Identification and Biocontrol of Cladosporium Mold Caused by Cladosporium cladosporioides on Wheat Spikes in Central China

Abstract

Wheat (Triticum aestivum L.) is one of the most agriculturally and economically important crops in the world. Wheat fungal diseases are becoming more severe and frequent due to global climate change, threatening wheat yields and security. While fungal diseases such as fusarium head blight, stripe rust, and powdery mildew have been extensively studied, the newly emerged fungal pathogens in wheat are still under-researched. In May 2023, black mold symptoms were observed on wheat spikes in Xinxiang City, Henan Province, China. However, the causal agent of this disease was not known. We employed a combination of morphological examination and molecular techniques to identify the pathogen. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1), and actin (act) genes of the fungus were partially sequenced (accession no. OR186209, PQ271633 and PQ271632) and showed 99.59–100% identity with the previously reported Cladosporium cladosporioides, which affects wheat, pokeweed, and black-eyed pea. The pathogenicity of this fungus was confirmed by fulfilling Koch’s postulates. Through a rigorous screening process, we found Simplicillium aogashimaense, Trichothecium roseum, and Bacillus velezensis as effective biocontrol agents, with B. velezensis demonstrating the most potent antagonistic activity against the Cladosporium mold. This discovery showed the potential of B. velezensis as a biocontrol agent for wheat disease management. The findings underscore the importance of the present study in advancing the control of this disease.

1. Introduction

Wheat (Triticum aestivum L.), one of the five major crops worldwide, contributes significantly to the global human calorie intake (FAO, http://www.fao.org/faostat/en, accessed on 1 July 2024). In addition, wheat is one of the most agriculturally and economically crucial crops, playing an important role in the development of civilization [1]. However, pests and pathogens significantly affect the quality and quantity of wheat plants and, thus, are an obstacle to wheat food security. The wheat losses worldwide and in China are estimated to be 21.5% and 28.1%, respectively [2]. It has been reported that in China, pests and pathogens reduced wheat yield by up to 16.29% in a main wheat planting region, the Yellow and Huai River valleys [3]. Therefore, securing the wheat production will remain a world priority due to outbreaks of various pests and pathogens.

Fungal pathogens, such as Fusarium spp., Puccinia spp., Blumeria graminis, and Rhizoctonia cerealis, notably affect the quality and quantity of wheat production in China [3]. While plant resistance genes and fungicides have been mined and utilized for controlling wheat pathogens, the newly emerging and/or resistant strains of pathogens will be suppressing wheat productivity [4,5,6,7,8,9,10,11,12]. In addition, mycotoxins that are produced by contaminating fungal pathogens are the most crucial risks related to wheat product consumption and are threats to animal and human health [13,14]. Identification and characterization of the causal agents of wheat diseases will support wheat management and, thus, help with the improvement of wheat production security.

The genus Cladosporium comprises more than 772 names [15]. Species of Cladosporium are widely distributed worldwide and are commonly isolated from various sources, such as soil, air, food, plants, fungi, insects, and other organic materials [16,17,18]. It has been reported that Cladosporium cladosporioides is a mycoparasitic fungus on a wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici [19]. In addition, it has been documented that C. atriellum, C. cladosporioides, C. graminum, C. herbarum, C. inversicolor, C. macrocarpum, C. malorum, C. oxysporum, C. perangustum, and C. pseudocladosporioides are found in wheat in many countries all over the world (https://fungi.ars.usda.gov/, accessed on 1 July 2024). However, in China, Cladosporium herbarum is the only species of Cladosporium which has been reported in wheat (https://fungi.ars.usda.gov/, accessed on 1 July 2024). Whether there are other Cladosporium spp. in wheat in China remains largely unknown.

Crop breeding and chemical fungicide application are capable of efficiently controlling fungal diseases in wheat [2,12]. However, there are new pathotypes and/or chemical fungicide-resistant isolates of pathogens emerging, and extensive chemical fungicide utilization is harmful to humans and the environment [7,20,21]. Therefore, the identification and screening of environment- and human-friendly alternatives, e.g., novel biocontrol agents (BCAs) and their metabolites, for managing wheat fungal pathogens are still in demand [18,20,22,23,24]. So far, in contrast to crop resistant gene mining and chemical fungicide screenings, fewer biocontrol agents have been identified and evaluated.

