Next Article in Journal
Sexual Dimorphism in Third Molar Agenesis in Humans with and without Agenesis of Other Teeth
Next Article in Special Issue
Bacillus spp. as Bioagents: Uses and Application for Sustainable Agriculture
Previous Article in Journal
Feeding Ecology of Odontaster validus under Different Environmental Conditions in the West Antarctic Peninsula
Previous Article in Special Issue
The Effect of Date Palm Genotypes on Rhizobacterial Community Structures under Saline Environments
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biology
Volume 11
Issue 12
10.3390/biology11121724
biology-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

Isolation and Characterization of Novel Biological Control Agent Clostridium beijerinckii against Meloidogyne incognita

by
Xinglong Lian
1,
Shuang Liu
1,
Lanyuwen Jiang
1,
Xinyu Bai
1 and
Yuanyuan Wang
1,2,*
1
College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, China
2
Nematology Institute of Northern China, Shenyang Agricultural University, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
Biology 2022, 11(12), 1724; https://0-doi-org.brum.beds.ac.uk/10.3390/biology11121724
Submission received: 27 October 2022 / Revised: 16 November 2022 / Accepted: 26 November 2022 / Published: 28 November 2022
(This article belongs to the Collection Plant Growth-Promoting Bacteria: Mechanisms and Applications)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Meloidogyne incognita is one of the most widely distributed and researched species of Meloidogyne, which has caused enormous economic losses for Chinese agriculture. Biological control is a nonhazardous method of pest and pathogen management. Nematophagous bacteria, which are ubiquitous and have extensive host ranges, are an efficient natural adversary of nematodes. Thus, to find novel, promising biological control agents for RKNs, we conducted trials to isolate and screen effective bacterial endophytes against M. incognita, and then we selected an effective strain using morphological and molecular approaches. Furthermore, a greenhouse experiment was conducted to assess their potential as biocontrol agents against M. incognita and to investigate the effect of several strains on increasing seed germination and tomato plant growth. Strain Sneb518 (Clostridium beijerinckii) was identified as having solid biocontrol activity against M. incognita. Sneb518 demonstrated significant inhibition against M. incognita, with J2 mortality reaching 90.73% at 12 h and with eggs hatching at a rate of 6.00% at 24 h. Additionally, Sneb518 was excellent for enhancing seed germination. The effectiveness and stability of M. incognita management by Sneb518 C. beijerinckii were further evaluated in a greenhouse. This research will offer insightful information on the application of Sneb518 as a biocontrol agent for RKN management.

Abstract

One of the most severe soil-borne pathogens in the world is the root-knot nematode (Meloidogyne incognita). Biological control is gaining more importance as environmental awareness increases. Thus, keeping this in mind, a total of 712 bacterial strains were isolated from 117 rhizosphere soil samples and investigated for potential biological control activity against M. incognita. Strain Sneb518 (Clostridium beijerinckii) was identified as having solid biocontrol activity against M. incognita. Sneb518 demonstrated significant inhibition against M. incognita, with J2 mortality reaching 90.73% at 12 h and with eggs hatching at a rate of 6.00% at 24 h, compared to a hatchability level of 29.07% for the control. Additionally, Sneb518 was excellent for enhancing seed germination. The seeds coated with a fermentation broth containing Sneb518 efficiently boosted the germination rate to 88.49%. The effectiveness and stability of C. beijerinckii Sneb518 against M. incognita were then further evaluated in a greenhouse. According to the pot experiment data, Sneb518 considerably (p < 0.05) reduced the number of root galls and egg masses on roots and also significantly (p < 0.05) increased tomato plant growth. C. beijerinckii Sneb518-treated tomato seedlings exhibited 50.26% biocontrol effectiveness compared to the control group. Our results demonstrate that C. beijerinckii Sneb518 can be a potential biological control agent against root-knot nematode disease and a biomass enhancer. This research will give new options for the sustainable control of root-knot nematode disease in tomatoes and other host plants.

1. Introduction

Plant-parasitic nematodes (PPNs) pose a considerable risk to a wide range of crops, causing global yield losses [1,2]. They may easily damage crops by feeding on them and forming associations with other microorganisms, posing a risk to agriculture globally, with an estimated annual loss of USD 100–150 billion [3,4,5]. Nematode control, as a result, is a significant obstacle to initiatives to ensure global food security. [6]. Furthermore, PPNs are more difficult to eradicate than are other pathogens because they live in the soil and typically target the subsurface parts of plants [7]. Root-knot nematodes (RKNs, Meloidogyne spp.) are widespread and cause significant yield losses in various crops, especially vegetables [8]. Meloidogyne incognita is one of the most widely distributed and researched species of Meloidogyne [9,10]. M. incognita has caused enormous economic losses for Chinese agriculture [11].
Chemical nematicides have been employed to suppress nematodes, but their continued and indiscriminate usage has adverse effects on humans and the environment [7]. Nematicides, such as ethylene dibromide (EDB), methyl bromide, and dibromochloropropane, are no longer widely available in local markets due to the hazards they pose to humans [12]. Nematode control methods are urgently required in light of the rising demand for organic and chemical-free crops [13]. Given the challenges posed by chemical nematicides, the research into innovative, environmentally acceptable alternatives for controlling PPN populations has become more crucial over the last 20 years [6]. The roles of many beneficial soil microorganisms are highly regarded as eco-friendly biological alternatives to synthetic chemical nematicides [14].
Biological control is a nonhazardous method of pest and pathogen management [15,16]. However, the most promising potential chemical nematicide substitutes are antagonists and nematophagous microorganisms [17]. There are few commercially available nematophagous bacteria and fungi for controlling plant-parasitic nematodes [18]. Microorganisms that develop in the rhizosphere protect roots from pathogen assaults and are appropriate for use as biocontrol agents [19]. Nematophagous bacteria are ubiquitous, have extensive host ranges, and are an efficient natural adversary of nematodes [6]. They directly decrease nematode activity by producing antibiotics, toxins, and enzymes; they also compete for resources and space by parasitizing, hence offering a defense response for plant development [20]. Biocontrol agents such as Clostridium, Alcaligenes, Serratia, Streptomyces, Pseudomonas, Desulfovibrio, Pasteuria, Bacillus, and Agrobacterium have demonstrated tremendous promise for biological nematode control [18]. Researchers have previously argued that nonparasitic rhizobacteria are harmful to nematodes, particularly when organic materials have been absorbed into the soil or under anaerobic circumstances [21,22,23]. Clostridium species have obvious plant-growth-promoting abilities [24]. Clostridium bacteria release toxins that kill nematodes [25]. Since Clostridium has a demonstrated biocontrol impact against a variety of pests and pathogens, it is likely that this species could be employed to control nematode infestations [26,27]. However, no studies have been conducted on using C. beijerinckii to suppress nematodes. Thus, to find novel, promising biological control agents against RKNs, we have conducted trials to isolate and screen effective bacterial endophytes against M. incognita, and then we selected the effective strain using morphological and molecular approaches. Furthermore, a greenhouse experiment was conducted to assess Sneb518′s potential as a biocontrol agent for M. incognita and to investigate the effect of several strains on increasing seed germination and tomato plant growth. This research will provide valuable data on using Sneb518 as a biocontrol agent for RKN management.

