Bioefficacy of Some Aqueous Phytoextracts Against Clavibacter Michiganensis Subsp. Michiganensis (Smith), the Cause of Bacterial Canker of Tomato

Tomato (Lycopersicon esculentum Mill.), a member of Solanaceae, is an important vegetable crop of Pakistan. The area under tomato cultivation in Pakistan is 40,238 hectares, with a total production of 566,043 tons while Khyber Pakhtunkhwa (KPK) province of Pakistan contributes an area of over 13,252 hectares with a production of 127,552 tons (Anonymous 2016). Cultivation of tomato crop has sharply increased in the country due to its growing demand. However, the demand-supply situation is still imbalanced mainly due to production constrains. Plant diseases, caused by fungi, viruses, nematodes and bacteria, are one of the major factors limiting tomato production (Agrios 2005). Among bacterial diseases, bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Smith), is a potentially severe disease of tomato that can occur in commercial plantings and home gardens. This infectious disease is capable of spreading rapidly resulting in devastating losses. Bacterial canker has caused huge economic losses in commercial tomato production throughout the world (Davis et al. 1984). The bacterium is aerobic, Gram-positive, non-motile, non-spore forming, curved rod (Bradbury 1986). The pathogen is seed-borne as well as plant-debris borne (Dutta et al. 2014).

Bacterial canker produces dark, necrotic lesions at the edges of older leaves. On the surface of young leaves and fruits, the bacteria multiply rapidly and produce tiny, raised, white blisters. Later, the centers of the white blisters turn brown, giving rise to “bird’s-eye” spots which is the characteristic symptom of bacterial canker. The vascular tissues of transversely cut stems of wilted plants near the nodes usually appear dark yellow to tan brown. On seedlings, initially, small, water-soaked lesions appear on stems and leaves which later turn into necrotic patches leading to stunting and wilting. On green-house-grown tomato plants, symptoms appear as chlorotic patches between veins that quickly become necrotic. On infected stems, light brown streaks or cankers appear which turn dark with the age. In many cases, plants affected by bacterial canker wilt and die (Bose et al. 2004). Warm temperatures (29–35 ºC) and high relative humidity favor the disease (Shenge et al. 2010).

The best disease control strategy to manage a hard-to-control disease, like bacterial canker, is the use of integrated disease management (IDM). Various components of IDM could include the use of resistant cultivars, pathogen-free seeds and transplants, green house disinfection, removal of infected plant debris, proper sanitation and rotation with non solanaceous plants for at least two years etc. (Gleason et al. 1993; Pradhanang et al. 2005). However, because of the complex nature of the pathogen, many of these strategies are of limited use (Hong et al. 2011). The scarcity of effective control measures and the magnitude of bacterial canker on economical crops, necessitate the search for new control options. One of such options is the use of dried powders or extracts of medicinal plants/weeds. A number of plants/weeds have been reported (Singariya et al. 2011; Singh and Kumar 2014; Bokaeian and Saeidi 2015) to have strong antibacterial properties. These plants have secondary metabolites, such as alkaloids, flavonoids, steroids, glycosides, saponins, and tannins, which possess antimicrobial properties (Cowan 1999). The goal of this study was to explore the bacterial-canker-control potential of those medicinal plants/weeds which have a long history as folk medicines and which are easily available in large amounts year round. We evaluated seven such medicinal plants/weeds including Withania somnifera, Melia azedarach, Peganum harmala, Allium sativum, Calotropis procera, Nerium oleander and Mentha piperita for their ability to inhibit the in vitro growth of the tomato canker bacterium using aqueous plant extracts and agar well diffusion method as well as the ability of some of these plants to reduce disease severity and enhance plant parameters in vivo.

