DOI: https://doi.org/10.21203/rs.3.rs-2751018/v1
This study was conducted to investigate the effectiveness of vermicompost (VC) in the control of tomato bacterial canker and wilt disease (Clavibacter michiganensis subsp. michiganensis), which causes significant yield losses in tomato production. In pot experiments, vermicompost was added to pot soil at 10, 20, 30 and 40% doses. In field experiments, vermicompost was applied to soil at a dose of 600 kg/da. In pot experiments, effect of vermicompost on disease severity varied between 53.4-90.8%. In 40% VC treatments, a low rate of lesions was detected in vascular bundles and the disease infection was suppressed by 66.77%. In field experiments, a low rate of disease was also detected in VC-treated plants and disease severity was affected by 66.6%. The infection rate in vascular bundles was suppressed by 69.9%. Present findings revealed that vermicompost significantly suppressed tomato bacterial canker and wilt disease and it could be used as an important component of integrated pests and disease management.
Tomato bacterial canker and wilt disease, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), significantly limit tomato production. Cmm is encountered in entire tomato production fields of the world (EPPO 2023) and causes significant yield losses in tomato production through systemic and local infections (Eichenlaub and Gartemann 2011; Sen et al. 2015; Chalupowicz et al. 2017). Seed-borne agent enters into the plant through wounds and natural openings (Bryan 1930; Carlton et al. 1998; de Leon et al. 2011). Especially in infections via seeds, symptoms that start with unilateral wilt progress along the vascular bundles and gradual plant death is encountered (Sharabani et al. 2013). Seedling stage is the most sensitive period for the disease (Nandi et al. 2018). A rapid die out is seen in young seedlings. Brownish spots surrounded by a small white halo, also so called as bird's eye, formed on fruits are typical symptoms of the disease (Medina-Mora et al. 2001; de Leon et al. 2011; Çetinkaya-Yıldız et al. 2019). Seeds are the first inoculum source of the disease agent and it survives in contaminated plant residues and contaminated soils (Chang et al. 1991; Gleason et al. 1991; Fatmi and Schaad 2002) and can easily spread in small areas through weeds, pruning shears, tools and equipment (Çetinkaya-Yıldız et al. 2019). Cultural practices may not be sufficient against tomato bacterial canker and wilt disease and there is no specific effective substance against the disease to be used in chemical control. Therefore, it is highly difficult to control the disease (Montenegro et al. 2018). Thus, alternative methods are being searched for effective control of the disease.
Vermicompost is the leading organic fertilizer used in farming practices. It plays a great role in enriching soil organic matter and nutrients. Just because of high nutrient and organic matter contents, vermicompost is widely used in agricultural practices. Vermicompost production is largely dependent on mesophilic composting, Advanced composting is achieved by worms and a rich final product that passes through the digestive system of the worms is obtained (Fracchia et al. 2006; Domínguez and Edwards 2011; Adhikary 2012). Cattle manure, vegetable wastes, domestic wastes and industrial wastes are commonly used in vermicompost production. Worm species, production technique, temperature, drying techniques used in vermicompost production significantly influence the quality of final product (Ceritoğlu et al. 2018). Vermicompost products have many advantages, from plant growth to soil regulating effects, from antioxidant effects to potential use of all kinds of (vegetable, animal, industrial, etc.) wastes in production. Besides increasing soil organic matter, vermicompost also provides the soil with plant nutrients, hormones, enzymes and humic substances. It increases microbial activity thus positively affects soil fertility and vitality (Ceritoğlu et al. 2018). Vermicompost obtained through digestive system of the worms has a significant potential to reduce the use of chemical pesticides and fertilizers (Simsek-Ersahin 2010). Vermicompost, an organic fertilizer, provides a balanced intake of essential nutrients for plants (Bellitürk 2018). Since it improves the uptake of plant nutrients, it has a special plant growth-enhancing effect (Szczech 1999; Atiyeh et al. 2000; Nagavallemma et al. 2004). Vermicompost contains coelomic fluid secreted from the bodies of worms. Enzymes and proteins such as agglutinin, chitinase, fetidin in the structure of coelom fluid are effective on some fungi, bacteria and pests (Wang et al. 2006; Yaviç et al. 2020).
