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

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.


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].
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.

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).
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).
Plants 2024, 13, x FOR PEER REVIEW

Molecular Characteristics and Phylogenetic Analysis
The ITS, tef1, tub2, cal, and his3 gene regions of rDNA from three isolate sequenced using their respective common primers.Sequences of the ITS regi EF1-α gene obtained from the three isolates showed 99-100% similarity with mo 40 or several reported D. aspalathi strains in GenBank by a BLAST analysis.The sequences of the five loci (ITS, tef1, tub2, cal, and his3) were cascaded, and 2 sequences of the three isolates and other sequences from reported Diaporthe/Ph spp.strains were involved in the dataset.
The phylogenetic tree was constructed using a concatenated sequence datase five gene regions (Figure 3).In the phylogenetic tree, the three isolates were group those of three reference strains of D. aspalathi, whose host was rooibos (Aspalathus from South Africa in GenBank (Figure 3).The phylogenetic analysis revealed t three isolates could be distinguished from other species of the Diaporthe/Phomop Altogether, the results of the molecular characteristics and phylogenetic analysis s support that the three isolates clearly belong to D. aspalathi [36].

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].

Pathogenicity and Host Range Tests
Firstly, the pathogenicity of the three D. aspalathi isolates was tested on the origina host.The results showed that all isolates were strongly pathogenic to the original host

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.

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 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.

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 multilocus phylogenetic analysis, the three isolates ZD36-1, ZD36-2, and ZD36-3 were identified as D. aspalathi.
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.

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.

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.

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-mmdiameter 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).

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 Plants 2024, 13, 1950 9 of 12 DNA, 2 µL of each primer, 25 µL of 2×Taq PCR Mastermix (TIANGEN, Beijing, China), and 17 µL ddH 2 O.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).

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.

Figure 1 .
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 .
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 3 .
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 th branching nodes (>50% out of 1000 bootstraps).The strain number, host, and origin of eac Diaporthe isolate were shown for each reference strain where available (The numbers mea neighbor-joining bootstrapping values).

Figure 3 .
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).