In May 2023, distinct symptoms of a mold disease were monitored on wheat grown at the campus of Henan Normal University. Black masses were observed on the wheat spikes. More than 90% of the observed wheat spikes exhibited symptoms of mold. However, the causal pathogen was unknown. Therefore, the aim of this study was (1) to identify the mold fungus based on morphological characteristics and molecular analysis and (2) to screen the biocontrol agents against the pathogen.

2. Materials and Methods

2.1. Plant and Fungal Materials

Wheat spikes with black mold symptoms were collected from wheat (Triticum aestivum L., cv. AK58) cultivated at the campus of Henan Normal University in Xinxiang City, Henan Province, China, and were then transported to the Xinxiang Key Laboratory of Plant Stress Biology at Henan Normal University for subsequent experiments. The fungal pathogen was isolated from wheat spikes and cultured on PDA medium. To obtain a pure isolate, individual spores of the pathogen were transferred onto PDA and incubated in the dark at 25 °C [25]. Isolation was conducted three times, and the purified isolate was used for identification.

2.2. Microscopic Observation

In order to observe morphological characteristics and identify the isolated pathogen, the fungal structures were first observed by a stereomicroscope (Olympus, SZ61) and then under light microscopy (Sunny Optical, EX30, Ningbo, Zhejiang, China).

2.3. DNA Extraction and Amplification

The total genomic DNA of the pathogen was extracted according to a previously reported method [5]. Briefly, the fungal structures were harvested and the gDNA was isolated with the extraction buffer (50 mM Tris/Cl pH 9.0, 150 mM NaCl, 5 mM EDTA, 5% SDS). The C. cladosporioides ribosomal ITS, translation elongation factor 1-alpha (tef1), and actin (act) gene sequences were amplified by polymerase chain reaction (PCR) with ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R primer pairs [26,27]. The PCR process was conducted on a C1000 TouchTM Thermal Cycler (Bio-Rad, Hercules, CA, USA) using the following program: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min, and final elongation at 72 °C for 5 min. Then, 1% agarose gel was used for electrophoresis detection of the PCR products. The amplicon was sequenced (Invitrogen, Shanghai, China), and the resulting sequence was deposited in GenBank with the accession number OR186209.

2.4. Phylogenetic Analysis

To further identify C. cladosporioides, sequences of Cladosporium spp. were retrieved from the National Center for Biotechnology Information (NCBI). A phylogenetic tree was built via MEGA software (version 10.1.8) with default parameters and the maximum likelihood method, with the options of the 1000 bootstrap method, the Tamura–Nei model, and 50% site coverage cut-off. The ITS sequences of Simplicillium aogashimaense and Trichothecium roseum were used as outgroups [4,6].

2.5. Pathogenicity Assay

To fulfill Koch’s postulates, pathogenicity tests were conducted to confirm the pathogenicity of the identified C. cladosporioides. Briefly, the spore suspension (1 × 10⁶ spores mL⁻¹) of C. cladosporioides was sprayed onto the surfaces of three independently detached healthy wheat spikes, and three detached healthy wheat spikes sprayed with sterile water were used as controls. Then, the spore-treated and control plants were placed into two growth chambers (light/dark, 16 h/8 h; temperature, 25 °C; humidity, 60%). The mold symptoms were checked every day after inoculation. The pathogenicity assays were repeated twice, and similar results were obtained. The moldy spikes were checked and the pathogenic fungus was re-isolated and compared with the initially isolated pathogen to confirm the same morphological features.

