Impaginato 47 1. Introduction The potato (Solanum tuberosum L.) has been con- sidered one of the most important food crops, along with rice, wheat, and maize (Ross, 1986; Douches et al., 2004). Potatoes grow in a variety of geo-environ- mental conditions. Developing countries cultivate potato to add nutritional balance to their food basket (Douches et al., 2004; Navarre et al., 2009). In Syria, more than 29,000 ha were planted with potato, pro- d u c i n g a b o u t 6 0 9 , 0 0 0 t o f t u b e r s i n 2 0 0 5 (Alammouri, 2008). However, severe damage may occur to potato crops at storage periods particularly in developing countries. Within the Lepidoptera order, potato tuber moth Phthorimaea operculella (Zeller) belongs to the Gelechiidae family and it has been reported in more than 90 countries, making it a cosmopolitan pest (Visser, 2005; Golizadeh and Esmaeili, 2012). It damages potato throughout the growing season by mining stems, petioles, leaves and tubers by larvae, with the latter considered the typi- cal damage. The procedure of potato damage begins when larvae penetrate the foliage, including leaves and stems. This insect can infest potato tubers stored and in field or it may develop on plants remaining in the field including tomatoes, aubergine or other solanaceous plants (Gilboa and Podoler, 1995; Coll et al., 2000; Alvarez et al., 2005). Farmers depend broadly on the use of insecticides and other varieties of farming practices (Clough et al., 2008); insecticides are widely used to control this pest. However, insecticides are costly, nonselective, unfriendly to the environment, and affective for only a short period of time (Simmons et al., 2006). Additionally, the phenomenon of resistance to insec- ticides in Lepidoptera has increased significantly (Gonzalez and Trevathan, 2001). Plant resistance, together with appropriate biological and farming practices in combination with insecticides may pro- vide the best management options (Rondon, 2010). Plants defeat pathogens through their active defense mechanisms that can be stimulated in some cases by plant growth-promoting rhizobacteria (PGPR) which ultimately reduce disease and render the host plant more resistant to any foreseeable pathogen attacks (Pieterse et al., 2002). Induction of such enhanced defensive capacity is systemic as root Adv. Hort. Sci., 2016 30(1): 47-52 DOI: 10.13128/ahs-18701 Induced resistance in potato plants by a non-pathogenic Pseudomonas putida BTP1 against potato tuber moth (Phthorimaea operculella Zeller) A. Adam (*), I. Idris, N. Khalil, K. Houssian Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria. Key words: biocontrol, plant resistance, potato tuber moth, P. putida BTP1, rhizobacteria. Abstract: Pseudomonas putida strain BTP1 is able to promote induced systemic resistance (ISR) in a wide spectrum of pathosystems. In this study, we investigated induced resistance in potato plants against potato tuber moth (Phthorimaea operculella Zeller) by non-pathogenic P. putida BTP1. Several physiological indicators in the life cycle of the potato tuber moth, such as survival rate, mean weight of pupae, and sex ratio were studied to assess the protective effect of P. putida BTP1. Our results showed that treatment of potato tubers by bacterial suspension of P. putida BTP1 caused evident disturbance to the development of P. operculella in potato plants. Survival rate of larvae feeding on treated plant leaves and mean weight of pupae decreased significantly. In addition, a clear deviation in the sex ratio in moths, in favor of males, resulted from larvae fed on bacteria-treated plants. This study preliminarily reports the ability of BTP1 to induce resistance in potato plants against potato tuber moth. Consequently, P. putida strain BTP1 could be a promising approach for potato tuber moth biocontrol. (*) Corresponding author: ascientific@aec.org.sy Received for publication 9 August 2015 Accepted for publication 8 February 2016 Copyright: © 2016 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Adv. Hort. Sci., 2016 30(1): 47-52 48 treatment with a PGPR was shown to trigger protec- tive effects on above-ground plant parts. These reac- tions are thought to typically result from the activa- tion of latent defense mechanisms that are over- expressed upon subsequent pathogen challenge (Van Loon et al., 1998; Ongena et al., 2002; Bakker et al., 2007). This induced systemic resistance (ISR) can be the basis for integrated plant disease management strategies (Ramamoorthy et al., 2001; Zehnder et al., 2001; Saravanakumar et al., 2007). Induced resis- tance in plants by non-pathogenic rhizobacteria against pests is a very important additional factor for the protection of agricultural crops. Several studies have indicated the ability of many strains of rhizobac- teria (PGPR) to induce systemic resistance against a large number of insect pests (Racke and Sikora, 1992; Zehnder et al., 1997). For example, in cucumber against Striped Cucumber beetle Acalymm avittatum (Zehnder et al., 1997, 2001), in cotton against American boll worm H e l i c o v e r p a a r m i g e r a (Vijayasamundeeswari et al., 2009), in tomato against whitefly Bemisia tabaci (Valenzuela-Soto et al., 2010) and also in cucumber against spider mites Tetranychus urticae (Tomczyk, 2006). A non-patho- genic BTP1 showed enhancement of resistance level in many plants including bean, cucumber, and toma- to against fungal pathogens (Ongena et al., 2002, 2004; Adam et al., 2008). In a previous study per- formed in vitro on grapevine rootstocks, we demon- strated the influence of P. putida BTP1 on reproduc- tion and development of grapevine phylloxera (Adam et al., 2013). However, to our knowledge no studies have been performed yet to assess the effects of PGPR on P. operculella in potato plants. Therefore, the present work aims to demonstrate the protective effect trig- gered by P. putida strain BTP1 against P. operculella in potato plants. The larvae survival rate, the mean weight of pupae and the sex ratio were studied as a biometers to detect the induced resistance. 