There are studies conducted by various researchers on the use of vermicompost for the control of plant diseases (Van Heerden et al. 1995; Pharand et al. 2002; Chaoui et al. 2002; Rose et al. 2003; Sarma et al. 2010; Asciutto et al. 2006; Soylu et al. 2020; Karnez et al. 2021). Few studies have been conducted about the effect of vermicompost on tomato bacterial canker and wilt disease (Utkhede and Koch 2004). Therefore, this study was conducted to investigate the effect of vermicompost on tomato bacterial canker and wilt disease.
The vermicompost used in this study was supplied from a private company (Tokat, Turkey). Vermicompost analyses were conducted in Ankara Soil and Fertilizer Research Institute (Turkey) and the analysis results are provided in Table 1.
| Analyses | Results |
|---|---|
| Organic matter, % | 64.19 |
| Total nitrogen, % | 2.60 |
| Moisture, % | 76.55 |
| pH | 7.70 |
| EC, ms cm− 1 | 4.75 |
| Total phosphorus, % | 1.59 |
| Total potassium, % | 1.43 |
| Total calcium, ppm | 17162 |
| Total magnesium, ppm | 7060 |
| Total iron, ppm | 5274 |
| Total copper, ppm | 76.16 |
| Total zinc, ppm | 149.02 |
| Total manganese, ppm | 289.02 |
In this study, Clavibacter michiganensis subsp. michiganensis isolate with the code PK-Cmm-1 isolated from diseased tomato plants in tomato production areas of Tokat province and identified by biochemical and molecular analyzes was used (Belgüzar et al. 2016). Bacterial culture is available as a stock culture in nutrient broth and glycerol at -20°C. Nutrient Agar (NA) medium was used for the development of pathogenic bacteria. Tomato seedlings of Alsancak RN F1 cultivar were used as the plant material of the study.
Pot experiments were conducted under controlled greenhouse conditions, Initially, a mixture of sterile soil, vermicompost (10%, 20%, 30%, 40%), peat and perlite were prepared. Resultant mixtures were placed into #5 pots (diameter: 34 cm; depth: 28 cm). Tomato seedlings were then transplanted into these pots. Pathogenic bacteria were inoculated on tomato plants 10 days after transplantation process. At this stage, a suspension of 1x106 cells/ml was prepared from the bacteria grown in NA medium and the pathogen was inoculated under the stem bark of the plants with the help of a sterile injector. Only the pathogen was applied to the plants in the growth environments where vermicompost was not mixed and they were used as a Positive Control (PC) group. In addition to PK, the plants to which the pathogen was not applied and sterile distilled water was given were used as a Negative Control (NC) group. Following the pathogen inoculation, plants were placed in a humidity chamber in a damp nylon bag for one day.
Symptom development was followed by daily checks on the plants removed from the humidity chamber. The study was established on 5 plants in 3 replications. The study was repeated twice. Considering the positive control plants, the disease was evaluated based on disease symptoms encountered on the plants. Disease severity was assessed over 0–5 scale defined by Francis et al. (2001). Scale values were determined according to wilt, leaf blight and canker formation in infected plants. If these three symptoms are seen at a moderate level in the plant, each symptom was scored as 1. Accordingly, the disease severity was defined as 3 in the plant exhibiting these 3 symptoms. In case each of these symptoms is severe or weak, 0.5 units are increased or decreased. In other words, if all three symptoms are weak in the plant, the scale value was defined as 1.5. If all three symptoms were severe, the scale value was defined as 4.5. If the plant is dead, the scale value was defined as 5. An assessment was also made through measuring plant height and lesion size in the vascular bundles.