2. Materials and Methods

2.1. Preparation of Bacterial Strain Culture and Nematodes

About 117 rhizosphere soil samples (5 samples were collected from each greenhouse and then combined) were collected randomly from various greenhouses in Liaoning Province to identify and purify a bacterial strain utilizing the soil dilution method. These strains were developed in a beef extract, peptone agar medium (NA) plate, suspended in sterile water, and adjusted to 1.0 × 108 cfu/mL using a hemocytometer under a microscope before being introduced to 50 mL of sterilized lysogeny broth (LB) [28]. Cultures were kept at 28 °C and stirred at 150 rpm for 48 h and were then centrifuged at 6000× g for 20 min. The J2s’ lethality and egg hatching bioassays were performed on fermentation filtrate using a 0.22 um filter.
M. incognita were maintained in greenhouses at Shenyang Agricultural University on the susceptible tomato cultivar L402. M. incognita egg masses were selected under a microscope and surface disinfected for 3 min with 0.5% sodium hypochlorite to decrease microbial contamination, and then rinsed three times with sterile water and incubated at 28 °C to allow the J2s to hatch.

2.2. Preliminary Screening on Mortality of Juveniles and Egg Hatchability Treated by Bacterial Strain Filtrate

Approximately 100 newly hatched M. incognita J2s were placed in 5 mL glass Petri dishes along with 2 mL of bacterial strain filtrate to determine their impact on J2′s mortality. The control treatment consisted of using sterile water. The tubes were incubated in the dark at 26 °C. The number of hatching J2s was counted using a stereomicroscope. The experiment was repeated three times, with three replicates in each experiment. The following formula was used to calculate the mortality percentage: Mortality (%) = the number of dead J2s/the total number of J2s × 100.
A similar method was used to investigate the influence of bacterial strains on egg hatchability. About 50 surface-sterilized eggs were incubated in 2 mL bacterial strain filtrate for 24 h. A control was performed using 100 surface-sterilized eggs in 2 mL of sterile distilled water. The eggs hatched in the dark at 26 °C. A stereomicroscope was used to count the number of hatching J2s. Each treatment comprised three duplicates, and the experiments were repeated three times. The hatchability of eggs was determined using the following formula:
Egg hatchability (%) = the number of hatched eggs/total number of eggs × 100.
Based on the findings above, the bacterial strain that strongly influenced J2 mortality and egg hatchability was chosen for future investigation.

2.3. Second Screening of Sneb518 on Mortality and Egg Hatchability In Vitro

To further investigate the effect of Sneb518 on M. incognita, the exposure duration of nematode J2s and eggs in the Sneb518 filtrate was prolonged. As a control, all of the treatments in 2 mL sterile distilled water were utilized. Each treatment had three replicates, and the experiments were repeated three times. The above-mentioned formula was used to calculate mortality and egg hatchability.

2.4. Identification of Strain Sneb518

According to Gerhart, et al. [29], physiological and biochemical tests were performed to identify strain Sneb518. Later, Sneb518 was identified further via the phylogenetic analysis of 16S rRNA gene sequences. To extract genomic DNA, standard techniques were used [30,31]. The 27F and 1492R primer pairs were used to amplify the 16S rRNA sequences [32,33]. A PGEM-T vector (Promega, Madison, WI, USA) was modified to include PCR products. The plasmid was extracted and sequenced by the Genewiz Biotechnology Co., Ltd. (Suzhou, China). The 16S rRNA gene sequence was identified using the GenBank database and then aligned with similar species using Clustal X 2.1 [34]. The neighbor-joining strategy was used in Mega 7.0 to create a phylogenetic tree based on the 16S rRNA at 1000 replications [35].

