75 1. Introduction Grape phylloxera (Daktulosphaira vitifoliae), aphid- like gall-forming parasite, is an economically important homopteran pest of grape vines Vitis vinifera L. world wide. In Syria, there are more than 70,000 ha planted with grapevine and the annual production is about 540,000 t. Grape phylloxera annually causes millions of dollars in losses in grape production. Grape phylloxera causes direct damage to grapevine by forming damaging root galls. The fleshy galls formed on immature roots, by swelling of the root cortex, are called “nodosities”, while on mature roots they are called “tuber- osities”; these latter are considered more damaging to the vine. These galls are metabolically active organs suited to match the nutritional requirements of phylloxera and can support populations with high reproductive rates, making this pest capable of destroying the root system of V. vinif- era vines (Granett et al., 2001). In most cases the swelling stops rootlet growth, and the affected portion dies. Root injuries reduce the vines’ ability to absorb nutrients and water, causing decline in vigor and productivity. Weak- ened plants probably become more susceptible to second- ary infections from fungal diseases and other insects, and to environmental stresses. Since there is not an effective control method for grape phylloxera, sanitation and quarantine can be considered required procedures to prevent the spread of this insect pest. Insecticides and hot water dips are used as quaran- tine treatments (Granett et al., 2001). Makee et al. (2010) proposed that gamma irradiation could be economically very useful in quarantine treatments against phylloxera. However, once phylloxera is in a vineyard, the use of re- sistant rootstocks is the most common and effective means of managing phylloxera. It should be mentioned that some rootstocks were more resistant than others to grape phyl- loxera (Makee et al., 2003). Moreover, for unknown rea- sons, the resistance of some rootstocks may break down and farmers must replant vineyards (Granett et al., 1983; Song and Granett, 1990; De Benedictis and Granett, 1993). Because replanting is costly in money, time, and labor, ad- ditional ways to control this pest should be considered. All plants have active defense mechanisms against patho- gen attacks. Some plant growth-promoting rhizobacteria (PGPR) are able to reduce disease through the stimulation of inducible plant defense mechanisms that render the host plant more resistant to further pathogen ingress. This in- duced systemic resistance (ISR) (Pieterse et al., 2002) can be the basis of integrated plant disease management strat- egies (Ramamoorthy et al., 2001; Zehnder et al., 2001; Saravanakumar et al., 2007). Many studies in plants on PGPR against pathogens have been performed. However, only a few of them de- termined the protective effect of PGPR against insects (Zehnder et al., 1997 a, b; Zehnder et al., 2001; Kloepper et al., 2004; Vijayasamundeeswari et al., 2009; Valenzu- ela-Soto et al., 2010). A non-pathogenic Pseudomonas putida strain (BTP1) was shown to enhance the level The influence of a non-pathogenic Pseudomonas putida strain BTP1 on reproduction and development of grape phylloxera A. Adam(1), H. Makee, I. Idris Department of Biotechnology, Atomic Energy Commission of Syria. P.O. Box 6091, Damascus, Syria. Key words: developmental time, fecundity, grape phylloxera, oviposition period, PGPR, Pseudomonas putida BTP1. Abstract: Some non-pathogenic rhizobacteria called Plant Growth Promoting Rhizobacteria (PGPR) possess the capac- ity to induce defense mechanisms effective in plant against pathogens. The effect of Pseudomonas putida BTP1 on repro- duction and development of phylloxera, which infested the roots of our local grape variety “Balady”, was evaluated. Our results showed that the life table of grape phylloxera was different between treated and control plants. The percentage of matured females, developmental time, fecundity and oviposition period were reduced when plants were treated with bacteria. The results showed that the phylloxera resistance was influenced by root soaking duration in P. putida BTP1 suspension. The present study provides good information on the possibility of using Pseudomonas putida BTP1 to increase the resistance of grape to phylloxera. Adv. Hort. Sci., 2012 26(2): 75-80 (1) Corresponding author: ascientific@aec.org.sy Received for publication 1 march 2012. Accepted for publication 7 June 2012. 