Next Article in Journal
Does Trema micranthum (L.) Blume Produce Cannabinoids?
Previous Article in Journal
Wild-Edible Allium Species from Highlands of Eastern Anatolia: Phytochemical Composition and In Vitro Biological Activities
Previous Article in Special Issue
Plant–Entomopathogenic Fungi Interaction: Recent Progress and Future Prospects on Endophytism-Mediated Growth Promotion and Biocontrol
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 13
Issue 14
10.3390/plants13141950
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China

by
Danhua Wang
†,
Dong Deng
†,
Junliang Zhan
,
Wenqi Wu
,
Canxing Duan
,
Suli Sun
* and
Zhendong Zhu
*
Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2024, 13(14), 1950; https://doi.org/10.3390/plants13141950
Submission received: 28 March 2024 / Revised: 4 July 2024 / Accepted: 11 July 2024 / Published: 16 July 2024
(This article belongs to the Special Issue Mycology and Plant Pathology)
Browse Figures
Review Reports Versions Notes

Abstract

:
Chickpea (Cicer arietinum L.) is an important legume crop worldwide. An emerging disease, basal stem rot with obvious wilt symptoms, was observed in the upper part of chickpea plants during the disease survey in Qiubei County of Yunnan Province. Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue of chickpea plants collected from the field. Those isolates were morphologically found to be similar to Diaporthe aspalathi. Molecular sequence analyses of multiple gene regions (ITS, tef1, tub2, cal, and his3) indicated that the three isolates showed a high identity with D. aspalathi. Pathogenicity and host range tests of the isolates were performed on the original host chickpea and eight other legume crops. The isolates were strongly pathogenic to chickpea and appeared highly pathogenic to soybean, cowpea, and mung bean; moderated or mild pathogenic to adzuki bean and common bean; however, the isolates did not cause symptoms on grass pea (Lathyrus sativus). Diaporthe aspalathi was previously reported as a main pathogen causing the southern stem canker in soybean. To our knowledge, this is the first report of D. aspalathi inducing basal stem rot on chickpea worldwide.

1. Introduction

Chickpea (Cicer arietinum L.) is a crucial legume crop cultivated for its nutritional significance, adaptability, and economic value [1]. Chickpea ranked third with an annual production of approximately 11.6 million tons, following beans and peas in worldwide pulse production in 2020 [2]. With a rich history of consumption dating back thousands of years, chickpea holds a prominent place in various cuisines, particularly in regions across the Mediterranean, Middle East, and South Asia. Beyond its culinary appeal, chickpea plays an important role in sustainable agriculture as a nitrogen-fixing crop, contributing to soil fertility and crop rotation practices [3].
Chickpea is affected by a range of pathogens, including fungi, bacteria, viruses, mycoplasma, nematodes, and Viroid [4,5]. Among these pathogens, fungal species are predominantly responsible for major chickpea diseases. Major fungal diseases causing substantial yield losses worldwide include Ascochyta blight, Fusarium wilt, powdery mildew dry root rot, Botrytis gray mold, and wet root rot. Therefore, controlling fungal disease is crucial to chickpea production [6].
Diaporthe/Phomopsis spp. are known to be associated with a wide range of host plants, about 167 plant species, as pathogens, endophytes, or saprobes [7,8,9]. The Diaporthe/Phomopsis complex (DPC) has been recognized as one of the major limiting factors on many crops worldwide due to inducing several devastating diseases [9,10]. The in-depth studies of Diaporthe/Phomopsis spp. were carried out in several host crops, especially those associated with soybean [10,11], citrus [12,13,14], sunflower [7,15], kiwifruit [16], and grape [17]. Diaporthe/Phomopsis spp. was first reported to cause pod and stem blight diseases on the most important legume crop, soybean, in the USA [18]. Since then, they occurred in other regions of the USA and almost all soybean-producing countries worldwide [15], Brazil [19], Argentina [20], Croatia [21], Korea [11], Canada [22], and China [23,24,25].
The Diaporthe/Phomopsis complex (DPC) induced severe diseases in soybean, including southern and northern stem canker, pod and stem blight, stem blight, and seed decay [11,22,23,24,26]. The typical symptoms of stem canker included foliar interveinal chlorosis and necrosis, reddish-brown and sunken lesions stems, protruding beaks perithecia [21,27]. The most obvious sign of pod and stem blight is the presence of small and black pycnidia in linear rows on infected material [25]. The typical characteristic symptoms of stem blight were dry rot with discoloration on the lower stem at the junction of a branch or petiole, which caused plant wilt and death finally [23]. The typical characteristic of Phomopsis seed decay is cracked and shriveled with white mold on the surface of the seeds; sometimes, infected seeds do not show any symptoms [11,22]. P. longicolla (Diaporthe longicolla) is the primary cause of Phomopsis seed decay [28,29]. Although Phomopsis seed decay is caused primarily by D. longicolla, other Diaporthe species were also reported to be associated with soybean seed decay, such as D. sojae (syn. D. phaseolorum var. sojae), D. caulivora, D. aspalathi, D. eres, D. novem (syn. D. pseudolongicolla), D. foeniculina, and D. rudis [30]. D. sojae is the primary causal agent of pod and stem blight [29]. P. longicolla can also induce pod and stem blight. The two forms of soybean stem canker, north stem canker and south stem canker, are caused by D. caulivora (syn. Diaporthe phaseolorum var. caulivora) and D. aspalathi (syn. Diaporthe phaseolorum var. meridionalis), respectively [11,19,27]. D. caulivora can also induce Phomopsis seed decay on soybean [11]. D. novem and D. eres showed strong virulence to soybean stems while showed moderate and no virulence to seeds, respectively [10]. Other DPC species, such as D. citri, D. melonis, and D. endophytica, have also been recorded to be obtained from soybean stems or seeds, but their virulence to soybean was still uncertain [21].
Identification of DPC species in history has been based on morphological differentiations, including the presence of an anamorph or a teleomorph, colonial and conidia morphology, disease symptoms, and virulence on soybean [31,32]. However, because of the diversity of DPC morphological features, physiological characteristics, interactions with the host, and variability in different survival conditions, it was difficult to comprehensively classify DPC species [33,34], and it was meaningless to distinguish DPC species only according to their host range [35]. Morphological differentiation was not enough to identify DPC at the species level. In order to clarify existing conflicts and increase the efficiency of Diaporthe/Phomopsis spp. identification, molecular methods were qualified for rapid and accurate identification of DPC species, particularly multi-locus phylogenetic analysis [7,9,21,29]. The related loci for identification of DPC species were mainly composed of the nuclear ribosomal internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1), β-tubulin (tub2), calmodulin (cal), and histone H3 (his3) gene regions, and any combination of them can be used to analyze and identify DPC species [9,29].
In China, Diaporthe/Phomopsis spp. has been reported to cause three diseases in soybean, including stem blight, stem canker, and seed decay [24,25]. During a field survey in Qiubei County, Yunnan Province, China, wilt symptoms on chickpea crops were observed. The objective of the present study was to identify the pathogen species inciting the chickpea disease observed by using morphology and molecular analysis.

