137 1. Introduction Grapevine phylloxera (Daktulosphaira vitifoliae) is a tiny aphid-like insect that feeds on grapevine (Vitis vi- nifera L.) roots and leaves, leading to stunted growth or death. It is considered the most destructive grapevine pest (Vidart et al., 2013). In Syria, there are more than 70,000 ha of grapevine with an estimated 540,000 ton annual pro- duction (Statistics of Syrian Agriculture Ministry, 2011). However, phylloxera causes millions of dollars in losses in grapevine production annually. Grapevine phylloxera forms damaging root galls which are metabolically active organs suited to meet the nutritional requirements of phyl- loxera and support its generation with high reproductive rates, making this pest capable of destroying the root sys- tem of V. vinifera vines. Root injuries reduce the vines’ ability to absorb nutrients and water, causing a decline in vigor and productivity. As a consequence, weakened plants probably become more susceptible to secondary in- fections by fungal diseases and other insects and are also vulnerable to environmental stresses (Granett et al., 2001). The use of resistant rootstocks is considered the most common and effective means to control phylloxera in the field. The vast majority of these rootstocks have been durably resistant for a long period. In Syria, the widely used resistant rootstocks are Ru140 (V. rupestris x V. Ber- landieri), R99 (V. rupestris x V. Berlandieri), and 3309C (V. riparia Michaux x V. rupestris) and B41 (V. vinifera x V. Berlandieri) (Makee et al., 2003). It is important to note that some rootstocks are more resistant than others to grapevine phylloxera. However, for yet unknown reasons, some rootstocks may lose their resistance to phylloxera. For example, AXR#1 (Vitis vinifera X V. rupestris Scheele hybrid) has failed to resist phylloxera in several parts of the world after many years of use (Granett et al., 1983). Likewise, rootstock B41 has remained resistant in France while it is not resistant in Californian vineyards, therefore farmers have to replant their vineyards with the appropri- ate resistant rootstocks (Song and Granett, 1990; De Bene- dictis and Granett, 1993). Plants have active defense mechanisms against patho- gen attacks. A group of microorganisms referred to as plant growth-promoting rhizobacteria (PGPR) are able to reduce disease through the induction of systemic resis- tance (ISR) that renders the host plant more resistant to further pathogen ingress (Pieterse et al., 2002). This phe- nomenon can occur in many plant species and was dem- onstrated to be effective against a broad spectrum of fun- gal, bacterial and viral diseases beside its effect on insect and nematode pests (Van Loon et al., 1998; Ramamoor- thy et al., 2001; Durrant and Dong, 2004; Verhagen et al., 2010; Weller et al., 2012). In addition to eliciting ISR against pathogens, protective effects of PGPR against in- sects have been noted (Zehnder et al., 1997 a, b; Zehnder et al., 2001; Kloepper et al., 2004; Vijayasamundeeswari et al., 2009; Valenzuela-Soto et al., 2010). However, to our knowledge no studies have been carried out to as- sess in vitro the effects of PGPR on grapevine phyl- loxera. In this context, a non-pathogenic Pseudomonas putida BTP1 strain has shown enhancement of the level of resistance in cucumber, bean and tomato against the fungal pathogens Pythium aphanidermatum and Botrytis In vitro Pseudomonas putida BTP1-induced systemic resistance in grapevine rootstocks against Phylloxera (Daktulosphaira vitifoliae) A. Adam (1), I. Idris, Z. Ayyoubi Department of Biotechnology, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria. Key words: B41, grapevine phylloxera, ISR, PGPR, Pseudomonas putida BTP1, Ru140. Abstract: This study investigates the systemic resistance induced by Pseudomonas putida strain BTP1 against phylloxera using an in vitro model in Ruggeri (Ru140) and B41 roostocks. Significant differences were found with regard to matured females, fecundity and oviposition period between untreated and bacteria-treated plants in both rootstocks. Treated Ru140 rootstocks were more resistant than treated B41 ones. BTP1 impacted negatively on the ability of phylloxera to develop, indicating an increase in grapevine resistance and tolerance toward this pest in bacteria-treated plants. This is the first known study of biocontrol of phylloxera in grapevine rootstocks by non-pathogenic P. putida strain BTP1 in vitro. Adv. Hort. Sci., 2013 27(4): 137-142 (1) Corresponding author: ascientific@aec.org.sv Received for publication 16 September 2013 Accepted for publication 10 December 2013 138 cinerea, respectively (Ongena et al., 1999; Ongena et al., 2004; Adam et al., 2008). In a previous study performed on fresh roots from local grape variety Helwani (V. vinif- era), we demonstrated the influence of P. putida BTP1 on reproduction and development of grapevine phylloxera (Adam et al., 2012). Implementation of in vitro dual culture assay has been used to evaluate the phylloxera/grapevine interaction (For- neck et al., 1996; Makee et al., 2003; Vidart et al., 2013). This method has several advantages for our designed ex- periments such as providing optimal conditions for phyl- loxera infestation, conducting experiments in small space, preventing the spreading of phylloxera and rhizobacteria, as well as reliable results in a relatively short period. The present work aims to demonstrate the ISR-related protective effect triggered by P. putida BTP1 in vitro in Ru140 and B41 rootstocks against grapevine phylloxera. The percentage of mature females, fecundity and oviposi- tion period of phylloxera were determined. 