76 of resistance in cucumber, bean and tomato against the fungal pathogens Pythium aphanidermatum and Botrytis cinerea, respectively (Ongena et al., 1999; Adam et al., 2008). These studies revealed that the disease-protective effect was associated with stimulation of defense mecha- nisms in host plant (Ongena et al., 2000, 2004; Adam et al., 2008). The main objective of the present study was to evalu- ate the ability of strain P. putida BTP1 to protect grape roots against grape phylloxera. Thus, the effect of concen- trations and root soaking duration in P. putida BTP1 sus- pension on percentage of matured females, developmental time, fecundity and oviposition period of local phylloxera strain were determined. 2. Materials and Methods Establishment of the phylloxera colony Grape phylloxera was originally collected from field- infested roots of the local grape varieties in southern parts of Syria. The phylloxera colony was established follow- ing 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 grape cultivar Helwani (V. vinifera) were taken and washed with tap water. Each piece was wrapped with moist cotton 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 hr darkness. The root pieces were re- placed when they desiccated, rotted or the phylloxera be- came crowded. Microbial strains 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 bioassays, two different concentrations (108 and 2x108 CFU/ml) of bacterial suspension were prepared. Effect of bacteria-treated roots on phylloxera Fresh root pieces were soaked for 3 hr in solutions with various P. putida concentrations: 0 (roots were soaked in distilled water as a control), 108, and 2x108 CFU/ml. All root pieces were then left to air-dry. For each concentra- tion five root pieces were taken. Each root piece was in- fested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°C, 70±5% RH and 24 hr darkness. Evaluation procedure A daily microscope inspection of all phylloxera stages on all root pieces was carried out. The number of eggs hatched, feeding nymphs and adults were detected to cal- culate the percentage of emerged mature females on each root piece at each concentration. Also, the mean develop- mental time (egg to egg) was determined. Fecundity (total number of eggs) of phylloxera was evaluated by randomly choosing five individuals of root-feeding phylloxera fe- males on each root piece at each concentration. Thus, at each tested concentration 20 females were examined. All eggs laid by each female were observed and counted till the female’s death. Additionally, the oviposition period (the time from the first laid egg to the natural death of in- dividual ovipositing females) was recorded for the females chosen for the fecundity measurement. Effect of root soaking duration in bacterial suspension on phylloxera Fresh root pieces were soaked in solutions with various P. putida concentrations: 0, 108, and 2x108 CFU/ml. At each tested concentration the root pieces were soaked for various periods: 0, 3, 5 and 15 hr. All root pieces were then left to air-dry. At each concentration and soaking duration, five root pieces were taken. Each root piece, at each concentra- tion and soaking duration, was infested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°C, 70±5% RH and 24 hr darkness. The same evaluation procedure as described above was followed to determine the percentage of emerged mature females, mean developmental time, fecundity and the ovi- position period of phylloxera. Statistical analysis All 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 variance (ANOVA) for the determination of differences in means between tested plants at each dose. Differences between means were tested for significance using Tukey HSD test. 3. Results Effect of bacterial treated roots on phylloxera Table 1 shows that when root pieces were treated with P. putida, the percentage of emerged matured females was significantly affected compared to the control (F=84.69; df=15, 64; P<0.05). However, the percentage emerged of matured females was not significantly increased by in- creasing P. putida concentrations. The result illustrates that the developmental time of phylloxera was significantly decreased by the application of P. putida (F= 15; df = 2, 42; P < 0.05). There was a sig- nificant reduction in developmental time as the concentra- tion of P. putida was increased (Table 1). There were significant differences in the mean number of laid eggs between all tested root pieces, regardless of concen- 77 tration (F = 19.9; df = 2, 42; P< 0.05). On untreated root pieces, the mean number of eggs was significantly higher than that on treated ones (Table 1). When phylloxera females were reared on P. Putida-treated root pieces, the mean number of eggs was markedly reduced by increasing P. putida