Following the pot experiments, a field trial was established in Ulaş village of Tokat province between June and August 2022. Experiments were conducted in randomized blocks design with 4 replications and 10 plants in each replicate. Treatments were planned as Positive Control (PC), Negative Control (NC) and Vermicompost (VC). In VC treatments, vermicompost was applied to soil as to have 600 kg/da. Seedlings were planted with 90 cm row spacing and 40 cm on-row plant spacing. Pathogen inoculation was performed on PC and VC plants one week after the transplantation process. As it was in pot experiments, a suspension of 1x106 cells/ml was prepared from the bacteria grown in the nutrient medium and inoculated under the stem bark of the plants with the help of a sterile injector (Fig. 1). NC plants were given only sterile distilled water. After inoculation, plants were examined daily and when disease symptoms were observed in positive control plants, disease severity was determined in all plants (Fig. 2). Plants were cut longitudinally and lesion length in the vascular bundles was measured.
When the pot and field experiments were terminated, re-isolates were obtained from plants with disease symptoms and it was proven that the disease was caused by Cmm.
The effect ratio (%) of vermicompost on bacterial canker and wilt disease was calculated with the use of Abbott’s formula (% effect = (control - treatment) / control) × 100) (Karman 1971). Experimental data were subjected to variance analysis with the use of SPSS statistical analysis software and significant means were compared with the use of Tukey’s multiple comparison test at P ≤ 0.05 significance level.
Pot experiments were carried out in the disease greenhouse of Plant Protection Department at the Research and Application Center between May-June 2022. Typical symptoms began to be observed about 30 days after inoculation in positive control plants to which Cmm was applied. Experiments were terminated 50 days after inoculation and assessments were made. In Positive Control plants, disease symptoms such as unilateral wilt at the beginning and general wilting in the later periods, recess in development, yellowing of the leaves, browning of the vascular bundles were observed and disease severity was determined as 5. No disease symptoms were observed in Negative Control plants and disease severity was determined as 0. Effect of vermicompost treatments at different doses on the severity of tomato bacterial canker and wilt disease varied between 53.4–90.8%. Partial wilting and yellowing were observed in the plants grown in 10% vermicompost mixture and disease severity was determined as 2.33. The 10% vermicompost treatment suppressed the disease by 53.4%. Disease severity of 20%, 30% and 40% vermicompost treatments was respectively determined as 1.36, 0.63 and 0.46. Disease suppression rate of these treatments was respectively calculated as 72.8%, 87.4% and 90.8%. As compared to PC, all treatments had significant effects on disease severity (Table 2; Fig. 3).
| Treatments | Disease severity | Effect ratio (%) | Lesion length (cm) | Effect ratio (%) | Plant height (cm) | Effect ratio (%) |
|---|---|---|---|---|---|---|
| NC* | 0.00 ± 0.00 a** | - | 0.00 ± 0.00 a | - | 66.00 ± 1.85 bc | - |
| PC | 5.00 ± 0.00 d | 0 | 43.73 ± 2.14 d | 0 | 43.73 ± 2.14 a | 0 |
| 10% VC | 2.33 ± 0.37 c | 53.4 | 28.73 ± 3.39 c | 34.30 | 55.73 ± 3.50 ab | 21.53 |
| 20% VC | 1.36 ± 0.26 b | 72.8 | 30.26 ± 3.24 c | 30.80 | 54.33 ± 3.45 ab | 19.51 |
| 30% VC | 0.63 ± 0.07 ab | 87.4 | 18.93 ± 1.71 b | 56.71 | 74.40 ± 3.54 cd | 41.22 |
| 40% VC | 0.46 ± 0.07 a | 90.8 | 14.53 ± 1.83 b | 66.77 | 81.26 ± 3.74 d | 46.18 |
| *NC: Negative Control, PC: Positive Control, VC: Vermicompost | ||||||
**Means indicated with the same letters in a column are not significantly different (Tukey’s multiple comparison test, p ≤ 0.05).