2. Materials and Methods Establishment of the phylloxera colony Grapevine phylloxera was originally collected from field-infested roots of the local grapevine varieties in southern parts of Syria. The phylloxera colony was estab- lished following similar procedures to those mentioned by Makee et al. (2003). Fresh and healthy pieces of roots (4-7 mm in diameter and 5-7 cm long) of local grapevine cultivar Helwani (V. vinifera) were taken and washed with tap water. Each piece was wrapped with moist cot- ton wool around one end, and then 10 to 15 phylloxera eggs were placed on each piece. The infested root pieces were then placed on a wet filter paper disk inside a plastic Petri dish (12 cm diameter). Each dish had three to four root pieces. For ventilation purposes the Petri dish lid was modified with a 1-1.5 cm cloth-screened hole. The edges of the dishes were sealed with parafilm and they were kept in plastic boxes with tightly fitting lids and incubated at 25±1°C, 70±5% RH and 24 h darkness. The root pieces were replaced when they desiccated, rotted or the phyl- loxera became crowded. Microbial strain and inoculum preparation P. putida strain BTP1, isolated from barley roots, was originally selected for its specific features regarding py- overdine-mediated iron transport (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 Casa- mino Acids medium (CAA) for 24 h on a rotary shaker (150 r.p.m.) at 28ºC. Cells were removed by centrifugation at 16500 g for 15 min at 4ºC and washed in sterile NaCl (5 g l–1). The final pellet was resuspended in an adequate volume of sterile distilled water to obtain a bacterial sus- pension at 108 CFU ml-1. In vitro culture of grapevine plants For in vitro culture of grapevine plants, we used a proto- col described by Makee et al. (2010). Wood cuttings having four to five nodes of Ru140 and B41 rootstocks were collect- ed from the field while the buds were still dormant. All cut- tings were washed in water, and then treated with gentami- cine sulphate 160 mg l-l. Thereafter, they were incubated in 0.5 g l-1 carbamate fungicide [Methyl-1-(butylcarbamoyl)- 2-benzimidazole-Carbamate 50%] (Bell®) for 24 h, and then grown in sterilized water at 25±1°C under 16 h pho- toperiod (140-150 μmol m-2 s-1) from daylight fluorescent tubes (Philips TLD 38/54). Shoots were grown in glass jars (1000 ml) when they became about 8 cm long; buds of 4 mm length were taken from the middle of each stem. These buds were dipped in a solution of 70% ethanol for 3 min, 1.5% commercial bleach for 15 min followed by 0.7% com- mercial bleach for 5 min (Charbaji and Nabulsi, 1999). Af- ter sterilization, they were washed three times with sterile water and planted in tubes containing 20 ml DSD1 medium (Da Silva and Doazan, 1995). The DSD1 media contains 100 mg l-1 NH 4 NO 3 , 1000 mg l-1 KNO 3 , 180 mg l-1 MgSO 4 . 7H 2 O, 100 mg l-1 KH 2 PO 4 , 500 mg l-1 Ca (NH 3 )4H 2 O, 27.5 mg l-1 MnSO 4 .7H2O, 37.5 mg l-1 Na 2 EDTA, 0.025 mg l-1 CuSO 4 5H 2 O, 0.025 mg l-1 CaCl 2 .6H 2 O, 1 mg l-1 H 3 BO 3 , 1 mg l-1 ZnSO 4 7H 2 O, 27.5 mg l-1 Fe SO 4 7H2O, 10 mg l-1 Myoinositol, 1 mg l-1 Acid Nicotinic, 1 mg l-1 Thiamine, and 1 mg l-1 Pyrodoxine. The pH of the medium was adjust- ed to 6.4 before adding agar and it was then autoclaved at 116°C for 25 min. The tubes were closed using cellophane paper and the edge of the tubes was sealed with parafilm to avoid contamination. All tubes were then incubated as described above. Experimental design Six-week-old grapevine plants were used to induced resistance; plantlets with two or three roots were selected. Due to the lack of phylloxera to infest the roots in the me- dium and to avoid the interaction between phylloxera and BTP 1 , one root of each plants was pulled out of the medium but kept within the tube while the other root remained in the medium. The second root was treated with 1 ml of bacterial suspension (108 CFU ml-1) of P. putida BTP1 on the root surface and inside the medium, or by distilled water for the control plantlets. The tubes were closed again as described above and incubated at 25°C under 16 h photoperiod. Seven days later, the second root was infested with sterile eggs of phylloxera according to Makee et al. (2003). Three-day-old eggs were taken from the colony and placed into 1.5 