2. Results

2.1. Disease Symptoms

During the field surveys conducted in Qiubei County, Yunnan Province, we found the incidence of a new disease in chickpea plants. Of the diseased plants with obvious symptoms, about 20% were distributed in a few spots in the infected field (Figure 1A). The aerial parts of affected plants exhibited leaf chlorosis and wilting (Figure 1A). When affected plants were removed from the soil and dissected, they exhibited typical symptoms of basal stem rot, the exodermis of the basal stems and roots being brown to black and rotted, but there was no discoloration of the pith or vascular tissues (Figure 1B).

2.2. Pathogen and Morphological Characteristics

Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue. The isolates produced white colonies with lanose mycelia that occasionally turned light tan with age on PDA (Figure 2A,B). After three weeks, black stromata with pycnidia were produced. Pycnidia released yellow cirrus extruding masses of α-conidia (Figure 2C). The α-conidia (5.8–7.9 × 2.1–3.8 µm) were hyaline, nonseptate, ellipsoidal, and biguttulate in each cell (Figure 2D). No perithecia of the teleomorphs were formed on the PDA. To observe the teleomorphs of the pathogen, the diseased tissue fragments were subjected to incubation in a moist petri dish for 7 days. Masses of beaked perithecia were produced and embedded in the epidermis of the diseased tissue (Figure 2E). Perithecia (230–390 × 175–380 µm) were black, subglobose to globose, and had long necks (210 to 360 µm in length) (Figure 2E). Eight-spored asci (31.0 to 42.5 × 4.6 to 9.1 µm) were elongate-clavate and unitunicate-walled, with a refractive ring at the apex (Figure 2F). Ascospores (8.1 to 13.5 × 2.1 to 4.3 µm) were hyaline, smooth, fusoid or elongate-ellipsoid, two-celled, widest at the septum, tapering towards both ends, medianly septate, not constricted at the septum, with 1–2 guttules per cell (Figure 2F). These morphological features are consistent with those of Diaporthe aspalathi (Syn. Diaporthe phaseolorum var. meridionalis) [27].

2.3. Molecular Characteristics and Phylogenetic Analysis

The ITS, tef1, tub2, cal, and his3 gene regions of rDNA from three isolates were sequenced using their respective common primers. Sequences of the ITS region and EF1-α gene obtained from the three isolates showed 99–100% similarity with more than 40 or several reported D. aspalathi strains in GenBank by a BLAST analysis. The aligned sequences of the five loci (ITS, tef1, tub2, cal, and his3) were cascaded, and 2655 bp sequences of the three isolates and other sequences from reported Diaporthe/Phomopsis spp. strains were involved in the dataset.
The phylogenetic tree was constructed using a concatenated sequence dataset of the five gene regions (Figure 3). In the phylogenetic tree, the three isolates were grouped with those of three reference strains of D. aspalathi, whose host was rooibos (Aspalathus linearis) from South Africa in GenBank (Figure 3). The phylogenetic analysis revealed that the three isolates could be distinguished from other species of the Diaporthe/Phomopsis spp. Altogether, the results of the molecular characteristics and phylogenetic analysis strongly support that the three isolates clearly belong to D. aspalathi [36].

2.4. Pathogenicity and Host Range Tests

Firstly, the pathogenicity of the three D. aspalathi isolates was tested on the original host. The results showed that all isolates were strongly pathogenic to the original host, chickpea. All inoculated plants showed typical symptoms similar to those observed in the field, including leaf chlorosis, wilting, and basal stem rot. The diseased plants were dwarfed and wilted, eventually resulting in plant death (Figure 4A). There were no disease symptoms in the control plants. The pathogen was re-isolated from inoculated plants, thus fulfilling Koch’s postulates.
Secondly, the host range of the D. aspalathi isolate ZD36-3 was tested on eight other legume crops. The isolate ZD36-3 showed strong virulence in soybean, cowpea, and mung bean, and the symptoms were similar to those on chickpea, eventually causing plant death (Figure 4B–D). The ZD36-3 caused moderate symptoms on adzuki bean and common bean, with slightly enlarged spots at the inoculation site and with yellowing and wilting of leaves (Figure 4E–G). The ZD36-3 showed no pathogenicity in the remaining three crops, pea, faba bean, and grass pea, and no symptoms were observed on these three legume crops (Figure 4H–J). Thus, these results indicated that D. aspalathi from chickpea may cause the related disease in other legume crops, soybean, cowpea, mung bean, adzuki bean, and common bean.