Lesions in the vascular bundles are the most typical symptoms of tomato bacterial canker and wilt disease. No signs of blackening were observed in the vascular bundles of NC plants treated with distilled water, thus disease severity was recorded as 0. In PC plants, on the other hand, darkening was observed in the vascular bundles and the lesion length was measured as 43.73 cm. Lesion length was measured as 34.30 cm in 10% VC treatments and as 30.80 cm in 20% VC treatments. In 30% and 40% VC treatments, a low rate of lesion was detected and the disease was suppressed in the vascular bundles by 56.71% and 66.77%, respectively. All treatments were found to be significant as compared to PC (Table 2). Effects of vermicompost treatments on plant heights were also evaluated in pot experiments. As compared to PC, 30% and 40% VC treatments increased plant height by 41.22% and 46.18%, respectively and they were placed into a significantly different group with the positive control (Fig. 4). In particular, despite Cmm treatments, 40% VC treatments increased plant height by 18% as compared to NC plants. On the other hand, in terms of the effects on plant height, 10% and 20% VC treatments were placed into the same group with PC plants (Table 2).
Field experiments lasted for 4 months. Pathogen inoculation was conducted on 10 June 2022 and typical disease symptoms were encountered 45 days after inoculation. While no disease symptoms specific to Cmm (disease severity: 0) were observed in the negative control plants, symptoms in the form of unilateral wilt were determined in the leaves and plants in the positive control plants. Disease severity was identified as 1.5. In addition, disease progression in plants remained limited. Wilting was observed on leaves and some shoots. No signs of wart or canker were found on the trunk. Such a case revealed that temperature was especially effective on disease progress. During the experimental period, July-August, temperatures were below regional averages, therefore it was concluded that the disease did not progress sufficiently in the plants. The disease severity of vermicompost-treated plants was determined as 0.5. Symptoms specific to bacterial wilt in the form of unilateral wilt were observed only on the leaves. Parallel to pot experiments, as compared to positive control, a lower rate of disease was detected and vermicompost suppressed the disease by 66.6% (Table 3).
Vermicompost also prevented 69.39% of lesion formation along the vascular bundles. While an average of 33.72 cm lesion was measured in PC plants, the lesion was measured at a lower rate (10.32 cm) in VC-treated plants. In general, an increase was observed in plant height. VC-treated plants were longer than the PC and NC plants (Table 3).
| Treatments | Disease severity | Effect ratio (%) | Lesion length (cm) | Effect ratio (%) | Plant height (cm) | Effect ratio (%) |
|---|---|---|---|---|---|---|
| NC* | 0.00 ± 0.00 a** | - | 0.00 ± 0.00 a** | - | 120.00 ± 3.36 b | - |
| PC | 1.50 ± 0.00 c | 0 | 33.72 ± 1.65 c | 0 | 105.10 ± 2.01 a | 0 |
| VC | 0.5 ± 0.07 b | 66.6 | 10.32 ± 1.32 b | 69.39 | 135.90 ± 3.40 c | 22.22 |
| *NC: Negative Control, PC: Positive Control, VC: Vermicompost | ||||||
**Means indicated with the same letters in a column are not significantly different (Tukey’s multiple comparison test, p ≤ 0.05).
Tomato bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis, is an important seed-borne pathogen that causes serious yield losses by damaging roots, stems, leaves, flowers and fruits of tomato plants both in greenhouse and open-field productions. Since it was first detected in tomatoes in 1909, several studies have been conducted in many countries on this disease agent. Since Cmm is a systemic spread disease agent, it is generally transmitted through infected seeds. It is also transferred to the next production season through diseased plant residues and contaminated soils, causing great crop losses. Since the disease agent is able to survive in different places, significant difficulties are encountered in control of tomato canker and wilt disease.
Vermicompost, obtained as a result of the vermiculture process and so called as black gold (Kangmin 2005; Patangray 2014), is the conversion of organic waste/wastes into fertilizer through digestive tracks of worms. It is used as a slow-release fertilizer and improves soil physical, chemical and biological properties (Yağmur and Eşiyok 2019). Vermicompost also provides soil aeration, increases water holding capacity and regulates soil microbial activity (Singh et al., 2003). Thus, an increase in product quality and quantity is ensured. Vermicompost is rich in microbial diversity and biomass. Therefore, it is also rich in enzymes and hormone-like chemicals produced by microorganisms. In addition, vermicompost ensures that plant nutrients can be used by the plant for a long time. This gives vermicompost a "slow-release fertilizer" feature (Şimşek-Erşahin 2013).
