in Shandong Province, China. Microorganisms. 2025; 13(9):2180. https://doi.org/10.3390/microorganisms13092180

Chicago/Turabian Style

Zhao, Qian, Ao Jia, Hongjuan Yang, Jinming Hu, Xuli Gao, Weiqin Zhao, Lifeng Zhou, Miao Zhang, Zhaoxia Li, and Weihua Zhang. 2025. "Unveiling Species Diversity of Plectosphaerellaceae (Sordariomycetes) Fungi Involved in Rhizome and Root Rots of Ginger in Shandong Province, China" Microorganisms 13, no. 9: 2180. https://doi.org/10.3390/microorganisms13092180

APA Style

Zhao, Q., Jia, A., Yang, H., Hu, J., Gao, X., Zhao, W., Zhou, L., Zhang, M., Li, Z., & Zhang, W. (2025). Unveiling Species Diversity of Plectosphaerellaceae (Sordariomycetes) Fungi Involved in Rhizome and Root Rots of Ginger in Shandong Province, China. Microorganisms, 13(9), 2180. https://doi.org/10.3390/microorganisms13092180

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