ml Ep- pendorf tubes for sterilization of the egg cuticle. One ml of formaldehyde (2.5%) was added to the eggs, gently shaken for 10 min, and left for 20 min. The sterilizing solution was then removed with a micro-pipette and the eggs were ex- tracted and placed on sterile filter paper. The sterile eggs were gently transferred and spread on the non-inoculated roots of in vitro cultured plants by using a 10 ml sterile loop (Kendall, USA). For each rootstock, five treated and five untreated plantlets were infested with 25 surface-sterile phylloxera eggs. The tubes were resealed with parafilm to 139 prevent contamination and to avoid the escape of phylloxera crawlers, and were then incubated at 25±1°C under 16 hr photoperiod (140-150 μmol m-2 s-1) from daylight fluores- cent tubes (Philips TLD 38/54). Evaluation procedure Stereo microscope inspection was carried out daily on treated and untreated plantlets maintained in closed tubes to observe distribution of the eggs. The number of eggs hatched, feeding nymphs and adults were recorded to de- termine the mean developmental time (egg to egg) for each tested plant. Five random of root-feeding phylloxera fe- males in each tube were inspected to determine the mean of oviposition period and the mean of fecundity (total number of eggs) of phylloxera. Thus, 25 females were ex- amined on each plantlet. All eggs laid by each female were observed daily and counted till the female’s death. Egg distribution during oviposition period (number of eggs per day), fecundity (total number of eggs) and female longev- ity were determined. Statistical analysis All statistical analyses were performed using GraphPad Prism 5 program at 5% level (P= 0.05). Data were sub- jected to analysis of variance (ANOVA) for the determina- tion of differences in means between tested plants of each treatment. Differences between means were tested for sig- nificance using Tukey HSD test. 3. Results Effect of P. putida BTP1 on grapevine resistance against phylloxera Percentage of matured females The result showed significant difference in phylloxera egg numbers that were able to hatch and develop to reach adult stage (matured females) on both rootstocks Ru140 and B41 (F= 79.6; df=3, 16; P<0.001) (Fig. 1). P. putida BTP1- treated plants emerged significantly percentage decreased of matured females in both rootstocks comparing to control plants. However, there was no significant difference in the percentage of emerged matured females between treated plants of B41 rootstock and plants of rootstock Ru140 no treated (Fig. 1). The percentage of matured females of phyl- loxera on treated B41 was significantly greater (33%) than that on treated Ru140 (16%) (Fig. 1). Fecundity There was a significant difference in the mean of fe- cundity between Ru140 and B41 rootstocks (F= 140.8; df=3.96; P<0.001) (Fig. 2), with it resulting greater (19.4 eggs) in Ru140 than in B41 (13.7 eggs). When plants were treated with BTP1, the mean number of eggs laid signifi- cantly decreased in both rootstocks, pointing to a signifi- cant decrease in the mean of fecundity of phylloxera in both rootstocks. However the decrease in this parameter was greater in treated Ru140 (5.4 eggs) than treated B41 rootstocks (9.9 eggs) (Fig. 2). Oviposition period A significant difference was observed in the mean ovi- position period of phylloxera between Ru140 and Fig. 1 - Effect of P. putida BTP1 on percentage of matured females of phylloxera in vitro in B41 and Ru140 rootstocks in com- parison with control plants. Each column represents data from 25 samples. Data were subjected to ANOVA analysis and the differences between means were tested for significance using Tukey HSD test (values with different letters are significantly different at P<0.001). Fig. 2 - Effect of P. putida BTP1 on fecundity of phylloxera in vitro in B41 and Ru140 rootstocks in comparison with control plants. Each column represent data from 25 samples. Data were sub- jected to ANOVA analysis and the differences between means were tested for significance using Tukey HSD test (values with different letters are significantly different at P<0.001). 140 B41 rootstocks (F= 38; df=3.96; P<0.001) (Fig. 3). The oviposition period was 7 and 6 days in Ru140 and B41, respectively. However, when plants were treated with P. putida BTP1, the oviposition period decreased in a signifi- cant way only in Ru140. No significant differences were observed in the mean oviposition period between treated and untreated B41 plants (Fig. 3). The mean oviposition period of phylloxera on treated B41 was significant: last- ing one day longer than treated Ru140 (6 and 5 days re- spectively) (Fig. 3). 