3. Discussion

Chickpea is a nutrient-dense food that provides an abundance of protein, dietary fiber, and certain dietary minerals as animal feed and human food worldwide [37,38,39]. The biotic stresses in chickpea, particularly pathogens, are the primary cause of yield loss, and some of them, such as Ascochyta rabiei [40,41] and Fusarium oxysporum f.sp. ciceris [42,43] can cause up to 90%. In recent years, new diseases caused by pathogens have emerged continuously in legume crops.
In this study, we discovered an emerging disease, basal stem rot, in chickpea from Qiubei County of Yunnan Province. It was found that basal stem rot of chickpea has been becoming one of the main diseases of chickpea in some regions.
Diaporthe represents a highly complex genus containing numerous cryptic species. Many Diaporthe species that are morphologically similar proved to be genetically distinct, and several Diaporthe isolates that were previously identified based on their host were shown to represent different taxa. The genera Diaporthe and Phomopsis have received much taxonomic attention. Molecular identification has gradually replaced morphological identification as the main identification method for DPC species [9,21,29] because molecular identification is accurate and efficient and can avoid many controversial parts of morphological identification. However, morphological identification is still an important auxiliary means for DPC species identification. Brumer et al. [27] isolated Diaporthe and Phomopsis strains from soybean stems and seeds across South America from 1989 to 2014. Genomic DNA from 26 isolates underwent PCR-RFLP, AFLP analysis, and ITS sequencing. The molecular analysis identified Phomopsis longicolla, D. phaseolorum var. sojae, D. caulivora, and D. aspalathi. Pathogenicity tests of 13 D. aspalathi isolates revealed at least three races in Brazil. Both molecular and phenotypic analyses showed clustering based on collection date and pathogenicity, indicating pathogen variability.
In this study, the disease plant tissues with typical symptoms were collected, and three isolates with Diaporthe/Phomopsis-like fungi were isolated. Morphological identification indicated that the colony, conidia, perithecia, asci, and ascospore morphology were the same as those of D. aspalathi. Molecular identification showed that the ITS, tef1, tub2, cal, and his3 gene regions of the three isolates were highly similar to previous D. aspalathi strains available in Genbank. The multi-locus phylogenetic analysis based on combining the five genes classified the three isolates into the D. aspalathi phylogenetic group (Figure 3). Therefore, based on the morphological characteristics, molecular characteristics, and multi-locus phylogenetic analysis, the three isolates ZD36-1, ZD36-2, and ZD36-3 were identified as D. aspalathi.
Diaporthe exhibits a wide geographic distribution and comprises pathogens, endophytes, and saprobes across diverse hosts [44,45]. Diaporthe species have been associated with economically significant hosts, such as Citrus sp. and Coffea sp., as well as ornamental plants, such as Clematis [44,46]. Some species have been reported as pathogens of die-backs, cankers, leaf spots, blights, melanoses, stem-end rot, and gummosis of various hosts [47,48,49,50,51]. For example, D. kochmanii and D. kongii have been associated with sunflower stem blight [7], while D. australafricana and D. viticola have been linked to cane and leaf spot disease of grapevine [52]. The D. aspalathi, formerly referred to as D. phaseolorum or D. phaseolorum var. meridionalis [53], was one of the most important DPC species and famous for causing southern stem canker on any growth stages of soybean with up to 100% yield loss worldwide, especially in South America [54], North America [28,53,55], and Africa [56,57]. Moreover, this pathogen, D. aspalathi, also induced die-back of rooibos (Aspalathus linearis), which caused substantial losses in South Africa [36]. Recently, D. aspalathi also induced die-back on honeybush (Cyclopia longifolia) [58].
In this study, nine legume crops were used in pathogenicity and host range tests. The three isolates were found to be strongly pathogenic to the original host chickpea. The representative isolate ZD36-3 showed highly pathogenic to other legume crops such as soybean, cowpea, and mung bean. It showed moderate or mild pathogenicity in adzuki bean and common bean, while no pathogenicity was observed in pea, faba bean, and grass pea. These results confirmed that D. aspalathi was not only a pathogen of soybean but also of chickpea, cowpea, mung bean, adzuki bean, and common bean.
Mostly, basal stem rot of plants is a soil-borne disease caused by soil-borne pathogens. But the pathogen D. aspalathi is also reported to be seed-borne, causing seed decay disease in soybean. Thus, the most possible reason for its severity is the extensive and transfer seed management [59]. The pathogen D. aspalathi causing basal stem rot on chickpea might come from preceding or fore-rotating crops, especially soybean, which is a main host of the pathogen.
Stanoeva Y and Beleva M [60] performed an investigation on the occurrence of Phomopsis/Diaporthe spp. on chickpea (Cicer arietinum) in Bulgaria. Their investigation is the first record of C. arietinum as a host of Phomopsis/Diaporthe spp. in Bulgaria. Unfortunately, Phomopsis/Diaporthe spp. was not identified at the species level only based on the cultural and morphological characteristics. Then, Thompson et al. [7] reported that Diaporthe novem of Phomopsis/Diaporthe complex was found to be associated with chickpea (Cicer arietinum) in Australia. In this study, we performed a detailed identification of the causal agent of basal stem rot from chickpea by morphology, molecular characteristics, multi-gene phylogenetic analysis, pathogenicity, and host range tests. Our results were sufficient to demonstrate that the three isolates from chickpea belong to D. aspalathi. This is the first report that D. aspalathi causes basal stem rot on chickpea in China and worldwide. Our study provided a foundation for disease control and screening of resistant germplasms in breeding programs of chickpea. And it also highlights that seed transport needs reinforcing quarantine and the disease of rotation legume crops needs reinforcing control.

4. Materials and Methods

4.1. Disease Survey

In late March 2017, a field disease survey of food legume crops was conducted by our research team in Yunnan Province, China. The survey discovered the wilt symptoms similar to those caused by Diaporthe/Phomopsis spp. of soybean plants in a chickpea field located in Qiubei County (23°63′ N, 103°34′ E). The incidence of the diseased plants with symptoms was about 20% in the infected field. The diseased plants were distributed in a few spots in the field. To confirm the causal agents, the diseased plants with typical symptoms of basal stem rot, such as leaf chlorosis and wilting, and the exodermis of the basal stems and roots being brown to black and rotted, were collected from these fields and used to subsequent pathogen isolation and identification.