2. Materials and Methods Establishment of the potato tuber moth colony Insects used in the experiments were reared on waxed potato slices as described by Rahalakar et al. (1985). The experiments were conducted at a con- stant temperature of 25±1˚C with 70±5% RH, and a photoperiod of 12:12 (L:D) h. Microbial strain and inoculum preparation Psudomonas putida strain BTP1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron trans- port (Jacques et al., 1995; Ongena et al., 2002). It was maintained and prepared for use in the ISR assays as previously described by Ongena et al. (2002). For the bioassays, BTP1 strain was grown in Erlenmeyer flasks (250 ml) containing 100 ml of Casamino Acids medium (CAA) for 24 h on a rotary shaker (150 rpm) at 28 oC. Cells were removed by centrifugation at 16500 g for 15 min at 4°C and washed in sterile NaCl (5g l–1). The final pellet was resuspended in an adequate volume of sterile dis- tilled water to obtain a bacterial suspension at 108 CFU ml-1. Assays for induced resistance “Draja” potato tubers were washed in sterile water, dipped separately in a suspension of P. putida strain BTP1 for 30 min, and air-dried, while control tubers were treated with sterile water. The tubers were then planted in 10 L plastic pots containing autoclaved, moistened soil (three tubers/pot) to exclude any microorganism could affect BTP1. The pots were placed in a greenhouse at 25±1°C (day) and 23±1°C (night) with daylight of 16 h and relative humidity of 85-95%. Both control and treated plants were under the same watering and fertilizing condi- tions during the planting period. Fresh leaves excised from potato plants (six to seven weeks old) were used for feeding the newly hatched larvae (24 h). For each treatment, 120 larvae in 10 (18 x 12 x 8 cm) plastic boxes (12 larvae/box) were fed on leaves until they reached the pupal stage. The boxes were resealed with parafilm to keep the larvae from escaping, and were then incubated at 25±1°C with daylight of 12 h and relative humidity of 70%. Each four-day-old pupae was weighed and placed separately within a small plastic tube. The pupae were classified into three groups according to their weights: small pupae (<5 mg), medium pupae (6-7 mg) and large pupae (>8 mg) to determine the larger sex. The number of pupae and the number of emerging moths (males or females) were recorded in order to calculate the survival rate of larvae and the sex ratio (the number of male/the number of female). The experiment was repeated three times. Isolation of bacteria from potato plant leaves Small leaf samples were taken from different parts of the potato plants treated with P. putida Adam et al. - Potato plant resistant to potato tuber moth by a non-pathogenic Pseudomonas putida 49 BTP1. The samples were sterilized with sodium hypochlorite solution (5%) for 3 min and washed three times for 3 min. Samples were left to dry on sterile paper. They were then grown on Petri dishes containing the Casamino acid (CAA) medium. The dishes were incubated at 30±1°C for 72 h. Statistical analysis Statistical analyses were performed using STATIS- TIC program version 6 (Statsoft, Inc. 2003) at 5% level (P = 0.05). Data were subjected to analysis of vari- ance (ANOVA) for the determination of differences between means. Differences between means of pupal weight were tested for significance using Tukey HSD test. Ratio Analysis test (Z-test) was used to compare the percentages of larval survival rate. 3. Results Isolation of bacteria from potato plant leaves The isolation of bacteria test on treated-potato plant leaves showed no bacterial colonies grew in the Petri dishes, indicating that P. putida BTP1 did not migrate through the plant (from the tubers to the leaves). There was no direct contact between bacte- ria and larvae. Effect P. putida BTP1 on potato plants against potato tuber moth Larvae survival rate. Induced resistance experi- ments showed the death of large numbers of larvae of P. operculella in different ages of development, particularly in P. putida BTP1-treated potato plants (Fig. 1.1). The larval survival rate decreased signifi- cantly (35%) when the larvae were fed on the excised leaves from P. putida BTP1-treated plants compared with the control plants (Fig. 1.2). Significant differ- ences between BTP1 and control were observed in all experiments. Effect on pupal weight. The results of three inde- pendent experiments showed that there was a nega- tive impact on mean pupal weight in P. putida BTP1- treated potato plants. Where mean of pupal weight was 7.77±0.12 mg in the control potato plants, it decreased significantly in P. putida BTP1-treated plants to 6.24±0.15 mg (Fig. 2). This implies an approximately 20% weight reduction of pupae in potato plants pre-inoculated with P. putida BTP1 as compared with the control. Significant differences between BTP1 and control were observed in all experiments. With regard to pupae weight, we observed that the pupae, which were classified into three groups according to their weight (small, <5 mg; medium, pupae 6-7 mg; large >8 mg), in control plants