2.5. Sneb518′s Effect on Tomato Seedling Growth

Tomato seeds were surface-sterilized with 70% ethanol for 30 s, rinsed three times with sterile distilled water, and air-dried [36]. Seeds were evenly coated with the Sneb518 fermentation at a 70:1 (g/mL) ratio, with distilled water used as a control treatment. The coated seeds were placed in a Petri dish with wet filter paper and cultured for 1 week at 28 ± l °C. The seed germination, shoot, and root length per 10 seeds were measured after 7 days of incubation. The vigor index and germination rate were calculated by Abdul-Baki [37].
Germination rate (%) = number of germinated seeds/the total number of seeds × 100
Vigor index (VI) = (shoot length + root length) × Germination percentage %

2.6. Effect of Sneb518 against M. incognita in the Pot Experiments

Tomato seeds were sterilized as above. Sterilized seeds were planted in a seedling tray (5 cm diameter each) and allowed to germinate at 25 °C. Tomato seedlings were transplanted 4 weeks later into pots (12 cm × 12 cm) filled with 500 g sterilized soil and sand (2:1). Plants were placed inside a lighted chamber (16 h photoperiod and temperature range of 23–26 °C). A total of four treatments were used. After 2 days of the plantation, the half-plant was inoculated with 20 mL 10× diluted Sneb518 fermentation (Sneb518), while the other half served as a control and was inoculated with sterilized water (CK). After 7 days of the plantation, the 2000 J2s were inoculated into half of the Sneb518 (Sneb518+J2) and half of the control plants (CK+J2). The nematodes were inoculated through four small, 2 cm deep holes around the roots of the plants. All pots were set in a randomized, complete block form and watered regularly (Figure 1).
Thirty days after inoculation, ten tomato plants were selected randomly for each treatment. The plant roots were rinsed under running water. Plant height, root length, and fresh and dry root weight were measured [38]. The root gall indices were evaluated as described by Barker [39]. The disease index of the root was graded on a scale of 0 to 5, with “0” indicating no gall, “1” suggesting 15% or fewer roots having galls, “2–4” indicating 16–25%, 26–50%, and 51–75% roots having galls, respectively, and “5” indicating >76% roots having galls [39]. The gall indices and biocontrol effectiveness were calculated using the following formulas:
Gall index = ∑No. of diseased plants in each grade × grade/ (total No. plants examined × the highest grade) × 100%
Biocontrol efficacy (%) = (gall index in the control group–gall index in the bacteria treated group)/gall index in the control group × 100.

2.7. Statistical Analysis

The statistical analysis was performed using SPSS software (version 20.0). Duncan’s multiple range test (p < 0.05) was used to determine the significant difference between treatments.

3. Results

3.1. Preliminary Screening on Mortality of Juveniles and Egg Hatching Treated by Bacterial Strains In Vitro

A total of 712 bacterial strains were obtained from 117 rhizosphere soil samples via the soil dilution method and screened for potential nematicidal activity against M. incognita J2. However, 11 bacterial isolates exhibited intense nematicidal activity (Table 1). At 12 h, the J2 mortality when treated with 11 strains was over 70%, 8 were over 80%, and 1 was over 90%. In terms of egg hatchability, these 11 strains dramatically lowered egg hatchability as well. The hatching rates of Sneb518, Sneb532, Sneb549, Sneb562, Sneb596, Sneb633, and Sneb687 were as low as 10% at 24 h. While Sneb518 showed distinctive inhibition against M. incognita, J2 mortality reached 90.73% at 12 h, with eggs hatching at 6.00% at 24 h, and the hatchability for the control was 29.07%. Based on these results, the strain Sneb518 was selected for further study.

3.2. Second Screening of Sneb518 for Nematode Mortality and Egg Hatchability

Sneb518 exhibited nematicidal activity against M. incognita J2; mortality was 91.55%, 93.99%, and 96.00% after 24 h, 48 h, and 72 h, respectively (Table 2). In contrast, the control group’s mortality rates at 24 h, 48 h, and 72 h were 1.34%, 3.47%, and 5.51%, respectively. M. incognita J2s had considerable time-dependent mortality. There was a significant difference between the Sneb518 treatment and the control for all three treatment-time periods.
Hatched juveniles were counted at 48 h, 72 h, and 96 h to determine the impact of Sneb518 on M. incognita egg hatching (Table 2). The hatchability of the control was 82.470% at 96 h; however, it was reduced dramatically to 24.12% with the Sneb518 treatment.

3.3. Identification of Strain Sneb518

The Sneb518 colonies that formed on the NA culture medium were rounded, with irregular edges and bulges in the middle, translucent, grayish white, creamy and sticky, and slightly shiny, with a diameter of about 2 mm at 48 h. The bacterial strain was Gram-positive and had rod-shaped cells (Figure 2).
Molecular identification of the bacteria Sneb518 showed that the length of the 16S rRNA sequence, as amplified with primers 27F/1492R, was 1418 bp. In addition, comparing the sequence to BLAST homologous sequences from the GenBank database revealed that the strain Sneb518 had a 98% similarity with Clostridium beijerinckii. The phylogenetic tree was constructed with Clostridium spp. sequences downloaded from the NCBI database (Figure 3). The strain Sneb518 is clustered on the same branch as C. beijerinckii (KF892544 and KX269863). Sneb518 was identified as C. beijerinckii based on the findings of morphological and molecular investigations. The sequence of C. beijerinckii Sneb518 was submitted to the GenBank database with accession number ON920936.

3.4. Sneb518′s Effect on Tomato Seedling Growth

Seed coating was used to evaluate the potential of strain Sneb518 to promote seed germination. Table 3 shows the effect of Sneb518 on tomato seedling growth. A significant difference (p < 0.05) was observed in coated seeds with Sneb518 fermentation broth, which increased germination by up to 88.49%. Shoot length and root length demonstrated the potential of Sneb518, which increased by 4% and 20%, respectively, compared to the control. Seed coating with C beijerinckii Sneb518 increased the seed vigor index by 60%.