4.2. Isolation of Pathogen from Diseased Plants

Diseased basal stems were washed under tap water and cut into several 2–3 mm sections from the margins of disease areas (1/3) or healthy parts (2/3). After being surface sterilized in 2% NaClO for 2 min, the tissues were subsequently rinsed three times with sterile distilled water and dried on sterilized filter paper. Every three to four pieces were cultivated on a potato dextrose agar (PDA; AoBoXing Biotech, Beijing, China). Plates were incubated at 25 °C under a 12 h photoperiod for 2–3 days. Fungal isolates were purified by transferring single hyphal tips to a fresh medium. The plates were incubated at 25 °C under a 12 h photoperiod. All isolates obtained were stored in 30% glycerol at −80 °C for long-term preservation.

4.3. Morphology Characteristics

Three isolates (ZD36-1, ZD36-2, ZD36-3) were used as representatives for morphological analysis. Five-millimeter-diameter mycelial plugs were cut from the edge of active colonies of each isolate by a puncher. The plugs were transferred to the center of a 90-mm-diameter plate containing PDA. Maintained at 25 °C under a 12 h light/12 h dark cycle. Teleomorph formation was induced from diseased chickpea tissue fragments, which were incubated in a moist petri dish. The conidia and ascospores produced were observed and measured under a light microscope (Olympus 31X, Tokyo, Japan) with the UV-C confocal imaging system (UVTEC).

4.4. Molecular Characteristics and Phylogenetic Analysis

The ZD36-1, ZD36-2, and ZD36-3 isolates were sub-cultured on cellophane-covered PDA for 10 days at 25 °C. The Mycelia of each isolate were scraped from the PDA plates with a sterilized blade. Genomic DNA was extracted by fusing the Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. Purified DNA was suspended in 1× TE buffer and stored at −20 °C for later use. PCR assays were performed using specific primer pairs targeting different genomic regions: ITS region (primers ITS4/ITS5) [61], tef1 gene (primers EF1-728F/EF1-986R) [62], tub2 gene (primers Bt2a/Bt2b) [63], cal gene (primers CAL-228F/CAL-737R) [62], and his3 gene (primers CYLH3F/H3-1b) [64]. PCR reactions were carried out using a Gene Amp 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) in 50 μL reaction mixtures containing 25 ng of DNA, 2 μL of each primer, 25 μL of 2×Taq PCR Mastermix (TIANGEN, Beijing, China), and 17 μL ddH2O. The cycling protocol was as follows: an initial denaturation for 5 min at 95 °C, followed by 35 cycles of denaturation at 94 °C for 45 s, 45 s at 55 °C–65 °C (depending on the primer-specific annealing temperature), and extension at 72 °C for 60 s, with a final extension of 72 °C for 10 min. All PCR products were analyzed using 1.5% agarose gel with the Gelgreen Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). Then, a cloning method was applied to generate a sequence from the obtained amplicons by the five primer pairs, respectively (Sangon Biotech, Shanghai, China). The resulting sequences for each isolate were aligned with Multalin http://multalin.toulouse.inra.fr/multalin/ (accessed on 25 June 2023). Phylogenetic analysis was performed based on the concatenated multiple gene regions of the nuclear sequence dataset (ITS-tef1-tub2-cal-his3) using the MEGA X with Neighbor-Joining method and Tamura-Nei distance model with 1000 bootstrap replicates [29]. And BLAST online tool in the GenBank database (National Center for Biotechnology Information; NCBI) to compare sequence similarities (Table S1).

4.5. Pathogenicity and Host Range Tests

Two pathogenicity trials were completed using chickpea seedlings (Cicer arietinum, original host), soybean (Glycine max), mung bean (Phaseolus radiatus), grass pea (Lathyrus sativus), common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), pea (Pisum sativum), adzuki bean (Vigna angularis), and faba bean (Vicia faba). The nine tested crops were sown in 500 mL paper cups filled with a mix of equal volumes of vermiculite and peat. The planted cups were randomly distributed on a greenhouse bench and incubated at 25 °C under a 12 h photoperiod and watered regularly every 3 to 4 days. The 12 to 14-day-old seedlings were inoculated using the modified hypocotyl puncture technique [11]. After the crops reached the stage of fully expanded true leaves, a sterile dissecting knife was used to make a light incision at hypocotyls of seedlings (not exceeding one-third of the stem diameter in width and approximately 1 cm in length), and hypocotyls of seedlings were inoculated with mycelial plugs. The plants inoculated with a sterile medium were used as negative controls. The treated plants were incubated in a misting room at 25 °C under high humidity (>90%) for 48 h. Misting occurred for 30 min every 2 h by an automatically controlled centrifugal humidifier, which maintained leaf wetness without excessive runoff. Then, the plants were removed to a greenhouse maintained at 25 °C until the plants were rated for disease symptoms. Plants were monitored every day for 4 weeks to detect symptoms. Pathogen was re-isolated from inoculated plants to verify Koch’s postulates. All tests were conducted twice.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13141950/s1, Table S1. Information of the Diaporthe isolates from diffident hosts used to constructing the phylogenetic tree this study.

