Research Article

Prevalence of (Clavibacter michiganensis subsp. Michiganensis) Causal Organism of Bacterial Canker in Weed Species in Tomato Fields of North West Pakistan

Ayesha Bibi*, Musharaf Ahmad and Shaukat Hussain

Department of Plant Pathology, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan.

Received | December 13, 2016; Accepted | January 15, 2018; Published | February 13, 2018

*Correspondence | Ayesha Bibi, Department of Plant Pathology, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan; Email: [email protected]

Citation | Bibi, A., M. Ahmad and S. Hussain. 2018. Prevalence of (Clavibacter michiganensis subsp. Michiganensis) causal organism of bacterial canker in weed species in tomato fields of North West Pakistan. Sarhad Journal of Agriculture, 34(1): 123-129.

DOI | http://dx.doi.org/10.17582/journal.sja/2018/34.1.123.129

Keywords | Tomato, Weed, Inoculum sources, Bacterial canker, Cmm

Introduction

Tomato (Solanum lycopersicum), is one of the major cash crops for small-scale farmers in Northwest Pakistan. In spite of being cultivated on a large area, the average per hectare yield of tomato in Pakistan is approximately 10 ton/ha (FAOSTAT, 2015) which ranks the country far below the world’s average (35 tons/ha).

Several factors contributes to this low yield and quality including abiotic and biotic stresses. Among biotic stresses, Bacterial canker of tomato (BCT) has become a major production constraint in the past couple of decadses in Pakistan. Bacterial canker of tomato, caused by bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) has a wide host range, from Lycopersicon species to some wild plants like Solanum douglasii, S. nigrum and S. triflorum. Furthermore, a number of solanaceous plants (Thyr et al., 1975); wheat, barley, rye, oats, sunflowers, watermelons and cucumbers (Stamova and Sotirova, 1987) are susceptible to the bacterium through artificial inoculation. The disease not only affect tomato in tunnels but also in open fields, either by killing the young plants or disfiguring the fruits. Experiments carried out in France have shown a yield loss of 20-30% (Rat et al., 1991). Vasinauskiene (2002) reported that disease incidence reached from 10% to even 80% of tomato plants grown in some greenhouses.

In Pakistan, the etiology of the disease was not investigated. We infer, from surveys conducted among tomato growers in Northwest Pakistan, that the diseases is prevalent in the major tomato growing areas for the last 15 years. Lack of proper quarantine measures and free movement of infected seeds might be responsible for the introduction of the outbreak of the disease causing significant yield losses.

Although, healthy seed plays a vital role in the disease management (Thyr et al., 1973). The broad host range of Cmm highlights the importance of the weeds commonly grown in tomato fields. These weeds acts as an alternate host and become a source of primary inoculums for the next crop, causing major losses. The bacterium can survive inside the alternative host species which can serve as sources of infection (Strider, 1969). Leaf-surface populations on alternative hosts and on non-host plants can also play a vital role (Chang et al., 1992). In the field, the bacterium can spread through insect vector, rain splashes or mechanical contact etc. (Ricker and Riedel, 1993). Once the bacteria reach the target plant, pathogen use chemical or mechanical injuries, stomata and trichomes or hydathodes to invade the plants (Strider, 1969; Gleason et al., 1993; Carlton et al., 1998) and approach towards the xylem vessels (Leyns and De Cleene, 1983) where it can cause lysigenous cavities. Within 3-5 days localized leaf symptoms initiates (Basu, 1966; Layne, 1967) which turns to blister-like spots and tan-coloured lesions with white halos (i.e. bird’s-eye spots) later on (Gleason et al., 1993; Carlton et al., 1998). Young plants are always more susceptible to the disease (Van Vaerenbergh and Chauveau, 1985).

Since, no research has been carried out to understand the epidemiology of the disease, we investigated the role of weeds as inoculum source for devising a better management strategy for bacterial canker disease in tomato. The research finding presented in this paper reports a comprehensive survey of weeds, for the presence of the bacterial canker pathogen, commonly found in tomato fields in Pakistan. We consistently identified a total of nine weeds as possible sources of primary inoculum of Cmm in tomato fields of Northwest of Pakistan.