concentration. Table 1 shows that the oviposition period of phyllox- era on untreated root pieces was significantly longer than that on treated ones, irrespective of the concentration of P. putida (F = 25.5; df = 2, 42; P < 0.05). There was a significant difference in the oviposition period between P. Putida-treated root pieces. The oviposition period on 108 CFU/ml-treated root pieces was significantly longer than that on 2x108 CFU/ml-treated ones. Effect of root soaking duration in bacterial suspension on phylloxera There was a significant effect of the soaking duration in P. putida suspension on the percentage emerged of ma- tured females, regardless of the concentration (F= 620.87; df= 11, 24; P<0.05) (Fig. 1). At each concentration, the percentage of emerged matured females on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. When the root pieces were soaked, the percentage of emerged matured females with 3 hr soaking duration significantly differed from that on 5 and 15 hr, whatever the P. putida concentration. At each P. putida concentration, there was no significant difference in the percentage of emerged matured females between 5 and 15 hr soaking duration (Fig. 1). There was a significant effect of soaking in P. putida suspension on the mean developmental time, regardless of the soaking duration and concentration (F= 16; df= 11, 168; P< 0.05) (Fig. 2). At each concentration, the mean developmental time on un-soaked root pieces was signifi- cantly higher than that on soaked ones, regardless of soak- ing duration. The result illustrates that at 2x108 CFU/ml the observed differences in mean development between soaking durations were not significant. There was a significant effect of the soaking duration in P. putida suspension on mean number of eggs, regardless of the concentration (F= 112; df = 11, 168; P<0.05) (Fig. 3). At each concentration, mean number of eggs on soaked root pieces was significantly higher than that on un-soaked ones, regardless of soaking duration. At concentration 0 (root pieces soaked with distilled water), there was no sig- nificant difference in the mean number of eggs between 3 and 5 hr soaking duration. However, the mean number of eggs was significantly reduced with 15 hr soaking dura- tion. At 108 and 2x108 CFU/ml concentrations, the mean number of eggs on 3 hr soaking duration was significantly higher than that on 5 and 15 hr (Fig. 3). Table 1 - Mean percentage of matured females, developmental time, fecundity and oviposition when phylloxera was reared on P.putida-treated grape root pieces Oviposition period/d (Mean±SE) Fecundity (Mean±SE) Developmental time/d (Mean±SE) % matured female (Mean±SE) Bacterial Con- centration CFU/ml 14.2±0.54 a 44.73±0.85 a 28.27±0.57 a 78.16±1.48 a 0 12.07±0.27 b 40.07±1.3 b 26.53±0.27 b 36.33±0.88 b 108 9.53±.53 c 34.2±1.3 c 24.53±0.54 c 32.03±1.49 b 2x108 Means, in a column, followed by the same letter are not significantly different at the P < 0.05 (Tukey HSD test). Fig. 1 - Effect of root soaking duration in bacterial suspension of per- centage emerged matured females of phylloxera. Fig. 2 - Effect of root soaking duration in bacterial suspension on mean developmental time of phylloxera. Fig. 3 - Effect of root soaking duration in bacterial suspension on mean number of eggs of phylloxera. 78 A significant effect of the soaking duration in P. putida suspension on mean oviposition period was found, regard- less of the concentration (F= 46; df = 11, 168; P<0.05) (Fig. 4). At each concentration, the mean oviposition period on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. At both 0 and 108 CFU/ml, the mean oviposition period with 3 hr soak- ing duration was significantly higher than that with 15 hr. At 0 concentration, there was no significant difference in the mean oviposition period between on 3 and 5 hr soaking duration, while at 108 CFU/ml there was. At 2x108 CFU/ml, there was no significant difference in the mean oviposition period between 3, 5 and 15 hr soaking duration. 