4. Discussion and Conclusions A recent study carried out on healthy pieces of roots of local grapevine cultivar Helwani showed the influ- ence of non-pathogenic P. putida BTP1 on reproduction and development of grapevine phylloxera (Adam et al., 2012). However, in the present work, the aim was to in- vestigate the ability of this bacteria to induce systemic resistance in two grapevine rootstocks against phylloxera by using in vitro cultured plants. For our module this ap- proach provided a strict separation condition between the inducer (bacteria) and the pathogen or pest (phylloxera) to induce systemic resistance (Ongena et al., 2002; Bak- ker et al., 2007). In agreement with a previous study (On- gena et al., 2002), the bacteria did not migrate through the plants, suggesting the observed decrease in the life cycle of phylloxera was due to induction of systemic re- sistance in the host plant. The present study confirmed that P. putida BTP1 had a protective effect on Ru140 and B41 rootstocks against phyl- loxera. The means of fecundity and oviposition period and emerged mature female percentage decreased significantly in both BTP1-treated rootstocks in comparison with con- trol plants. These results are consistent with similar previ- ous studies that demonstrated the ability of some strains of PGPR to induce systemic resistance in tomato against white- fly, where the percentage of matured females decreased in treated plants (Hanafi et al., 2007; Valenzuela-Soto et al., 2010). In addition, similar results were reported when cu- cumber beetles and American bollworm fed on PGPR-treat- ed cucumber plants and cotton bolls, respectively (Zehnder et al., 1997) (a, b; Vijayasamundeeswari et al., 2009). Other studies also indicated that changes in the feeding behavior of Leaffolder and decreases in the weight of larvae and pu- pae were observed in rice plants treated with rhizobacteria (Radjacommare, 2002). On the other hand, our results showed that there was a significant difference in reproduction and development of grapevine phylloxera between BTP1-treated B41 and Ru140 rootstocks. In comparison, the percentage of ma- tured females and the means of fecundity and oviposi- tion period decreased significantly by up to 50%, 45% and 12% respectively in treated Ru140 rootstock versus treated B41 rootstock (Figs. 1, 2 and 3). This is consistent with results of previous studies indicating the presence of a type of gradient from the resistant plant to sensitive plant (Granett et al., 1983; Makee et al., 2010). These results show that phylloxera laid a large number of eggs on sen- sitive varieties, more than on resistant varieties. It is be- lieved that poor nutrition or the inability to colonize good locations for feeding could directly affect the number of eggs and ultimately the ratio of hatching. Thus, the resis- tance of grapevine to phylloxera could be a reflection of the strong relationship between poor nutrition and a de- cline in the productivity of the insect (Granett et al., 1983). In addition, the mechanism of defense in these rootstocks may be due to toxic effects against phylloxera, such as the accumulation of some phenolic compounds in the cells of resistant plants leading to an increase in the death rate (Omer et al., 1999). Other workers illustrated that there is a positive relationship between resistance/susceptibil- ity characteristics against aphids and flavonoid glycoside content (Quercetin and Isorhamentin) of cowpea lines as these compounds possess a good inhibitory rate for aphid reproduction (Lattanzio et al., 2000). Therefore, the re- sistance of Ru140 and B41 rootstocks to phylloxera may be attributed to an ability to produce such toxic phenolic compounds. In conclusion, understanding the mechanisms of de- fense induced by some strains of PGPR in plants is very important to develop systemic resistance in plants. The current study provides evidence that P. putida strain BTP1 has the ability to stimulate a systemic resistance in grape- vine rootstocks against phylloxera. We suggest that P. pu- tida BTP1 treatment leads to an alteration in the plant’s metabolic pathway eliciting the induction of plant defense compounds. These substances would have a negative in- fluence on phylloxera feeding and development in treated plants. However, more research in the field must be done before implementing this technique on a large scale. Fig. 3 - Effect of P. putida BTP1 on oviposition period of phylloxera in vitro in B41 and Ru140 rootstocks in comparison with con- trol plants. Each column represent data from 25 samples. Data were subjected to ANOVA analysis and the differences between means were tested for significance using Tukey HSD test (val- ues with different letters are significantly different at P<0.001). 141 To our knowledge, this work is the first study interested in biocontrol of phylloxera in grapevine by PGPR strains in vitro. Furthermore, this investigation supplies important information about the possibility of implementing this strain to stimulate systemic resistance against plant pests. Moreover, this study illustrates the effectiveness of using in vitro dual culture in evaluating the phylloxera/grapevine and grapevine/rhizobacteria interactions. 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