Materials and Methods

Survey and sampling

In order to collect weeds plant samples from tomato field, a comprehensive survey was conducted in tomato-growing areas of Khyber Pakhtunkhwa (KP) and Federally Administrated Tribal Areas (FATA, agencies) from April to August, 2012. Khyber Pakhtunkhwa was divided into five Agro-ecological zones based upon weather, temperature, precipitation and geographic location etc. (Table 1) i.e. Northern KP; consisting of Malakand, Buneir, Mingora, Swat, Shangla, Dir upper, Dir lower, Chitral and Kalam, Southern KP consisting of D. I. Khan, Bannu, Hangu, Kohat, Karak, Lakki Marwat and Tank, Eastern KP consisting of Abbottabad, Naran, Kaghan, Mansehra, Haripur and Battagram and Western KP consisting of Khyber agency, Orakzai agency, Mohamand agency and North & South Waziristan and the most important Central KP consisting of Mardan, Nowshera, Peshawar, Swabi and Charsadda.

Table 1: Agro-ecological zones of Khyber Pakhtunkhwa surveyed for weeds sampling during April to August, 2012.

Plants were excised into small pieces, surface sterilized by dipping first in 20% Clorox solution for one minute and then rinsed twice with sterilized distilled water (SDW). These pieces were then homogenized in an equal amount of 0.85% TBE buffer. Homogenate was filtered through a muslin clothe and 100 µl of filtrate was platted directly on NA (Nutrient Agar) medium and the growing bacterial colonies were purified on YDC (Yeast extract-Dextrose-CaCO3) medium. Inoculated petri plates were incubated at 29oC for 72 hours for bacterial growth (Wilson et al., 1967).

PCR confirmation of Cmm in weeds samples

Polymerase chain reaction (PCR) was used to confirm the identity of isolates. Cmm-specific primers CMM-5; 5’GCGAATAAGCCCATATCAA3’ and CMM-6; 5’CGTCAGGAGGTCGCTAATA3’ from plasmid-bornepat-1 gene producing a 614bp amplification product, were used for this purpose (Ozdemir, 2005).

DNA extraction

For extracting DNA from Cmm isolates, the modified procedure of Li and De Boer (1995) was used. The whole bacterial DNA (to be used as template) was extracted with this procedure. Bacteria were grown overnight at 27oC in 5ml LB broth in a shaking incubator. A small amount (1.5ml) of liquid culture was transferred to Eppendorf tube, and pelleted by centrifugation at 7000 rpm for 10 minutes at 4oC. Supernatants were discarded and the pellets were frozen at -20oC for one hour and thawed at room temperature. Next, the pellets were treated with 100µl of cold acetone (-20oC) for 10 min, re-suspended in 0.5 ml of TE (10mM Tris hydrochloride, 1mM EDTA, pH 8.0) buffer, followed by addition of 50 µl sodium dodecyl sulfate (14%) and 10µl of 0.1% protinase-K (sigma). The mixture was then incubated for 1 hour at 55oC. An equal volume of 7.5M ammonium acetate was added to precipitate cell debris which were removed by centrifugation at 5000 rpm for 10 min. DNA in the supernatant was precipitated with cool isopropanol (-20oC) for 30 min. Then DNA (40-60ng) was pelleted by centrifugation at 13,000 rpm for 5 min and isopropanol was discarded. Pelleted DNA was washed (with 70% ethanol), vacuum dried and dissolved immediately in 100µl sterile distilled water.

PCR Amplification with species-specific primers

PCR for all 34 candidate isolates was performed in MJ mini the rmocycler (Bio-rad, USA). The 25 µl PCR reaction mixture contained 2.25 µl of 1x reaction buffer (20mM Tris-HCl, pH 8.4, 50mM KCl), 2 µl of 1.0 mM MgCl2, 2.5 µl of 100 µM of each dNTP, 1 µl of 0.2 µM of each primer (forward and reverse), 1 µl of Taq DNA polymerase (Fermentas) and 3µl of (approx; 1ng) of template DNA. The PCR conditions used were: 94oC for 3 min followed by 30 reaction cycles of 94oC for 30s (denaturation), 55oC for 20s (primer annealing) and 72oC for 45s (primer extension). After the final reaction cycle the mixture was held at 72oC for 5 min and stored at 4oC. A negative control without template DNA was included in PCR amplification.

Gel electrophoresis

PCR product (25 µl aliquot per isolate per well) was electrophoresed through 2% agarose (0.667 gm agarose dissolved in 30 ml TBE buffer) gel. Agarose was dissolved in TBE buffer by heating in an oven for 2 minutes and after cooling down to 45-50oC, it was poured into gel electrophoresis apparatus tray and allowed to solidify at room temperature.

Table 2: Isolation of Cmm (on YDC medium) from weeds commonly growing in commercial tomato fields of KP, Pakistan.