2.6. Screenings of Biological Control Agents against C. cladosporioides

To screen the efficient biocontrol agents against C. cladosporioides, the previously identified fungi with antagonistic activities were applied [4,5,6]. A Bacillus velezensis strain, ZM202201 (NCBI accession number OP210007), that was isolated from tomatoes (Solanum lycopersicum L.) was used as a bacterial antagonistic agent. The antagonists were cultivated on potato dextrose agar (PDA) in darkness for 7 days. Then, the PDA plates were inoculated with a PDA plug containing C. cladosporioides (in the plate center) and biocontrol agents, i.e., B. velezensis, T. roseum, and S. aogashimaense, and then cultivated in darkness. The colony sizes of C. cladosporioides were measured at 3, 6, 9, and 12 days post-inoculation (dpi).

2.7. Effects of B. velezensis Exudates on Cladosporium Mold In Vivo and In Vitro

To determine whether B. velezensis exudates impair C. cladosporioides developments on wheat spikes and on PDA, B. velezensis were incubated in a centrifuge tube with Luria–Bertani broth (LB) and incubated on a shaker (150 rpm) at 20 °C for 3 days. Next, total LB was filtered through filter paper (Ø = 7 cm, Newstar, Hangzhou, China) and a 0.22 μm syringe filter (Jinteng, Tianjin, China). The resulting solution containing B. velezensis exudates was stored at 4 °C for further experiments. Wheat spikes were sprayed with B. velezensis exudates or water (control group) and then were inoculated with a spore suspension of C. cladosporioides (1 × 10⁶ spores mL⁻¹). The disease symptoms were observed at 5 dpi. PDA plates were sprayed with water (control group) or B. velezensis exudates and then inoculated with C. cladosporioides. The colony sizes of C. cladosporioides were measured from 2 dpi to 8 dpi.

2.8. Statistical Analysis

The basis for statistical analyses of colony sizes was n = 3 independent biological experiments. Significant differences (p < 0.05) were tested with the SPSS software (version 25.0, IBM Corporation, Armonk, NY, USA) using the one-way ANOVA test followed by a Tukey post hoc test between multiple datasets. Alternatively, they were tested using the Student’s t-test between two datasets.

3. Results

3.1. Morphological Characteristics of the Black Mold Fungus on Wheat Spikes

The spikes with black mold symptoms were collected, and then the morphological characteristics of the fungus were examined (Figure 1A,B). It was shown that the colony on potato dextrose agar (PDA) had a grey to green color, and was dense and floccose-felty (Figure 1C). Conidiophores (n = 30) were branched, and conidia (n = 50) in long branched chains varied in size, ranging from 3 to 6 µm × 2 to 3 µm (length × width). Secondary ramoconidia (n = 50) were subcylindrical to cylindrical–oblong and measured 10 to 30 µm × 2 to 4 µm (length × width). The morphological characteristics of this mold fungus were quite similar to the reported C. cladosporioides.

3.2. Molecular Identification of the Fungal Pathogen

To further confirm the causal agent of the black mold, ITS, translation elongation factor 1-alpha (tef1), and actin (act) genes were amplified with universal primers, and the resulting sequences were deposited into NCBI (accession number OR186209, PQ271633 and PQ271632). BLASTn analysis revealed that OR186209 100% (592/592 bp) was identical to the previously reported C. cladosporioides (HM148042) on wheat in South Africa [28]. PQ271633 and PQ271632 were, respectively, 99.59% (244/255 bp) and 100% (228/228 bp) identical to C. cladosporioides (HM148260 and HM148507) on pokeweed (Phytolacca americana) and on black-eyed pea (Vigna unguiculata subsp. Unguiculata) in South Korea. In addition, the phylogenetic analysis clearly demonstrated that the identified fungus and reported C. cladosporioides were clustered in the same clade (Figure 2). Therefore, based on the molecular analysis, the pathogen was identified and confirmed as C. cladosporioides.

3.3. Pathogenicity of C. cladosporioides on Wheat

To fulfill Koch’s postulates, the pathogenicity of C. cladosporioides on wheat spikes was tested (Figure 3). Five days after inoculation, black mold symptoms were found on inoculated spikes, while no mold symptoms were observed on controls. The observed symptoms were morphologically identical to those on the originally infected wheat spikes. Therefore, C. cladosporioides was the causative agent of black mold on wheat.