Keeping in view the ever-increasing demand and the economic importance of tomatoes in Pakistan, the potential threat of bacterial canker to profitable tomato crop, the lack of our resource-poor farmers to afford the high costs of disease control inputs, the possible effectiveness and the year round, free of cost, large-scale availability of these medicinal plants, we tested the potential of these for the control of bacterial canker of tomato. Both in vitro and in planta experiments were carried out. The hypothesis was that the aqueous extracts of some of these medicinal plants would be inhibitory to the in vitro growth of the bacterial canker pathogen and this inhibitory effect would be equally effective for the control of the disease on tomato plants grown under screen house conditions. Moreover, we hoped that the in vivo use of the dried powders of some of these medicinal plants, if found effective, would be very economical for our small land-holding farmers on one hand, and would eliminate or at least reduce the environment-polluting chemicals such as antibiotics or copper fungicides used to control plant bacterial diseases, on the other hand. In addition to being environment-friendly, the use of dried powder soil amendments will also deter the appearance of chemical-resistant bacteria (Cowan 1999) as well as enhance tomato yield by controlling the disease and improving the physical and chemical properties of soil. The objectives of this study were (i) to investigate the potential of the aqueous extracts of these selected plants to inhibit the in vitro growth of the tomato canker pathogen, and (ii) to correlate the in vitro bacterial growth inhibition to in planta disease control and plant growth parameters enhancement.

Organic amendment of soil using different plants/weeds for the control of plant diseases is an active research domain. In addition to being environment friendly, plant products are effective against many plant pathogens, both soil-borne and phylloplane-borne. Different formulations such as dried powders, green manures (Naz et al. 2015a, 2015b), foliar sprays or soil drenches with aqueous extracts (Deberdt et al. 2012) have been reported to have successfully controlled different biotic diseases. This practice is expected to replace or reduce the amount of different chemicals used for the control of plant diseases, resulting in the reduction of environmental pollution and human health hazards. In our current soil organic amendment research, we explored the plant-disease control bioefficacy of some abundantly and free-of cost available plant species, which are well-known locally for their use as folk medicine. These plants included: (i) W. somnifera is a medium-sized Solanaceous shrub having bioactive plant substances. Organic extracts of leaves of this plant were found to have high antibacterial activity against some human’s and plant’s bacterial pathogens (Singariya et al. 2012). (ii) M. azedarach, a member of the family Meliaceae, is one of the most useful traditional medicinal plants (Biswas et al. 2002). Methanol extracts of its seeds, fruits, leaves as well as flowers are very effective against some plant bacterial pathogens (Viqar et al. 2008; Neycee et al. 2012). Leaves of M. azedarach were found to have all major plant secondary metabolites, i.e. alkaloids, saponins, tannins, phenols, glycosides, steroids and flavonoids (Fazil et al. 2012). (iii) P. harmala is an important medicinal plant which is used in global folk medicine (Sharaf et al. 1997; Jumaa 2005). It has good antibacterial and antifungal properties (Lala et al. 2004). (iv) C. procera is a salt tolerant, small compact, desert shrub which is well known for its traditional medicinal properties (Murti et al. 2010). (v) M. piperita is a perennial medicinal plant containing 0.5–4% volatile oil (Dew and Evans 1984). Volatile oil, aqueous extracts as well as organic extracts of M. piperita have been reported (Saeed et al. 2006; Bupesh et al. 2007; Al-sum and Al-Arfaj 2013) to have good antibacterial properties. The presence of many of these bioactive plant substances was confirmed by our test results. Our tests, however, are not analytical and show only rough estimates of the substances under investigation.