were mostly large pupae (approximately 61%) while the Fig. 1 - Example of potato leaves infested by potato tuber moth showing larvae feeding on P. putida BTP1-treated plant leaves (1A), and control plant leaves (1B). (1C): profile of the dead larvae in different stages because of malnutri- tion. (2): Influence of potato tuber treatment by the bacterial suspension of P. putida BTP1 on the survival rate of larvae of the potato tuber moth. Three separate experiments were carried out (120 larvae per treatment and per experiment were used). Data were subjected to ANOVA and the differences between means were tested for significance using Tukey HSD test (values with differ- ent letters are significantly different at P<0.001). Adv. Hort. Sci., 2016 30(1): 47-52 50 small pupae was almost absent (3%) (Fig. 3). In con- trast, in BTP1-treated plants, the percentage of the large pupae decreased significantly to reach (36%), while the percentage of the small pupae increased significantly to reach (27%) (Fig. 3). Sex ratio of P. operculella The sex ratio of moths (number of males/number of females) emerged from control (180 moths) and P. putida BTP1-treated potato plants (91 moths) was calculated. A clear deviation was observed in the sex ratio in favor of males from feeding on BTP1-treated plants: 1.84:1, compared to 1:1 in control plants. 4. Discussion and Conclusions Our study has shown that the larval survival rate and the mean of pupal weight were significantly decreased in BTP1-treated potato plants compared to control. These results are consistent with previous studies conducted on whitefly, which showed a sig- nificant decrease in survival rate (number of nymphs which are able to develop and reach the adult stage) in tomato plants treated by rhizobacteria (Valenzuela-Soto et al., 2010). In addition, similar results were found in PGPR-treated cotton bolls with mortality of larval, malformation of pupal and adult with decreased adult emergence of American boll- worm H. armigera (Vijayasamundeeswari et al., 2009). Moreover, changes in dietary behavior of the rice leaf roller (Cnaphalocrocis medinalis) was observed, and there was a decrease in the larval and pupal weight in treated rice leaves by rhizobacteria (Radjacommare, 2002). The reduction of larval sur- vival rate and pupal weight in BTP1-treated potato could be attributed to the inability of larvae to feed on treated plant leaves. It is well known that the growth of phytophagous larvae is affected indirectly by chemical or physical conditions or even both which characterize their host plants. For instance, PGPR-treated plants may have a decrease in essential nutrients or have compounds that inhibit growth, or both (Reese and Field, 1986; Bong and Sikorowski, 1991; Yaman et al., 1999). On the other hand, treatment of potato tubers with P. putida BTP1 also caused a clear deviation in sex ratio in favor of males. Quezada-Garcia et al. (2014) proved that nutritional variation causes differ- ential mortality to the larger sex and the most sensi- tive to nutritional stress (female) in spruce budworm (Choristo neural fumiferana (Clemens); Lepidoptera). In contrast, House et al. (2011) demonstrated that offspring mortality in the dung beetle (Onthopha g u s t a u r u s; Coleoptera) vitally depends on the amount of resources that females have provisionally. In addition, they also showed that males have greater nutritional demands than females during development, which ultimately leads to higher mor- tality in the male population (the larger sex and the Fig. 2 - Influence of potato tuber treatment with bacterial suspension of P. putida BTP1 on mean of pupal weight of the potato tuber moth. Three separate experiments were carried out. Each column represents the weight mean of 25 pupae. Data were subjected to ANOVA and the differences between means were tested for signifi- cance using Tukey HSD test (values with different letters are significantly different at P<0.001). Fig. 3 - Influence of potato tuber treatment with bacterial suspension of P. putida BTP1 on percentages of pupal weight of the potato tuber moth (75 pupae/treatment). Data were subjected to test ratio analysis (Z-test) to determine the significant differences at P<0.05 (values with different letters are significantly different). Adam et al. - Potato plant resistant to potato tuber moth by a non-pathogenic Pseudomonas putida 51 most sensitive to nutritional stress) (House et al., 2011; Quezada-Garcia et al., 2014). These findings are consistent with our results which showed that females were larger (16.74%) than males. In conclusion, understanding the mechanisms of induced defense by P. putida BTP1 is very important to enhance the resistance in potato plants. The cur- rent study provides evidence that P. putida strain BTP1 has a protective effect in potato plants against potato tuber moth. Based on similar studies that illustrated that the accumulation of some toxic phe- nolic compounds in the cells of resistant plants led to an increase in the death rate in insects, we believe that the treatment of potato tubers with P. Putida BTP1 leads to secondary metabolic changes in treat- ed plant cells which elicit the production of defense compounds (Lattanzio et al., 2000; Zehnder et al., 2001; Arimura e t a l . , 2005; Melvin and Muthukumaran, 2008). This study preliminarily reports the ability of P. putida strain BTP1 to induce resistance in potato plants against potato tuber moth. This bacterial strain could be a promising agent for potato tuber moth biocontrol. 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