3.4. Screening of Biocontrol Agents against Cladosporium Mold In Vitro

To screen the biocontrol agents against C. cladosporioides, in vitro antagonistic activities of Bacillus velezensis, Trichothecium roseum, and Simplicillium aogashimaense were tested (Figure 4). It was shown that B. velezensis, T. roseum, and S. aogashimaense significantly inhibited the colony expansion in vitro. The colony area in control groups increased from about 1.7 cm² at 3 dpi to 15 cm² at 12 dpi, while the areas of B. velezensis- and T. roseum-antagonized colonies were less than 2 cm² from 3 dpi to 12 dpi, indicating that B. velezensis and T. roseum were the most effective biocontrol agents.

3.5. Exudates of B. velezensis Inhibit Cladosporium Mold In Vivo and In Vitro

B. velezensis is known as a biocontrol agent and was described to produce antifungal compounds. To test whether exudates of B. velezensis would suppress the development of Cladosporium black mold, wheat spikes or PDA were inoculated with B. velezensis exudates and then suspensions of C. cladosporioides conidia (Figure 5). It was illustrated that exudates of B. velezensis inhibited black mold disease development on wheat spikes (Figure 5A,B) and impaired the colony expansion on PDA (Figure 5C,D). Comparing to the colony sizes of water-treated (control) C. cladosporioides, the colony sizes of exudates-treated C. cladosporioides were notably decreased. The colony sizes in control group were 1.2 and 1.6 times bigger than those in the exudates-treated group at 2 dpi and 8 dpi, respectively.

4. Discussion

As one of the most agriculturally and economically important crops, wheat (Triticum aestivum L.) notably contributes to the global human calorie intake (FAO, http://www.fao.org/faostat/en, accessed on 1 July 2024) and significantly supports the development of civilization [1]. However, the quality and quantity of wheat plants are negatively affected by pests and pathogens. In China, the wheat loss was estimated as 28.1% [2]. Wheat diseases, such as rust, powdery mildew, scab, and sharp eyespot, are obstacles to wheat food security in China [3]. Since fungi-produced mycotoxins are harmful to animal and human health, the determination and identification of pathogen contamination in wheat are crucial to wheat production, consumption, and security [13,14]. Species of Cladosporium are isolated from various sources, such as soil, air, food, plants, fungi, insects, and other organic materials [16,17,18]. While some Cladosporium spp. in wheat has been documented in many countries all over the world (https://fungi.ars.usda.gov/, accessed on 1 July 2024), there is only one species, C. herbarum, that has been reported on wheat in China. In this study, we observed the black mold disease on wheat spikes and identified the causal pathogen as C. cladosporioides based on morphological and molecular analysis (Figure 1 and Figure 2). It was determined that C. cladosporioides is one of the fungal contaminants in stored wheat [29]. Therefore, one may speculate that the spores of C. cladosporioides on wheat spikes will be transported and will be a contaminant in stored wheat after harvest and storage. However, more research is needed to survey whether there is C. cladosporioides contaminating wheat in China.

C. cladosporioides has previously been reported as the causal agent of plant diseases (such as Cladosporium rot in grapes, blossom blight in strawberry and red powder puff Calliandra haematocephala, black mold in post-harvest tomato, and fruit rot in sweet pepper) [30,31,32,33,34,35]. In addition, C. cladosporioides has been shown to cause human and animal diseases, such as cutaneous phaeohyphomycosis, pulmonary phaeohyphomycosis, and pneumonia [36,37,38,39,40,41]. Therefore, C. cladosporioides on wheat spikes may be the inoculum for its further infection and, thus, plays crucial roles in the epidemiology of this pathogen. In our study, we tested the infection of C. cladosporioides on wheat spikes and found that this pathogen formed visible symptoms at 5 days post-inoculation (Figure 3). These findings will support the control of C. cladosporioides at pre- and post-infection stages. However, due to the lack of chromosome-scale genome assembly of C. cladosporioides, the infection mechanisms of this fungus are still obscure. It is not known whether C. cladosporioides produces mycotoxins on plants and thus affects human health. More studies are needed which illustrate the infection mechanisms and metabolites of C. cladosporioides in the future.