Our in vitro results indicated that the aqueous extracts of P. harmela and M. piperita, M. azedarach, W. somnifera and C. procera were highly effective in inhibiting the growth of Cmm, the causal agent of bacterial canker of tomato. These results suggest the presence of anti-microbial secondary metabolites in these plants. Our results are similar to those of Sarpeleh et al. (2009) and Amel et al. (2012). Sarpeleh et al. (2009) chopped fresh P. harmela into small pieces and buried the plant material into bacteria- and fungi-infested soil two-three months prior to plantation. They found that the buried plants or plant parts slowly released water-soluble anti-microbial substances which killed pathogens present in soil. Moreover, some of these water soluble substances were absorbed by plant roots resulting in killing of pathogens present inside host plant tissue. The use of finely ground dried powder of P. harmala, in case of our studies, confirms the good results for this species. Amel et al. (2012) found P. harmala to be effective even against antibiotic-resistant bacteria and some other microbes. Under in vitro studies, it effectively restricted the growth of Candida albicans and Lactobacillus. Four important alkaloids including harmine, marmaline, harmalol and peganin were found in P. harmela. Harmine has been reported to have strong antibacterial and antifungal activities (Amel et al. 2012). Using in vitro agar well diffusion assay, Bupesh et al. (2007) found that aqueous as well as organic extracts of M. piperita leaves had strong antibacterial activity against a range of pathogenic bacteria. Saeed et al. (2006) tested the antibacterial activity of M. piperita against eleven human pathogenic bacteria, using disc diffusion method. They reported that the essential of peppermint exhibited the highest activity (11.78 mm mean ZI) followed by its juice (10.41 mm mean ZI). Similarly, 1:1, 1:5, 1:10 and even 1:20 dilutions of aqueous extracts of Mentha species were found to be effective against six human pathogenic bacteria (Al-sum and Al-Arfaj 2013). Water extracts of M. azedarach have been reported to enhance the defense system of cucumber against Meloidogyne incognita (Cavoski et al. 2012). The researchers found that the activities of catalase and peroxidase enzymes involved in host H₂O₂ detoxification were reduced, suggesting enhanced defense system of the host plant. Soil organic amendment with dried powder of Withania somnifera and W. coagulan had a significant effect on improving plant growth characters in vivo, reducing bacterial load g⁻¹ soil and affecting bacterial wilt disease severity by decreasing the AUDPC values (Khan et al. 2019a; Najeeb et al. 2019). Saponins and steroids were also reported to be present in significant amounts in leaves of W. somnifera (Panchal et al. 2016). Finely ground powder of C. procera, added to soil (40 g kg⁻¹soil) was found to be about 60% as effective as the antibiotic in reducing bacterial wilt (BW) severity and as effective as the antibiotic in enhancing yield and yield parameters (Din et al. 2016). The researchers concluded that the dried powder of this plant had the potential to become an effective component of the integrated disease management against BW of tomato.

Contrary to the results of some other researchers (Jeyaseelan et al. 2010; Al-Obaidi 2014), however, the aqueous of A. sativum and N. oleander, according to our findings, did not perform as good as those of the other plants that we tested. Jeyaseelan et al. (2010) reported that both, organic solvent-extracts and water extracts of A. sativum bulbs inhibited the growth of plant pathogenic bacteria such as Xanthomonas axonopodis and Ralstonia solanacearum under in vitro conditions. This difference in results might be explained on the basis of formulation of the medicinal plant used, type of the solvent used, concentration of the extract used, and different nature of the bacterial pathogens involved. For example, we used finely ground dried powder of garlic to prepare its aqueous extract, whereas the other researchers used fresh garlic bulbs for preparing water extracts. Fresh garlic bulbs have some volatile compounds which are not present in the stored dried powder. Similarly, ethyl acetate-extracts of garlic were found to be twice as effective as aqueous extracts against bacterial pathogens (Jeyaseelan et al. 2010) indicating more solubility of anti-microbial chemicals of garlic in organic solvents. Likewise, Al-Obedi (2014) found that the aqueous extract of N. oleander significantly reduced the in vitro growth of Staphylococcus aureus and Escherichia coli. As little concentrations as 25 mg ml⁻¹ produced growth inhibition zones of 22 mm and 19 mm, respectively, of the two bacteria. In this case, the difference in results might be due to different concentrations used and different nature of the pathogens.

The observed lower disease severity, enhanced yield and yield-contributing plant growth characters of the inoculated tomato plants treated with 40 g/kg soil of P. harmela, M. piperita and the other medicinal plants could be based upon many factors. One possibility is the presence of various anti-microbial compounds in the finely ground dried powders of the medicinal plants added to soil. Our results indicated that all the major anti-microbial compounds (i.e., alkaloids, tannins, saponins, flavonoids, and glycosides) were present in the aqueous extracts prepared from dried powders of P. harmela, M. piperita, M. azedarach, W. somnifera and C. procera. These antimicrobial compounds have diverse mechanisms of action against pathogens. For example, alkaloids inhibit vital enzymes, such as topoisomerase. Others damage DNA by intercalating into it (Tanaka et al. 2006). Similarly, some tannins directly kill bacteria by damaging their cell membranes (Mainasara et al. 2012). Other tannins bind to bacterial adhesins, resulting in the prevention of bacterial attachment to host cells (Wang 2014). Steroidal saponins damage cell membrane by disabling its sterol component, resulting in the death of bacterial cell (Wang et al. 2000). The phenolic compounds, flavonoids, produced in response to pathogen’s attack, coagulate bacterial proteins and enzymes of vital pathways (Al-Obaidi 2014). The anti-microbial action of these bioactive plant compounds is, however, dose- and pathogen-contact dependent (Regnault-Roger et al. 2005; Naz et al. 2015b; Khan et al. 2019b). In our studies, the doses and amounts in soil amended with plant powder are most likely much lower than when pure substances are considered. Furthermore, there is no true contact between the pathogen in the plant and these substances in soil; unless some of them are directly absorbed by plant roots and go inside plant tissue to kill the pathogen as reported by Sarpeleh et al. (2009).