Author Contributions

Z.Z. conceived and designed the experiments. D.W., D.D., J.Z. and W.W. performed the experiments. D.D. wrote the manuscript. D.W. analyzed the data. C.D. and S.S. supplied the disease samples and common cultivars. Z.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (31401695), the Modern Agro-industry Technology Research System (CARS-09), the National Crop Germplasm Resources Center (NCGRC-2023-09), the Agricultural Science and Technology Program for Innovation Team from Chinese Academy of Agricultural Sciences (CAAS).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We sincerely thank Xuxiao Zong at the Institute of Crop Sciences, CAAS, and Yuhua He at Yunnan Academy of Agricultural Sciences for providing samples and legume crop cultivars.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hossain, M.B.; Hasan, M.M.; Sultana, R.; Bari, A.K.M.A. Growth and yield response of chickpea to different levels of boron and zinc. Fundam. Appl. Agric. 2016, 1, 82–86. [Google Scholar]
  2. FAOSTAT. Food and Agriculture Organization of the United Nations. Statistical Database. 2020. Available online: https://www.fao.org/faostat/en/#data/QCL (accessed on 30 September 2021).
  3. Esfahani, M.N.; Sulieman, S.; Schulze, J.; Yamaguchi Shinozaki, K.; Shinozaki, K.; Tran, L.S.P. Mechanisms of physiological adjustment of N2 fixation in Cicer arietinum L. (chickpea) during early stages of water deficit: Single or multi-factor controls. Plant J. 2014, 79, 964–980. [Google Scholar] [CrossRef] [PubMed]
  4. Pirovano, W.; Miozzi, L.; Boetzer, M.; Pantaleo, V. Bioinformatics approaches for viral metagenomics in plants using short RNAs: Model case of study and application to a Cicer arietinum population. Front. Microbiol. 2014, 5, 790. [Google Scholar] [CrossRef] [PubMed]
  5. Rocha, F.S.; Sharma, M.; Tarafdar, A.; Chen, W.; Azevedo, D.M.; Castillo, P.; Costa, C.A.; Chobe, D.R. Diseases of Chickpea. In Handbook of Vegetable and Herb Diseases; Springer International Publishing: Cham, Switzerland, 2023; pp. 1–44. [Google Scholar] [CrossRef]
  6. Yan, J.; Yuan, S.; Jiang, L.; Ye, X.; Bun, T.; Wu, Z. Plant antifungal proteins and their applications in agriculture. Appl. Microbiol. Biotechnol. 2015, 99, 4961–4981. [Google Scholar] [CrossRef]
  7. Thompson, S.M.; Tan, Y.P.; Neate, S.M.; Grams, R.M.; Shivas, R.G.; Lindbeck, K.; Aitken, E.A.B. Diaporthe novem Isolated from Sunflower (Helianthus annuus) and Other Crop and Weed Hosts in Australia. Eur. J. Plant Pathol. 2018, 152, 823–831. [Google Scholar] [CrossRef]
  8. Suryanarayanan, T.S.; Devarajan, P.T.; Girivasan, K.P.; Govindarajulu, M.B.; Kumaresan, V.; Murali, T.S.; Rajamani, T.; Thirunavukkarasu, N.; Venkatesan, G. The Host Range of Multi-Host Endophytic Fungi. Curr. Sci. 2018, 115, 1963–1969. [Google Scholar] [CrossRef]
  9. Guo, Y.S.; Crous, P.W.; Bai, Q.; Fu, M.; Yang, M.M.; Wang, X.H.; Du, Y.M.; Hong, N.; Xu, W.X.; Wang, G.P. High Diversity of Diaporthe Species Associated with Pear Shoot Canker in China. Persoonia—Mol. Phylogeny Evol. Fungi 2020, 45, 132–162. [Google Scholar] [CrossRef]
  10. Hosseini, B.; El-Hasan, A.; Link, T.; Voegele, R.T. Analysis of the Species Spectrum of the Diaporthe/Phomopsis Complex in European Soybean Seeds. Mycol. Prog. 2020, 19, 455–469. [Google Scholar] [CrossRef]
  11. Sun, S.; Van, K.-J.; Kim, M.-Y.; Min, K.-H.; Lee, Y.-W.; Lee, S.-H. Diaporthe phaseolorum Var. caulivora, a Causal Agent for Both Stem Canker and Seed Decay on Soybean. Plant Pathol. J. 2012, 28, 55–59. [Google Scholar] [CrossRef]
  12. Huang, F.; Hou, X.; Dewdney, M.M.; Fu, Y.; Chen, G.; Hyde, K.D.; Li, H. Diaporthe Species Occurring on Citrus in China. Fungal Divers. 2013, 61, 237–250. [Google Scholar] [CrossRef]
  13. Udayanga, D.; Castlebury, L.A.; Rossman, A.Y.; Chukeatirote, E.; Hyde, K.D. Insights into the Genus Diaporthe: Phylogenetic Species Delimitation in the D. eres Species Complex. Fungal Divers. 2014, 67, 203–229. [Google Scholar] [CrossRef]
  14. Guarnaccia, V.; Crous, P.W. Species of Diaporthe on Camellia and Citrus in the Azores Islands. Phytopathol. Mediterr. 2018, 57, 307–319. [Google Scholar] [CrossRef]
  15. Hulke, B.S.; Markell, S.G.; Kane, N.C.; Mathew, F.M. Phomopsis Stem Canker of Sunflower in North America: Correlation with Climate and Solutions through Breeding and Management. OCL 2019, 26, 13. [Google Scholar] [CrossRef]
  16. Thomidis, T.; Exadaktylou, E.; Chen, S. Diaporthe neotheicola, a New Threat for Kiwifruit in Greece. Crop Prot. 2013, 47, 35–40. [Google Scholar] [CrossRef]
  17. Baumgartner, K.; Fujiyoshi, P.T.; Travadon, R.; Castlebury, L.A.; Wilcox, W.F.; Rolshausen, P.E. Characterization of Species of Diaporthe from Wood Cankers of Grape in Eastern North American Vineyards. Plant Dis. 2013, 97, 912–920. [Google Scholar] [CrossRef] [PubMed]
  18. Lehman, S.G. Pod and Stem Blight of Soybean. Ann. Mo. Bot. Gard. 1923, 10, 111–178. [Google Scholar] [CrossRef]