3.5. Effect of Sneb518 on the Control of M. incognita in the Pot Experiments

A pot experiment assessed the potential biological control ability of C. beijerinckii Sneb518 against M. incognita. Thirty days after M. incognita inoculation, CK tomato plants exhibited 77.2 root galls, whereas C. beijerinckii Sneb518-treated tomato seedlings exhibited 50.26% biocontrol effectiveness as compared to the control (Table 4). Overall, the findings provided strong evidence that C. beijerinckii Sneb518 could effectively control M. incognita on tomato plants under greenhouse conditions. Moreover, in the present research, C. beijerinckii Sneb518 efficiently boosted the growth of plants.

4. Discussion

Clostridium is the second-largest genus after Streptomyces, with over 100 species. It is one of the common bacteria that produce acetate, acetoin, butyrate, hydrogen gas, lactate, carbon dioxide, butanol, acetone, acetyl methyl carbonyl, and ethanol during fermentation [40,41]. Moreover, Clostridium species have obvious plant-growth-promoting abilities [24,42]. Toxins produced by Clostridium bacteria destroy nematodes [25,43]. Clostridium beijerinckii species are abundant in nature and are commonly isolated from soil [44]. Numerous plants and pathogens have reported Clostridium’s biocontrol effect against various pests and pathogens, indicating that this genus can be used to control nematode infection [26,27,45]. However, no such research had been performed on the application of C. beijerinckii to control nematodes. Keeping that in mind, a novel research study was developed to investigate the nematode-reducing potential of C. beijerinckii through seed coating and to increase biomass. Our findings could be a theoretical basis for creating a valuable and marketable biocontrol agent.
Plant-growth-promoting rhizobacteria (PGPR) are a hot issue in the hunt for plant protection against various diseases [46]. Many PGPR strains have been used effectively to treat RKNs [47]. Effective and safe RKN disease control techniques are desperately needed in greenhouses [48,49]. C. beijerinckii Sneb518 isolates were selected for further study from 712 bacterial strains found to be effective against RKNs. Sneb518 inhibited juvenile and egg hatching significantly more than other strains. This strain was also tested for its ability to boost plant growth and its biocontrol effectiveness against RKNs through seed treatment in both pot and field experiments. The findings indicated that this Sneb518 strain exhibited biological control ability against M. incognita. The biocontrol effect of C. beijerinckii Sneb518 might be the consequence of strains promoting plant growth and secreting chemicals that destroy the J2s of nematodes and impede egg hatching (Table 1 and Table 2). Colonization is thought to be a crucial phase in the biological control action of biocontrol bacteria. Secondary metabolites such as enzymes, antibiotics, and poisonous chemicals have been frequently linked to nematicidal activity [50,51]. However, the nematicidal secondary metabolites generated by C. beijerinckii Sneb518 have yet to be characterized.
Seed biopriming is the process of coating seeds with microorganisms to protect seedlings against pathogens [52]. Seed treatment was employed as a general approach for applying PGPR that might impart resistance to RKNs in agricultural plant production [53,54]. Seed treatment has been extensively utilized to manage M. incognita, M. javanica, and Rotylenchulus reniformis in various crops, including tomatoes, soybeans, and cotton [55,56,57]. PGPRs showed significant potential as biocontrol agents against M. incognita in tomatoes, efficiently promoting plant growth [58]. This study revealed that the application of C. beijerinckii Sneb518 as a seed treatment helped to reduce nematode disease and enhance plant growth (Table 3 and Table 4). These data suggested that seed treatment might be an effective and cost-effective economic approach for Sneb518 to control M. incognita.

5. Conclusions

In conclusion, our findings demonstrated that treating tomato seeds with C. beijerinckii Sneb518 considerably decreased the amount of M. incognita galls in pot experiments. Furthermore, C. beijerinckii Sneb518 killed J2s and reduced egg hatchability in in vitro experiments. Moreover, C. beijerinckii Sneb518 also exhibited plant growth characteristics and nematicidal potential in a greenhouse experiment. The findings indicate that C. beijerinckii has the potential for microbial application and commercial use as a biocontrol agent in the field. This research will give new options for the sustainable control of root-knot nematode disease in tomatoes and other host plants. Further research is required to assess its biochemical characterization before recommending it as a commercial nematicide. Future research will continue to explore the mechanisms of Sneb518 as a biological control agent for RKNs. This may provide a theoretical basis for the better prevention of RKN disease.