Vermicompost contains active substances like plant growth regulators. It is used as organic fertilizer, soil conditioner and in disease and pest control (Edwards and Bohlen 1996). Rich plant nutrients and microbial composition of vermicompost increase plant resistance against pests and diseases. With the aid of vermicompost, plants whose roots are covered by beneficial microorganisms are less affected by pathogens and population growth of soil pathogens and pests is limited. Thus, pathogens cannot cause disease or damage remains below the economic damage threshold.
It was proved in previous studies that vermicompost was quite effective in suppression of plant diseases and pests. Recently, studies on this subject have been gaining momentum. In present study, it was determined that vermicompost significantly suppressed bacterial canker and wilt disease. In pot experiments, different doses of vermicompost were used and the disease severity decreased the most in 40% vermicompost treatments and the disease was suppressed by 90%. The lesion size in vascular bundles also significantly decreased (66%). Parallel to pot experiments, vermicompost suppressed the disease significantly in the field experiments. Vermicompost suppressed the disease by 66% in field experiments. The disease remained at a limited level in the vascular bundles.
Present findings comply with the findings of earlier studies. In this sense, it was reported that compost tea prepared from the vermicompost supplied from Abbsfort (Canada) suppressed bacterial canker and wilt disease by 63%. In that study by Utkhede and Koch (2004), chemical and biological applications were made to tomato plants of Dombito variety. Cmm was applied to the plant in the form of spray through the applications made to the cotyledon leaves. Evaluations were made 42 days after inoculation, the infection rate was 71.6 in the plants to which the disease agent was applied, while a low infection rate (26.6) was determined in the vermicompost treatments. Yogev et al. (2009) studied the effect of compost consisting of tomato and pepper waste combined with poultry and cattle manure and reported that compost reduced Cmm disease intensity up to 100% in both natural infection and artificial inoculations and Cmm population of the compost decreased within 15–20 days. Tutar (2013) indicated that chloroform extracts of vermicompost had a strong effect against Pseudomonas syringae, Xhantomonas carotae, Sclerotinia sclerotiorum, Fusarim oxysporum, Aspergillus humicola, A. fumigatus and a weak effect against Erwinia chrysanthemi, Pseudomonas fluorescens and Penicillium brevicompactum. On the other hand, ethanol extracts had a strong effect against Pseudomonas syringae, Xhantomonas campestris and Aspergillus fumigatus and a weak effect against Erwinia herbicola, E. chrysanthemi and S. sclerotiorum. It was reported that vermicompost products obtained from some organic residues or wastes suppressed plant fungal diseases of Phythium, Fusarium, Rhizoctonia ve Phytophthora spp., Plectosporium tabacinum, Botrytis cinerea, Verticillium wilt, Sclerotonia rolfsii (Szczech 1999; Edwards and Arancon, 2004; Şimsek-Erşahin et al. 2008).
Good agricultural practices including organic farming, integrated pests and disease management and sustainable agriculture have recently been developed to minimize chemical inputs in agricultural practices. These practices aim to obtain sufficient and quality products at an affordable cost. Good agricultural practices also ensure more efficient use of agricultural lands and protection of environment and natural resources. Vermicompost is an excellent biofertilizer. It increases soil fertility, supports plant growth and reduces the negative effects of plant pathogens. Present findings revealed that vermicompost significantly suppressed tomato bacterial canker and wilt disease. Thus, vermicompost could be used as a part of integrated disease management plans developed against the disease.
Acknowledgment
The vermicompost used in this study was supplied from a private company (Tokat, Turkey). Thank you to the company.
Also, I thank Zeki Gökalp (Erciyes University) for his support and critical evaluation of the manuscript.
Conflict of interest
The author declares that there is no confict of interest for this submission.
Ethics approval and consent to participate
Not applicable.
Consent for participate and publication
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Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Competing interests
The author declare that she has no competing interests.
Informed consent
This manuscript is new and not being considered elsewhere.
Funding
There are no funds for the research.
Authors’ contributions
SB performed the experiment, analysis of data, writing, original draft preparation.