  19. Costamilan, L.M.; Yorinori, J.T.; Almeida, Á.M.R.; Seixas, C.D.S.; Binneck, E.; Araújo, M.R.; Carbonari, J.A. First Report of Diaporthe phaseolorum Var. caulivora Infecting Soybean Plants in Brazil. Trop. Plant Pathol. 2008, 33, 381–385. [Google Scholar] [CrossRef]
  20. Pioli, R.N.; Morandi, E.N.; Martínez, M.C.; Lucca, F.; Tozzini, A.; Bisaro, V.; Hopp, H.E. Morphologic, Molecular, and Pathogenic Characterization of Diaporthe phaseolorum Variability in the Core Soybean-Producing Area of Argentina. Phytopathology® 2003, 93, 136–146. [Google Scholar] [CrossRef]
  21. Santos, J.M.; Vrandečić, K.; Ćosić, J.; Duvnjak, T.; Phillips, A.J.L. Resolving the Diaporthe Species Occurring on Soybean in Croatia. Persoonia—Mol. Phylogeny Evol. Fungi 2011, 27, 9–19. [Google Scholar] [CrossRef]
  22. Abdelmagid, A.; Hafez, M.; Lawley, Y.; Rehal, P.K.; Daayf, F. First Report of Pod and Stem Blight and Seed Decay caused by Diaporthe longicolla on Soybean in Western Canada. Plant Dis. 2021, 106, 1061. [Google Scholar] [CrossRef]
  23. Cui, Y.-L.; Duan, C.-X.; Wang, X.-M.; Li, H.-J.; Zhu, Z.-D. First Report of Phomopsis longicolla Causing Soybean Stem Blight in China. Plant Pathol. 2009, 58, 799. [Google Scholar] [CrossRef]
  24. Shan, Z.; Li, S.; Liu, Y.; Yang, Z.; Yang, C.; Sha, A.; Zhou, X.A. First report of Phomopsis seed decay of soybean caused by Phomopsis longicolla in South China. Plant Dis. 2012, 96, 1693. [Google Scholar] [CrossRef] [PubMed]
  25. Zhao, X.; Li, K.; Zheng, S.; Yang, J.; Chen, C.; Zheng, X.; Wang, Y.; Ye, W. Diaporthe diversity and pathogenicity revealed from a broad survey of soybean stem blight in China. Plant Dis. 2022, 106, 2892–2903. [Google Scholar] [CrossRef] [PubMed]
  26. Baird, R.E.; Abney, T.S.; Mullinix, B.G. Fungi Associated with Pods and Seeds during the R6 and R8 Stages of Four Soybean Cultivars in Southwestern Indiana. Phytoprotection 2005, 82, 1–11. [Google Scholar] [CrossRef]
  27. Brumer, B.B.; Lopes-Caitar, V.S.; Chicowski, A.S.; Beloti, J.D.; Castanho, F.M.; Gregório Da Silva, D.C.; De Carvalho, S.; Lopes, I.O.N.; Soares, R.M.; Seixas, C.D.S.; et al. Morphological and Molecular Characterization of Diaporthe (Anamorph phomopsis) Complex and Pathogenicity of Diaporthe aspalathi Isolates Causing Stem Canker in Soybean. Eur. J. Plant Pathol. 2018, 151, 1009–1025. [Google Scholar] [CrossRef]
  28. Li, S.; Darwish, O.; Alkharouf, N.W.; Musungu, B.; Matthews, B.F. Analysis of the Genome Sequence of Phomopsis longicolla: A Fungal Pathogen Causing Phomopsis Seed Decay in Soybean. BMC Genom. 2017, 18, 688. [Google Scholar] [CrossRef] [PubMed]
  29. Udayanga, D.; Castlebury, L.A.; Rossman, A.Y.; Chukeatirote, E.; Hyde, K.D. The Diaporthe sojae Species Complex: Phylogenetic Re-Assessment of Pathogens Associated with Soybean, Cucurbits and Other Field Crops. Fungal Biol. 2015, 119, 383–407. [Google Scholar] [CrossRef] [PubMed]
  30. Petrović, K.; Riccioni, L.; Vidić, M.; Đorđević, V.; Balešević-Tubić, S.; Đukić, V.; Miladinov, Z. First report of Diaporthe novem, D. foeniculina, and D. rudis associated with soybean seed decay in Serbia. Plant Dis. 2016, 100, 2324. [Google Scholar] [CrossRef]
  31. Morgen-Jones, G. The Diaporthe/Phomopsis complex of soybean, morphology. In Proceedings of the Conference on Diaporthe/Phomopsis Disease Complex of Soybean; United States Department of Agriculture: Beltsville, MD, USA, 1985; pp. 1–7. [Google Scholar]
  32. Sinclair, J.; Backman, P. Compendium of Soybean Diseases, 3rd ed.; The American Phytopathological Society: St. Paul, MN, USA, 1989. [Google Scholar]
  33. Morgan-Jones, G. The Diaporthe/Phomopsis complex, taxonomic considerations. In World Soybean Research Conference IV; Pascale, A., Ed.; Orientación Gráfica: Buenos Aires, Argentina, 1989; pp. 1699–1706. [Google Scholar]
  34. Vanderaa, H.; Noordeloos, M.; Degruyter, J. Species concepts in some larger genera of the Coelomycetes. Stud. Mycol. 1990, 32, 3–19. [Google Scholar]
  35. Mostert, L.; Crous, P.W.; Kang, J.-C.; Phillips, A.J.L. Species of Phomopsis and a Libertella Sp. Occurring on Grapevines with Specific Reference to South Africa: Morphological, Cultural, Molecular and Pathological Characterization. Mycologia 2001, 93, 146–167. [Google Scholar] [CrossRef]
  36. Van Rensburg, J.C.J.; Lamprecht, S.C.; Groenewald, J.Z.; Castlebury, L.A.; Crous, P.W. Characterisation of Phomopsis spp. Associated with Die-Back of Rooibos (Aspalathus linearis) in South Africa. Stud. Mycol. 2006, 55, 65–74. [Google Scholar] [CrossRef] [PubMed]
  37. Milán-Carrillo, J.; Valdéz-Alarcón, C.; Gutiérrez-Dorado, R.; Cárdenas-Valenzuela, O.G.; Mora-Escobedo, R.; Garzón-Tiznado, J.A.; Reyes-Moreno, C. Nutritional Properties of Quality Protein Maize and Chickpea Extruded Based Weaning Food. Plant Foods Hum. Nutr. 2007, 62, 31–37. [Google Scholar] [CrossRef] [PubMed]
  38. Bampidis, V.A.; Christodoulou, V. Chickpeas (Cicer arietinum L.) in Animal Nutrition: A Review. Anim. Feed Sci. Technol. 2011, 168, 1–20. [Google Scholar] [CrossRef]