Author Contributions

Conceptualization, Y.W. and X.L.; methodology, X.L. and S.L.; software, L.J. and X.B.; formal analysis, Y.W.; investigation, X.L.; resources, X.L. and L.J.; data curation, L.J.; writing—original draft preparation, X.L. and S.L.; writing—review and editing, Y.W.; funding acquisition, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Shenyang Science and Technology Bureau Seed Industry Innovation Project (21–110-3–11); National Parasitic Resources Centre (NPRC-2019–194-30).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Abd-Elgawad, M.M.; Askary, T.H. Impact of Phytonematodes on Agriculture Economy; CABI: Wallingford, UK, 2015. [Google Scholar]
  2. Sikandar, A.; Khanum, T.A.; Wang, Y. Biodiversity and community analysis of plant-parasitic and free-living nematodes associated with maize and other rotational crops from Punjab, Pakistan. Life 2021, 11, 1426. [Google Scholar] [CrossRef]
  3. Kim, T.Y.; Jang, J.Y.; Jeon, S.J.; Lee, H.W.; Bae, C.-H.; Yeo, J.H.; Lee, H.B.; Kim, I.S.; Park, H.W.; Kim, J.-C. Nematicidal activity of kojic acid produced by Aspergillus oryzae against Meloidogyne incognita. J. Microbiol. Biotechnol. 2016, 26, 1383–1391. [Google Scholar] [CrossRef]
  4. Sikandar, A.; Zhang, M.; Wang, Y.; Zhu, X.; Liu, X.; Fan, H.; Xuan, Y.; Chen, L.; Duan, Y. In vitro evaluation of Penicillium chrysogenum Snef1216 against Meloidogyne incognita (root-knot nematode). Sci. Rep. 2020, 10, 8342. [Google Scholar] [CrossRef] [PubMed]
  5. Sikandar, A.; Zhang, M.; Wang, Y.; Zhu, X.; Liu, X.; Fan, H.; Xuan, Y.; Chen, L.; Duan, Y. Review article: Meloidogyne incognita (root-knot nemtaode) a risk to agriculture. Appl. Ecol. Environ. Res. 2020, 18, 1679–1690. [Google Scholar] [CrossRef]
  6. Nyaku, S.T.; Affokpon, A.; Danquah, A.; Brentu, F.C. Harnessing useful rhizosphere microorganisms for nematode control. In Nematology-Concepts, Diagnosis and Control; Shah, M.M., Mahamood, M., Eds.; IntechOpen: London, UK, 2017; p. 153. [Google Scholar]
  7. Pulavarty, A.; Egan, A.; Karpinska, A.; Horgan, K.; Kakouli-Duarte, T. Plant parasitic nematodes: A review on their behaviour, host interaction, management approaches and their occurrence in two sites in the Republic of Ireland. Plants 2021, 10, 2352. [Google Scholar] [CrossRef] [PubMed]
  8. Bahadur, A. Nematodes diseases of fruits and vegetables crops in India. In Nematodes-Recent Advances, Management and New Perspectives; Bellé, C., Kaspary, T.E., Eds.; IntechOpen: London, UK, 2021; pp. 1–18. [Google Scholar]
  9. Rusinque, L.; Nóbrega, F.; Cordeiro, L.; Lima, A.; Andrade, S.; Inácio, M.L. Root-knot nematode species associated with horticultural crops in the Island of Azores, Portugal. Horticulturae 2022, 8, 101. [Google Scholar] [CrossRef]
  10. Sikandar, A.; Zhang, M.; Yang, R.; Liu, D.; Zhu, X.; Liu, X.; Fan, H.; Duan, Y.; Wang, Y. Analysis of gene expression in cucumber roots interacting with Penicillium chrysogenum strain Snef1216 through seed coating, which induced resistance to Meloidogyne incognita. Nematology 2021, 24, 121–135. [Google Scholar] [CrossRef]
  11. Na, J.; Hui, X.; Li, W.-J.; Wang, X.-Y.; Qian, L.; Liu, S.-S.; Pei, L.; Zhao, J.-L.; Heng, J. Field evaluation of Streptomyces rubrogriseus HDZ-9-47 for biocontrol of Meloidogyne incognita on tomato. J. Integr. Agric. 2017, 16, 1347–1357. [Google Scholar]
  12. Onkendi, E.M.; Kariuki, G.M.; Marais, M.; Moleleki, L.N. The threat of root-knot nematodes (Meloidogyne spp.) in A frica: A review. Plant Pathol. 2014, 63, 727–737. [Google Scholar] [CrossRef] [Green Version]
  13. Mwamula, A.O.; Kabir, M.F.; Lee, D. A review of the potency of plant extracts and compounds from key families as an alternative to synthetic nematicides: History, efficacy, and current developments. Plant Pathol. J. 2022, 38, 53–77. [Google Scholar] [CrossRef]
  14. Abd-Elgawad, M. Biological control agents of plant-parasitic nematodes. Egypt. J. Biol. Pest Control 2016, 26, 423–429. [Google Scholar]
  15. Sharma, A.; Diwevidi, V.; Singh, S.; Pawar, K.K.; Jerman, M.; Singh, L.; Singh, S.; Srivastawav, D. Biological control and its important in agriculture. Int. J. Biotechnol. Bioeng. Res. 2013, 4, 175–180. [Google Scholar]
  16. Sikandar, A.; Zhang, M.; Zhu, X.; Wang, Y.; Ahmed, M.; Iqbal, M.; Javeed, A.; Xuan, Y.; Fan, H.; Liu, X. Efficacy of Penicillium chrysogenum strain SNEF1216 against root-knot nematodes (Meloidogyne incognita) in cucumber (Cucumis sativus L.) under greenhouse conditions. Appl. Ecol. Environ. Res. 2019, 17, 12451–12464. [Google Scholar] [CrossRef]
  17. Hu, H.; Gao, Y.; Li, X.; Chen, S.; Yan, S.; Tian, X. Identification and nematicidal characterization of proteases secreted by endophytic bacteria Bacillus cereus BCM2. Phytopathol. 2020, 110, 336–344. [Google Scholar] [CrossRef]