4. Discussion and Conclusions Several studies reported the use of rhizobacteria as a bi- ological control of pests (Racke and Sikora, 1992; Zehnder et al., 1997 a, b). To our knowledge, the application of rhizobacteria against phylloxera has not been investigat- ed. Our study indicates that when P. putida BTP1-treated roots were infested by phylloxera eggs, the percentage of matured females was negatively influenced (Table 1). The result illustrates that the percentage of matured females was significantly decreased when the concentration and the soaking duration were increased (Fig. 1). The reduc- tion of the number of matured females on the treated roots could be attributed to the inability of phylloxera to feed. Thus, nymphs fed on P. putida BTP1-treated roots showed antifeeding behaviour. When Heliothis zeae (Boddie) diet was contaminated with the bacterium Pseudomonas maltophila, a 60% reduction in adult emergence and high pupal and adult malformations were observed (Bong and Sikorowski, 1991). Moreover, when chestnut was treated with Pseudomonas fluorescens, 20% chestnut weevil mor- tality was recorded (Yaman et al., 1999). Therefore, the antifeeding behaviour of phylloxera nymphs on the PGPR treatments could be related to the reduction in the feeding stimulant. Consequently, the developmental time was altered leading to early emergence of matured females (Table 1). A comparison between P. putida BTP1-treated and untreated roots showed that the phylloxera development on treated roots was faster than that on untreated ones. Similar results were reported when cucumber beetles and American bollworm were fed on PGPR-treated cucumber plants and cotton bolls, respectively (Zehnder et al., 1997 a, b; Vijayasamundeeswari, 2009). Our current study indi- cates that the developmental time was significantly shorter on treated root pieces soaked for 15 hr (Fig. 2). Qingwen et al. (1988) detected a reduction of relative growth rate, consumption rate and digestibility of feed when Heliothis armigera fed on P. gladioli-treated cotton plants. It is known that fecundity could be considered an es- sential factor in assessing the effect of P. putida BTP1on phylloxera. Phylloxera fecundity on P. putida BTP1-treated root pieces was distinctly lower than that on untreated ones. When phylloxera females were reared on treated roots they were unable to produce a normal number of eggs compared to the control, especially at high concentration (Table 1). Nevertheless, when soaking duration in P. putida BTP1 suspension was prolonged, a defective reproductive capac- ity was obtained (Fig. 3). The average number of eggs was 65.3, 40, 32 and 21.6 eggs on 108 CFU/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, respectively. While on 2x108 CFU/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, the average number of eggs was 63.7, 34.2, 23.6 and 17.5 eggs, respectively. Inadequate nutrition and inability to establish good feeding sites could directly affect the num- ber of eggs laid. Therefore, phylloxera resistance could be reflected in a strong relationship between poor feeding and the reduction of insect reproduction (Granett et al., 1983). Thus, phylloxera produced more eggs on untreated roots compared with treated ones. Correspondingly, the oviposition period of phylloxera on treated roots was markedly shorter than that on untreat- ed ones (Table 1). When the concentration of P. putida BTP1 was increased a great reduction in the oviposition period was obtained. Such reduction was noticeably in- creased by prolonging the root soaking duration in P. pu- tida BTP1 (Fig. 4). It is known that the resistance mechanisms of phyllox- era could be related to several factors including: 1) reduc- tion of phylloxera fitness (antibiosis); 2) decrease in plant attractiveness to phylloxera (antixenosis) (Granett et al., 2001). Zehnder et al. (2001) mentioned that the PGPR treatment led to alteration in the plant metabolic pathway which elicited the induction of plant defense compounds. Qingwen et al. (1998) reported that polyphenol and ter- penoid content were increased with cotton treated with P. gladioli. The synthesis and formation of such materials in P. putida BTP1-treated roots would have a negative influ- ence on phylloxera feeding and development. The results illustrate that the soaking of grape roots with P. putida BTP1 suspension for 5 hr and 15 hr, regard- less of concentration, led to a great reduction in matured females, developmental time, fecundity and oviposition Fig. 4 - Effect of root soaking duration in bacterial suspension on mean oviposition period of phylloxera. 79 period. Such reduction might be attributed to increased bacterial cell concentration by prolonging the soaking du- ration. Therefore, roots pieces soaked with 2x108 CFU/ml for 15 hr showed more resistance to phylloxera than lower concentrations and a shorter soaking duration. These results provide essential information about the relationship between P. putida BTP1 and phylloxera re- sistance of grape plants. Phylloxera encounters serious difficulties in surviving, feeding and reproducing on P. pu- tida BTP1-treated roots. P. putida BTP1 reduced the sus- ceptibility of roots to phylloxera. 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