After solidification, enough TBE buffer was added to cover the top of the gel. The PCR product (25 µl; after mixing with the blue tracking dye) of each isolate was loaded into the wells of the gel. 1Kb DNA ladder was used for size comparison. Electrophoresis was performed according to standard procedures (200 volts, 30 mints). The gel was stained in ethidium bromide (0.5µg/ml) solution for 15 minutes to visualize the amplified DNA and washed with sterile distilled water for 15 min. The ethidium bromide-stained bands were observed under UV light in UV tech machine (ESSENTIAL, D-55-20-M-Auto., UK) and the images were saved (Figure 1).

Results and Discussion

Twenty tomato weeds, occurring commonly in tomato fields from all the five agro-ecological zones were tested for isolation of the pathogen. Out of total 100 samples 52 produced bacterial colonies on NA medium. These 52 different colonies were then purified by sub-culturingon YDC medium. Gram staining and 3% KOH test, when performed for these isolates, 34 out of 52 proved to be Gram positive. The remaining 18 (Gram negative) were discarded, while the others were subjected to PCR for their genetic confirmation with Cmm-specific primers. 27 out of 34 isolates were confirmed to be Cmm as they successfully amplified 614bp band from plasmid-born pat-1 gene (Figure 1).

Data (Table 2) showed that Solanum nigrum L (Black nightshade) was the only weed from which pathogen was consistently isolated (regardless of the collection zone). In case of Amaranthus blitoides, pathogen was recovered from all such weeds except those collected from Southern KP. Same way, Lactuca serriola weeds harbored the pathogen except those collected from central KP. However, pathogen was not recovered from Sonchus oleraceus, Tribulus terrestris, Sorghum halepense, Chenopodium album, Chenopodium murale, Portulaca oleracea, Physalis acutifolia, Convolvulus arvensis, Digitaria sanguinalis, Poa annua and Cyperus esculentus.

Some of the weeds having biology like tomato and producing compounds which provide a good microenvironment for the existence of the bacterium, can serve as alternate host for Cmm. Solanaceous weeds serve as asymptomatic hosts on which the pathogen can multiply during the course of a growing season (Holmstrom, 2015). In general the main host of Cmm which is of economic importance is tomato, but the pathogen has also been reported on other Lycopersicon spp. and on the wild plants like Solanum douglasii, S. nigrum and S. triflorum. Natural infections have also been found on Capsicum annuum and C. frutescens (Strider, 1969; Lai, 1976; Moffett and Wood, 1984; Latin et al., 1995; Lewis-Ivey and Miller, 2000; Yim et al., 2012), and several solanaceous weeds (e.g. Solanum nigrum, S. douglasii and S. triflorum) (Bradbury, 1986). Among other solanaceous plants, aubergine (S. melongena) is susceptible upon artificial inoculation (Thyr et al., 1975). In this study nine weeds in tomato fields including; Solanum nigrum, Solanum americanum, Solanum Sarrachoides, Amaranthus blitoides, Amaranthus albus, Lactuca serriola, Amaranthus retroflexus, Malva parviflora and Sisymbrium irio were found to serve as an alternate host for the pathogen however, the presence of symptomless pathogen supported the findings of Bradbury (1986), Holmstrom (2015) other scientists.

In the present study in some of the weeds, the pathogen was not detected in some particular area but in other are they do carry the bacteria. This phenomenon might be attributed to genetic makeup of the pathogen as well as the plant which have slight variations due to their geographical distribution which include all other factors e.g. weather, temperature, precipitation and topography etc. Andreote et al. (2010) also believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Plant growth stage was the most important factor influencing the composition of the bacterial communities. Bacterial inoculation of plants may lead to shifts in the composition of plant-associated bacterial communities (Andreote et al., 2006; Viebahn et al., 2005) and this on its turn may also lead to differences in plant metabolism. Cultivar effects (which is generally not studied in weeds) might having effect on bacterial survival. With the above justification our result are in line with the findings of Andreote et al. (2010). A particular weed cannot be attributed as host for the pathogen due the presence of the pathogen. It might be quite possible that the pathogen is instantly transferred during sampling or insect vector activity. The significance of these epiphytic populations is not fully understood, although they appear to contribute to infections through pruning wounds (Carlton et al., 1994).

Conclusion and Recommendation

From the present research it can be concluded that the naturally grown local weeds in tomato fields can serve as an alternate host for the pathogen and play an important role of primary inoculum in disease development. To reduce the amount of primary inoculum, weeds inside and around tomato fields should be controlled thoroughly.

Acknowledgments

The authors extends their thanks to Higher education commission of Pakistan (HEC) for funding the current research through indigenous PhD fellowship program.

Author’s Contribution

Bibi, A. performed the experiment; Bibi, A. and Ahmad, M. wrote the first draft of the manuscripts. Hussain, S. designed the experimentation and Bibi, A. analysed the data. All authors read and approved the final manuscript.

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