  18. Abd-Elgawad, M.M.; Askary, T.H. Fungal and bacterial nematicides in integrated nematode management strategies. Egypt. J. Biol. Pest Control 2018, 28, 74. [Google Scholar] [CrossRef] [Green Version]
  19. Khan, N.F.; Rasool, A.; Mansoor, S.; Saleem, S.; Baba, T.R.; Haq, S.M.; Rehman, S.A.; Adetunji, C.O.; Popescu, S.M. Potential applications of rhizobacteria as eco-friendly biological control, plant growth promotion and soil metal bioremediation. In Sustainable Crop Production; Meena, V., Choudhary, M., Yadav, R.P., Meena, S.K., Eds.; IntechOpen: London, UK, 2022. [Google Scholar]
  20. Tian, B.; Yang, J.; Zhang, K.-Q. Bacteria used in the biological control of plant-parasitic nematodes: Populations, mechanisms of action, and future prospects. FEMS Microbiol. Ecol. 2007, 61, 197–213. [Google Scholar] [CrossRef] [Green Version]
  21. Moosavi, M.R. Potential of soil amendment with organic matters in controlling phytonematodes. In New and Future Developments in Microbial Biotechnology and Bioengineering; Elsevier: Cambridge, MA, USA, 2022; pp. 315–344. [Google Scholar]
  22. Mcsorley, R. Overview of organic amendments for management of plant-parasitic nematodes, with case studies from Florida. J. Nematol. 2011, 43, 69–81. [Google Scholar] [PubMed]
  23. Raaijmakers, J.M.; Paulitz, T.C.; Steinberg, C.; Alabouvette, C.; Moënne-Loccoz, Y. The rhizosphere: A playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 2009, 321, 341–361. [Google Scholar] [CrossRef] [Green Version]
  24. Febri, D.; Anizan, I.; Che, R.; Wan, N.; Abidah, A.; Eka, S.; Wan, M. Enhanced rice seedling growth by Clostridium and Pseudomonas. Biotechnology 2014, 13, 186–189. [Google Scholar]
  25. Num, S.M.; Useh, N.M. Clostridium: Pathogenic roles, industrial uses and medicinal prospects of natural products as ameliorative agents against pathogenic species. Jordan J. Biol. Sci. 2014, 7, 81–94. [Google Scholar]
  26. Khabbaz, S.E.; Ladhalakshmi, D.; Babu, M.; Kandan, A.; Ramamoorthy, V.; Saravanakumar, D.; Al-Mughrabi, T.; Kandasamy, S. Plant Growth Promoting Bacteria (PGPB)—A versatile tool for plant health management. Pest Manag. Sci. 2019, 1, 1–25. [Google Scholar] [CrossRef]
  27. Ueki, A.; Takehara, T.; Ishioka, G.; Kaku, N.; Ueki, K. Production of β-1, 3-glucanase and chitosanase from clostridial strains isolated from the soil subjected to biological disinfestation. AMB Express 2019, 9, 114. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Zhou, Y.; Chen, J.; Zhu, X.; Wang, Y.; Liu, X.; Fan, H.; Duan, Y.; Chen, L. Efficacy of Bacillus megaterium strain Sneb207 against soybean cyst nematode (Heterodera glycines) in soybean. Pest Manag. Sci. 2021, 77, 568–576. [Google Scholar] [CrossRef] [PubMed]
  29. Gerhart, P.; Murray, R.; Costilow, R.; Nester, E.; Wood, W.; Krieg, R.; Philips, G. Manual of Methods for Gen. Bacteriol; American Society for Microbiolology: Washington, DC, USA, 1981. [Google Scholar]
  30. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1989. [Google Scholar]
  31. Zhao, J.; Liu, D.; Wang, Y.; Zhu, X.; Xuan, Y.; Liu, X.; Fan, H.; Chen, L.; Duan, Y. Biocontrol potential of Microbacterium maritypicum Sneb159 against Heterodera glycines. Pest Manag. Sci. 2019, 75, 3381–3391. [Google Scholar] [CrossRef]
  32. Edwards, U.; Rogall, T.; Blöcker, H.; Emde, M.; Böttger, E.C. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 1989, 17, 7843–7853. [Google Scholar] [CrossRef]
  33. Lane, D.J. 16S/23S rRNA Sequencing; John Wiley and Sons: Chichester, UK, 1991; pp. 115–175. [Google Scholar]
  34. Thompson, J.D.; Gibson, T.J.; Higgins, D.G. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinform. 2002, 2, 1–22. [Google Scholar] [CrossRef]
  35. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Hosseini, P.; Matthews, B.F. Regulatory interplay between soybean root and soybean cyst nematode during a resistant and susceptible reaction. BMC Plant Biol. 2014, 14, 300. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Abdul-Baki, A.A.; Anderson, J.D. Vigor determination in soybean seed by multiple criteria 1. Crop Sci. 1973, 13, 630–633. [Google Scholar] [CrossRef]
  38. Adam, M.; Heuer, H.; Hallmann, J. Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants. PLoS ONE 2014, 9, e90402. [Google Scholar] [CrossRef] [Green Version]
  39. Barker, K. Design of Greenhouse and Microplot Experiments for Evaluation of Plant Resistance to Nematodes; University of Massachusetts Agricultural Experiment Station: Amherst, MA, USA, 1985; pp. 103–113. [Google Scholar]
  40. Yao, X.; Zhang, Q.; Fan, Y.; Xu, X.; Liu, Z. Butanol–isopropanol fermentation with oxygen-tolerant Clostridium beijerinckii XH29. AMB Express 2022, 12, 57. [Google Scholar] [CrossRef]