  39. Wallace, T.; Murray, R.; Zelman, K. The Nutritional Value and Health Benefits of Chickpeas and Hummus. Nutrients 2016, 8, 766. [Google Scholar] [CrossRef]
  40. Gan, Y.T.; Siddique, K.H.M.; MacLeod, W.J.; Jayakumar, P. Management Options for Minimizing the Damage by Ascochyta Blight (Ascochyta rabiei) in Chickpea (Cicer arietinum L.). Field Crops Res. 2006, 97, 121–134. [Google Scholar] [CrossRef]
  41. Manjunatha, L.; Saabale, P.R.; Srivastava, A.K.; Dixit, G.P.; Yadav, L.B.; Kumar, K. Present Status on Variability and Management of Ascochyta rabiei Infecting Chickpea. Indian Phytopathol. 2018, 71, 9–24. [Google Scholar] [CrossRef]
  42. Navas-Cortés, J.A.; Hau, B.; Jiménez-Díaz, R.M. Effect of Sowing Date, Host Cultivar, and Race of Fusarium Oxysporum f. sp. Ciceris on Development of Fusarium Wilt of Chickpea. Phytopathology® 1998, 88, 1338–1346. [Google Scholar] [CrossRef]
  43. Sharma, M.; Ghosh, R.; Tarafdar, A.; Rathore, A.; Chobe, D.R.; Kumar, A.V.; Gaur, P.M.; Samineni, S.; Gupta, O.; Singh, N.P.; et al. Exploring the Genetic Cipher of Chickpea (Cicer arietinum L.) Through Identification and Multi-Environment Validation of Resistant Sources against Fusarium Wilt (Fusarium oxysporum f. Sp. Ciceris). Front. Sustain. Food Syst. 2019, 3, 78. [Google Scholar] [CrossRef]
  44. Dissanayake, A.J.; Phillips, A.J.L.; Hyde, K.D.; Yan, J.Y.; Li, X.H. The current status of species in Diaporthe. Mycosphere 2017, 8, 1106–1156. [Google Scholar] [CrossRef]
  45. Bhunjun, C.S.; Niskanen, T.; Suwannarach, N.; Wannathes, N.; Chen, Y.J.; McKenzie, E.H.; Maharachchikumbura, S.S.; Buyck, B.; Zhao, C.L.; Fan, Y.G.; et al. The numbers of fungi: Are the most speciose genera truly diverse? Fungal Divers. 2022, 114, 387–462. [Google Scholar] [CrossRef]
  46. Phukhamsakda, C.; McKenzie, E.H.C.; Phillips, A.J.L.; Gareth Jones, E.B.G.; Jayarama Bhat, D.; Stadler, M.; Bhunjun, C.S.; Wanasinghe, D.N.; Thongbai, B.; Camporesi, E.; et al. Microfungi associated with Clematis L. (Ranunculaceae) and integrated approach to delimiting species boundaries. Fungal Divers. 2020, 102, 1–203. [Google Scholar] [CrossRef]
  47. Gomes, R.R.; Glienke, C.; Videira, S.I.R.; Lombard, L.; Groenewald, J.Z.; Crous, P.W. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia 2013, 31, 1–41. [Google Scholar] [CrossRef]
  48. Gopal, K.; Lakshmi, L.M.; Sarada, G.; Nagalakshmi, T.; Sankar, T.G.; Gopi, V.; Ramana, K.T.V. Citrus melanose (Diaporthe citri Wolf): A review. Int. J. Curr. Microbiol. App. Sci. 2014, 3, 113–124. [Google Scholar]
  49. Manawasinghe, I.S.; Dissanayake, A.J.; Li, X.; Liu, M.; Wanasinghe, D.N.; Xu, J.; Zhao, W.; Zhang, W.; Zhou, Y.; Hyde, K.D.; et al. High genetic diversity and species complexity of Diaporthe associated with grapevine dieback in China. Front. Microbiol. 2019, 10, 1936. [Google Scholar] [CrossRef] [PubMed]
  50. Zapata, M.; Palma, M.A.; Aninat, M.J.; Piontelli, E. Polyphasic studies of new species of Diaporthe from native forest in Chile, with descriptions of Diaporthe araucanorum sp. nov., Diaporthe foikelawen sp. nov. and Diaporthe patagonica sp. nov. Int. J. Syst. Evol. Microbiol. 2020, 70, 3379–3390. [Google Scholar] [CrossRef]
  51. Caio, G.; Lungaro, L.; Caputo, F.; Zoli, E.; Giancola, F.; Chiarioni, G.; De Giorgio, R.; Zoli, G. Nutritional Treatment in Crohn’s Disease. Nutrients 2021, 13, 1628. [Google Scholar] [CrossRef]
  52. van Niekerk, J.M.; Groenewald, J.Z.; Farr, D.F.; Fourie, P.H.; Halleer, F.; Crous, P.W. Reassessment of Phomopsis species on grapevines. Australas. Plant Pathol. 2005, 34, 27–39. [Google Scholar] [CrossRef]
  53. Fernández, F.A.; Hanlin, R.T. Morphological and RAPD Analyses of Diaporthe Phaseolorum from Soybean. Mycologia 1996, 88, 425–440. [Google Scholar] [CrossRef]
  54. Pioli, R.N.; Morandi, E.N.; Bisaro, V. First report of soybean stem canker caused by Diaporthe phaseolorum var. caulivora in Argentina. Plant Dis. 2001, 85, 95. [Google Scholar] [CrossRef]
  55. Backman, P.A.; Weaver, D.B.; Morgan-Jones, G. Soybean stem canker, an emerging disease problem. Plant Dis. 1985, 69, 641–647. [Google Scholar] [CrossRef]
  56. Jaccoud-Filho, D.S.; Lee, D.; Reeves, J.C.; Yorinori, J.T. Characterization of the seedborne fungal pathogen Phomopsis phaseoli f.sp. meridionalis, the agent of soybean stem canker. In Seed Health Testing; Hutchins, J.D., Reeves, J.C., Eds.; CAB International: Wallingford, UK, 1997; pp. 147–152. [Google Scholar]
  57. Asante, S.N.A.; Wolffhechel, H.; Neergaard, E.D. First report of Diaporthe phaseolorum var. meridionalis on soybean seeds from Ghana. Plant Dis. 1998, 82, 1401. [Google Scholar] [CrossRef] [PubMed]
  58. Smit, L.; Langenhoven, W.E.; Petersen, Y. Diaporthe Species Associated with Dieback on Cyclopia (Honeybush). Eur. J. Plant Pathol. 2021, 161, 565–578. [Google Scholar] [CrossRef]
  59. West, J.S.; Kharbanda, P.D.; Barbetti, M.J.; Fitt, B.D.L. Epidemiology and Management of Leptosphaeria maculans (Phoma Stem Canker) on Oilseed Rape in Australia, Canada and Europe. Plant Pathol. 2001, 50, 10–27. [Google Scholar] [CrossRef]