Biocontrol agent applications are regarded as one of the most environment- and human-friendly methods for wheat disease control [4,6,18,20,22,23,24]. Biocontrol agent identification and subsequent screenings are a prerequisite for the biocontrol of wheat diseases. Therefore, in this study, we utilized previously identified biocontrol agents, i.e., B. velezensis, T. roseum, and S. aogashimaense, to determine their antagonistic activities against C. cladosporioides. It was found that these fungi and the bacterium were capable of efficiently inhibiting the colony development of C. cladosporioides, and that T. roseum and B. velezensis were the effective biocontrol agents (Figure 4). In addition, exudates of B. velezensis were able to significantly inhibit disease occurrence and colony development on wheat spikes and PDA (Figure 5), indicating the potential of B. velezensis application for further control of C. cladosporioides. The antagonistic fungi were able to notably suppress fungal phytopathogens; however, one may speculate that the antagonistic fungi may lead to contamination in wheat and/or produce harmful compounds. Previously, the metabolites of Bacillus spp. were repeatedly shown as antifungal compounds against phytopathogens [42,43]. However, it is not clear which compounds are capable of efficiently inhibiting C. cladosporioides development. While the genomes of B. velezensis strains were repeatedly sequenced and assembled, the molecular mechanisms and effective anti-fungal metabolites of B. velezensis were still underestimated. Thus, more research is needed demonstrating the mechanisms of activities of B. velezensis against plant fungal pathogens and supporting the effort regarding phytopathogen control.

5. Conclusions

In this study, we employed morphological characteristics and molecular analysis to identify the causal agent of black mold on wheat spikes in central China. C. cladosporioides was identified and confirmed as the pathogen. The pathogenicity and infection time points were tested. Through biocontrol agent screening, B. velezensis and its metabolites were validated to be capable of significantly suppressing the development of C. cladosporioides. Our findings expand the knowledge about the hosts of C. cladosporioides and provide fundamental information for developing effective black mold management strategies in wheat.

Author Contributions

M.Z. and F.H. conceptualized the research program, designed the experiments, and critically edited the whole manuscript. H.B. and W.Z. performed material preparation, analyzed the data, and wrote up the initial manuscript. S.Z. and Z.Q. designed the experiments and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Dataset is available upon request from the authors.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Footnotes

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Figures

Figure 1. Morphological characteristics of black mold caused by Cladosporium cladosporioides on wheat spikes. (A,B) Black mold signs and symptoms. (C,D) Morphological characteristics of C. cladosporioides structures.

Figure 2. Phylogenetic analysis of the identified C. cladosporioides. The phylogenetic tree was constructed with the ITS sequences of C. cladosporioides and other Cladosporium spp. using the MEGA software.

Figure 3. Pathogenicity of identified C. cladosporioides on wheat spikes. (A,B) Infected wheat spikes with black fungal mass. (C) A fungal colony on a wheat spike. The images of infected wheat spikes with different magnifications were taken at 5 days post inoculation. The scale bars in (B,C) are 4 mm and 0.5 mm, respectively.

Figure 4. In vitro antagonistic activities of B. velezensis (Bv), T. roseum (Tr), and S. aogashimaense (Sa) against C. cladosporioides. Colonies of C. cladosporioides treated with water (A), B. velezensis (B), T. roseum (C), and S. aogashimaense (D). (E) Colony area of C. cladosporioides antagonized with different biocontrol agents on PDA. In E, each value is given as mean ± SD. Significant differences were determined using a one-way ANOVA with post hoc Tukey test. Different letters indicate significant differences (p [less than] 0.05).

Figure 5. In vivo and in vitro inhibiting effects of B. velezensis exudates on development of C. cladosporioides. Disease development of C. cladosporioides in water (A) and B. velezensis exudates and (B) treated wheat spikes. (E), Colony sizes of C. cladosporioides treated with water or B. velezensis exudates on PDA from 2 dpi to 8 dpi. In (E), each value is given as mean ± SD. Significant differences were determined using Student’s t test; different letters indicate significant differences (p [less than] 0.05).

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