  41. Narueworanon, P.; Laopaiboon, L.; Laopaiboon, P. Capability of immobilized Clostridium beijerinckii TISTR 1461 on lotus stalk pieces to produce butanol from sugarcane molasses. Processes 2021, 9, 573. [Google Scholar] [CrossRef]
  42. Doni, F.; Al-Shorgani, N.K.N.; Abuelhassan, N.N.; Isahak, A.; Zain, C.R.C.M.; Yusoff, W.M.W. Microbial involvement in growth of paddy. Curr. Res. J. Biol. Sci. 2013, 5, 285–290. [Google Scholar] [CrossRef]
  43. Dong, H.; Zhang, Y.; Dai, Z.; Li, Y. Engineering Clostridium strain to accept unmethylated DNA. PLoS ONE 2010, 5, e9038. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Makut, M.; Obiekezie, S.; Owuna, G. Isolation and screening of biobutanol producing Clostridium species from the soil environment of Keffi Metropolis. South Asian J. Res. Microbiol. 2018, 1, 1–6. [Google Scholar] [CrossRef]
  45. Ueki, A.; Takehara, T.; Ishioka, G.; Kaku, N.; Ueki, K. Degradation of the fungal cell wall by clostridial strains isolated from soil subjected to biological soil disinfestation and biocontrol of Fusarium wilt disease of spinach. Appl. Microbiol. Biotechnol. 2017, 101, 8267–8277. [Google Scholar] [CrossRef]
  46. Tyagi, S.; Kumar, R.; Saharan, B.; Nadda, A.K. Plant-Microbial Interactions and Smart Agricultural Biotechnology; CRC Press: Boca Raton, FL, USA, 2021. [Google Scholar]
  47. Aioub, A.A.; Elesawy, A.E.; Ammar, E.E. Plant growth promoting rhizobacteria (PGPR) and their role in plant-parasitic nematodes control: A fresh look at an old issue. J. Plant Dis. Prot. 2022, 129, 1305–1321. [Google Scholar] [CrossRef]
  48. Cheng, W.; Yang, X.; Xue, H.; Huang, D.; Cai, M.; Huang, F.; Zheng, L.; Yu, Z.; Zhang, J. Reproductive toxicity of furfural acetone in Meloidogyne incognita and Caenorhabditis elegans. Cells 2022, 11, 401. [Google Scholar] [CrossRef]
  49. Ji, X.; Li, J.; Dong, B.; Zhang, H.; Zhang, S.; Qiao, K. Evaluation of fluopyram for southern root-knot nematode management in tomato production in China. Crop Prot. 2019, 122, 84–89. [Google Scholar] [CrossRef]
  50. Lahlali, R.; Ezrari, S.; Radouane, N.; Kenfaoui, J.; Esmaeel, Q.; El Hamss, H.; Belabess, Z.; Barka, E.A. Biological control of plant pathogens: A global perspective. Microorganisms 2022, 10, 596. [Google Scholar] [CrossRef]
  51. Migunova, V.D.; Sasanelli, N. Bacteria as biocontrol tool against phytoparasitic nematodes. Plants 2021, 10, 389. [Google Scholar] [CrossRef] [PubMed]
  52. Mitra, D.; Mondal, R.; Khoshru, B.; Shadangi, S.; Mohapatra, P.K.D.; Panneerselvam, P. Rhizobacteria mediated seed bio-priming triggers the resistance and plant growth for sustainable crop production. Curr. Res. Microb. Sci. 2021, 2, 100071. [Google Scholar] [CrossRef] [PubMed]
  53. Backer, R.; Rokem, J.S.; Ilangumaran, G.; Lamont, J.; Praslickova, D.; Ricci, E.; Subramanian, S.; Smith, D.L. Plant growth-promoting rhizobacteria: Context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. Front. Plant Sci. 2018, 9, 1473. [Google Scholar] [CrossRef] [Green Version]
  54. Rizvi, A.; Ahmed, B.; Khan, M.S.; El-Beltagi, H.S.; Umar, S.; Lee, J. Bioprospecting plant growth promoting rhizobacteria for enhancing the biological properties and phytochemical composition of medicinally important crops. Molecules 2022, 27, 1407. [Google Scholar] [CrossRef] [PubMed]
  55. Devindrappa, M.; Kamra, A.; Grover, M.; Gawade, B. Nematicidal rhizobacteria with plant growth-promoting traits associated with tomato in root-knot infested polyhouses. Egypt. J. Biol. Pest Control 2022, 32, 51. [Google Scholar] [CrossRef]
  56. Messa, V.; Nunes, J.; Mattei, D. Seed Treatment with Bacillus amyloliquefaciens for the control of Meloidogyne javanica “in vivo” bean culture and its direct effect on the motility, mortality and hatching of M. javanica “in vitro”. Agron. Sci. Biotechnol. 2019, 5, 59. [Google Scholar] [CrossRef] [Green Version]
  57. Subedi, P.; Gattoni, K.; Liu, W.; Lawrence, K.S.; Park, S.-W. Current utility of plant growth-promoting rhizobacteria as biological control agents towards plant-parasitic nematodes. Plants 2020, 9, 1167. [Google Scholar] [CrossRef] [PubMed]
  58. El-Mageed, A.; Taia, A.; El-Mageed, A.; Shimaa, A.; El-Saadony, M.T.; Abdelaziz, S.; Abdou, N.M. Plant growth-promoting rhizobacteria improve growth, morph-physiological responses, water productivity, and yield of rice plants under full and deficit drip irrigation. Rice 2022, 15, 16. [Google Scholar] [CrossRef] [PubMed]
Biology 11 01724 g001 550
Figure 1. Effect of Sneb518 on the control of M. incognita in the pot experiments. A total of four treatments were used. After 2 days of the plantation, the half-plant was inoculated with 20 mL 10× diluted Sneb518 fermentation (Sneb518), while the other half served as a control and was inoculated with sterilized water (CK). After 7 days of the plantation, the 2000 J2s were inoculated into half of the Sneb518 (Sneb518+J2) and half of the control plants (CK +J2).