  60. Stanoeva, Y.; Beleva, M. First record of Phomopsis sp. on chickpea (Cicer arietinum) in Bulgaria. In Proceedings of the Sixth International Scientific Agricultural Symposium” Agrosym 2015”, University of East Sarajevo, Jahorina, Bosnia and Herzegovina, 15–18 October 2015; pp. 850–853. [Google Scholar]
  61. White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. 1990, 18, 315–322. [Google Scholar]
  62. Carbone, I.; Kohn, L.M. A Method for Designing Primer Sets for Speciation Studies in Filamentous Ascomycetes. Mycologia 1999, 91, 553–556. [Google Scholar] [CrossRef]
  63. Glass, N.L.; Donaldson, G.C. Development of Primer Sets Designed for Use with the PCR to Amplify Conserved Genes from Filamentous Ascomycetes. Appl. Environ. Microbiol. 1995, 61, 1323–1330. [Google Scholar] [CrossRef]
  64. Crous, P.W.; Groenewald, J.Z.; Risède, J.-M.; Simoneau, P. Calonectria Species and Their Cylindrocladium Anamorphs: Species with Sphaeropedunculate Vesicles. Stud. Mycol. 2006, 55, 213–226. [Google Scholar] [CrossRef]
Plants 13 01950 g001
Figure 1. Symptoms of basal stem rot caused by Diaporthe/Phomopsis spp. on chickpea plants in the field. (A) Chickpea plants infected by Diaporthe/Phomopsis spp. showed leaf chlorosis and wilting; (B) exodermis of basal stems and roots become brown to black and rotted.
Figure 1. Symptoms of basal stem rot caused by Diaporthe/Phomopsis spp. on chickpea plants in the field. (A) Chickpea plants infected by Diaporthe/Phomopsis spp. showed leaf chlorosis and wilting; (B) exodermis of basal stems and roots become brown to black and rotted.
Plants 13 01950 g001
Plants 13 01950 g002
Figure 2. Morphological characteristics of the Diaporthe aspalathi isolates from chickpea. (A) Upper colony surface; (B) lower colony surface; (C) pycnidia extruding masses of conidia; (D) α-conidia with biguttulate; (E) embedded perithecia with visible beaks; (F) mature asci and ascospores with two-celled (Scales: 10 μm).
Figure 2. Morphological characteristics of the Diaporthe aspalathi isolates from chickpea. (A) Upper colony surface; (B) lower colony surface; (C) pycnidia extruding masses of conidia; (D) α-conidia with biguttulate; (E) embedded perithecia with visible beaks; (F) mature asci and ascospores with two-celled (Scales: 10 μm).
Plants 13 01950 g002
Plants 13 01950 g003
Figure 3. The Neighbor-Joining tree was generated from the analysis of the combined ITS, tef1, tub2, cal, and his3 gene regions. The neighbor-joining bootstrapping values are shown above the branching nodes (>50% out of 1000 bootstraps). The strain number, host, and origin of each Diaporthe isolate were shown for each reference strain where available (The numbers mean neighbor-joining bootstrapping values).
Figure 3. The Neighbor-Joining tree was generated from the analysis of the combined ITS, tef1, tub2, cal, and his3 gene regions. The neighbor-joining bootstrapping values are shown above the branching nodes (>50% out of 1000 bootstraps). The strain number, host, and origin of each Diaporthe isolate were shown for each reference strain where available (The numbers mean neighbor-joining bootstrapping values).
Plants 13 01950 g003
Plants 13 01950 g004
Figure 4. Pathogenicity and host range tests of Diaporthe aspalathi isolate causing chickpea stem canker. (A) Control and induced symptoms by inoculating the three isolates ZD36-1, ZD36-2, and ZD36-3 on chickpea seedlings. Control and induced symptoms on soybean (B), cowpea (C), mung bean (D), adzuki bean (E), common bean (F,G) (red box indicates inoculated stem showing extended lesion), pea (H), faba bean (I), and grass pea (J) by inoculating isolate ZD36-3.
Figure 4. Pathogenicity and host range tests of Diaporthe aspalathi isolate causing chickpea stem canker. (A) Control and induced symptoms by inoculating the three isolates ZD36-1, ZD36-2, and ZD36-3 on chickpea seedlings. Control and induced symptoms on soybean (B), cowpea (C), mung bean (D), adzuki bean (E), common bean (F,G) (red box indicates inoculated stem showing extended lesion), pea (H), faba bean (I), and grass pea (J) by inoculating isolate ZD36-3.
Plants 13 01950 g004
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, D.; Deng, D.; Zhan, J.; Wu, W.; Duan, C.; Sun, S.; Zhu, Z. An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China. Plants 2024, 13, 1950. https://doi.org/10.3390/plants13141950

AMA Style

Wang D, Deng D, Zhan J, Wu W, Duan C, Sun S, Zhu Z. An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China. Plants. 2024; 13(14):1950. https://doi.org/10.3390/plants13141950

Chicago/Turabian Style

Wang, Danhua, Dong Deng, Junliang Zhan, Wenqi Wu, Canxing Duan, Suli Sun, and Zhendong Zhu. 2024. "An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China" Plants 13, no. 14: 1950. https://doi.org/10.3390/plants13141950

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

Article Metrics

Back to TopTop