The possible activation of innate defense mechanism(s) of plants by natural elicitor compounds present in the dried powders of the medicinal plants (Walters et al. 2005) is another major fascinating scenario responsible for the observed lower disease severity and enhancement of yield and yield-contributing factors of the treated plants. Some of the components of the activated plant defense include reactive oxygen species or ROS-mediated oxidative burst, strengthening of cell walls, synthesis of anti-microbial compounds, changes in the levels of defense-related enzymes and priming of host cells etc. (Daayf et al. 2000; Kuc 2006; Cavoski et al. 2012). A coffee-leaf extract formulation, when sprayed on plants, induced defense genes in tomato against the bacterial pathogen Xanthomonas vesicatoia (Medeiros et al. 2009). By soil drenching potato plants with aqueous extracts Hibiscus sabdariffa, Punica granatum and Eucalyptus globulus, Hassan et al. (2009) demonstrated the elicitation of systemic resistance in potato against BW. Pre-inoculation foliar application of leaf extracts of Datura metel induced systemic resistance in rice plants, as evident from the accumulation of pathogenesis-related proteins and other defense-related compounds, resulting in reduced incidence of sheath blight and bacterial blight disease of rice under greenhouse conditions (Kagale et al. 2004). Interestingly enough, Graham and Myers (2011) discovered that the application of SAR (systemic acquired resistance) activators in the form of soil drench was more effective and the effect was more prolonged than their foliar sprays to control citrus canker bacterium, a non-soil-borne pathogen of citrus. The longevity of the activity of soil-applied SAR activators was attributed to their gradual release, uptake by the plant roots, and translocation to the actively growing shoots (Sur and Strok 2003; Graham and Myers 2011). Moreover, these researchers also concluded that soil-applied SAR inducers could deter the appearance of copper resistance in bacteria. Other researchers (Graham and Leite 2007; Francis et al. 2009) also recommended soil application of SAR inducers to control citrus canker.

Our in vivo results of the higher doses (40 g/kg soil) of the dried powders of P. hamela, and M. piperita are very encouraging. The finely ground powders of these plants added to soil proved to be about 80% as effective as the antibiotic in terms of reducing the disease severity, and 73% as effective as the antibiotic in enhancing yield and yield-contributing plant growth characters. This suggests that the dried powders of these plants or their extracts could be used as an effective component of the integrated disease management against bacterial canker of tomato or possibly other plant diseases. Taking advantage of the small-scale agricultural systems prevalent in Pakistan, dried powders of these medicinal plants could be directly applied to the root-zones of tomato plants with practically no increase in the input costs. Additionally, these plants are evergreen, available in large amounts free of cost which make them an attractive disease control option. Moreover, infested seed-bed soils could be treated with dried powders or green manures of these plants to make them disease-free which in turn could be used to produce disease-free tomato transplants. Many anti-microbial plant’s secondary metabolites are more soluble in organic solvents than in water (Al-Obaidi 2014). Further research is needed for the evaluation of different organic solvents for the extraction of these anti-microbial metabolites, elucidation of chemical structures in case of discovery of new compounds, and testing the disease-control properties of these natural compounds against other plant pathogens. Moreover, the disease-control ability of these medicinal plants could even be further enhanced. For example, more complete physical disruption of the plant tissue by producing and using very fine powders versus coarse powders; mulching of the powder on the surface of soil versus mixing in soil; addition of small amount of some chemical to the powders etc. Other possible future research could include the identification and purification of the possible SAR inducer(s) present in the dried powders of these plants.