Figure 1. Effect of Sneb518 on the control of M. incognita in the pot experiments. A total of four treatments were used. After 2 days of the plantation, the half-plant was inoculated with 20 mL 10× diluted Sneb518 fermentation (Sneb518), while the other half served as a control and was inoculated with sterilized water (CK). After 7 days of the plantation, the 2000 J2s were inoculated into half of the Sneb518 (Sneb518+J2) and half of the control plants (CK +J2).
Biology 11 01724 g001
Biology 11 01724 g002 550
Figure 2. Morphological characteristics of bacteria isolate Sneb518. Left: The morphological characteristics of Sneb518 on NA plate. Right: Gram-positive staining of Sneb518.
Figure 2. Morphological characteristics of bacteria isolate Sneb518. Left: The morphological characteristics of Sneb518 on NA plate. Right: Gram-positive staining of Sneb518.
Biology 11 01724 g002
Biology 11 01724 g003 550
Figure 3. Phylogenetic tree of strain Sneb518 based on the partial nucleotide sequences of 16S rRNA. The tree was constructed using the neighbor-joining method based on a bootstrap analysis of 1000 replicates.
Figure 3. Phylogenetic tree of strain Sneb518 based on the partial nucleotide sequences of 16S rRNA. The tree was constructed using the neighbor-joining method based on a bootstrap analysis of 1000 replicates.
Biology 11 01724 g003
Table
Table 1. Preliminary screening results of bacterial strains against M. incognita.
Table 1. Preliminary screening results of bacterial strains against M. incognita.
TreatmentsJ2 Mortality (%)Egg Hatchability (%)
CK0.99 ± 1.36 a29.07 ± 3.42 f
Sneb51890.73 ± 0.74 b6.00 ± 1.94 a
Sneb52883.75 ± 5.10 c11.20 ± 1.19 cd
Sneb53286.54 ± 6.76 d6.40 ± 1.21 a
Sneb54987.87 ± 7.77 c7.60 ± 1.80 ab
Sneb56275.91 ± 6.83 de8.00 ± 1.05 ab
Sneb57677.29 ± 4.81 ef13.20 ± 2.76 de
Sneb59681.30 ± 8.59 c7.87 ± 1.59 ab
Sneb63381.60 ± 3.23 de7.60 ± 2.79 ab
Sneb67683.67 ± 4.68 f11.20 ± 2.02 cd
Sneb68782.68 ± 4.76 e9.60 ± 1.21 bc
Sneb73774.37 ± 2.38 d14.13 ± 1.73 e
Note: Different letters indicates that values are significantly different according to Duncan’s multiple range test at p < 0.05.
Table
Table 2. Second screening of Sneb518 for J2 mortality and egg hatchability.
Table 2. Second screening of Sneb518 for J2 mortality and egg hatchability.
TreatmentsJ2 Mortality (%)Egg Hatchability (%)
24 h48 h72 h48 h72 h96
CK1.34 ± 1.59 b3.47 ± 3.02 b5.51 ± 3.11 b58.93 ± 2.67 a66.72 ± 3.78 a82.47 ± 2.71 a
Sneb51891.55 ± 1.06 a93.99 ± 0.87 a96.00 ± 1.16 a10.27 ± 1.80 b16.42 ± 3.21 b24.12 ± 4.34 b
Note: Different letters indicates that values are significantly different according to Duncan’s multiple range test at p < 0.05.
Table
Table 3. Effect of Sneb518 on the seedling growth of tomato plants.
Table 3. Effect of Sneb518 on the seedling growth of tomato plants.
TreatmentsGermination RateShoot LengthRoot LengthSeed Vigor Index
CK82.75 ± 3.89 b5.73 ± 0.46 a4.87 ± 0.50 a8.77 ± 1.03 b
Sneb51888.49 ± 3.03 a6.50 ± 0.50 a5.80 ± 0.26 a13.90 ± 2.08 a
Note: Different letters indicates that values are significantly different according to Duncan’s multiple range test at p < 0.05.
Table
Table 4. The biocontrol effect of Sneb518 against M. incognita in the pot experiments.
Table 4. The biocontrol effect of Sneb518 against M. incognita in the pot experiments.
TreatmentsPlant Length (cm)Root Length (cm)Root Dry Weight (g)Root Fresh Weight (g)Gall Index (%)Biocontrol
Efficacy (%)
CK+J234.82 ± 3.45 b15.72 ± 1.14 b0.31 ± 0.05 b2.66 ± 0.31 b77.2 b-
Sneb518+J237.06 ± 3.19 a16.50 ± 0.75 a0.34 ± 0.05 ab2.85 ± 0.09 a38.4 a50.26
Note: Different letters indicates that values are significantly different according to Duncan’s multiple range test at p < 0.05.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Lian, X.; Liu, S.; Jiang, L.; Bai, X.; Wang, Y. Isolation and Characterization of Novel Biological Control Agent Clostridium beijerinckii against Meloidogyne incognita. Biology 2022, 11, 1724. https://0-doi-org.brum.beds.ac.uk/10.3390/biology11121724

AMA Style

Lian X, Liu S, Jiang L, Bai X, Wang Y. Isolation and Characterization of Novel Biological Control Agent Clostridium beijerinckii against Meloidogyne incognita. Biology. 2022; 11(12):1724. https://0-doi-org.brum.beds.ac.uk/10.3390/biology11121724

Chicago/Turabian Style

Lian, Xinglong, Shuang Liu, Lanyuwen Jiang, Xinyu Bai, and Yuanyuan Wang. 2022. "Isolation and Characterization of Novel Biological Control Agent Clostridium beijerinckii against Meloidogyne incognita" Biology 11, no. 12: 1724. https://0-doi-org.brum.beds.ac.uk/10.3390/biology11121724

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