14 1. Introduction Grapevine phylloxera (Daktulosphaira vitifoliae Fitch) is considered the most destructive grapevine insect pest in Syria where more than 70,000 ha of this crop, annually producing approximately 540,000 t of grapes, are planted (Makee et al., 2010). Grape phylloxera invaded Syria on nursery stock from bordering countries during the 1920s where it quickly spread in Syrian vineyards among the non-resistant local Vitis vinifera L. grape (Makee et al., 2003). In Syria, the most commonly used resistant rootstocks are Ru140 (V. rupestris x V. berlandieri), R99 (V. rupestris x V. berlandieri), 3309C (V. riparia Michaux x V. rupestris) and 41 B (V. vinifera x V. Berlandieri Planchon). However, local phylloxera demonstrate an ability to develop and re- produce on all American rootstocks (Makee et al., 2010). In addition, the susceptibility of Ru140 rootstock was found to be higher than that of R99 and 3309C rootstocks (Makee et al., 2003). The rootstocks often allow limited growth and reproduction of phylloxera, but they may also reduce the growth vigor of grafted varieties because of in- compatibility phenomena. Moreover, most Syrian grape- vines are planted in areas of 300-500 mm rainfall, thus they are unirrigated and such conditions are inappropriate for resistant rootstocks (Makee et al., 2010). Granett et al. (1996) reported that once a vineyard is infested with grape phylloxera it is expected to cease its production in about two to five years. Generally, spring phylloxera is found primarily on the small feeder roots that proliferate during root flush. However, during summer when the climate is dry and the top of the plant is grow- ing, phylloxera is found extensively on the infected ma- ture older roots. In winter, phylloxera is found on mature roots due to the disappearance of feeder roots (Omer et al., 1997). Once the phylloxera’s proboscis is inserted into root bark cells, the parenchyma cells below the suberized outer layer, they inject saliva that, in turn, trigger the root cells to increase in size and number. These galls provide a feeding site from which stored nutrients such as sugars and amino acids can be extracted by developing phyllox- era, which leads to a decrease in vine vigor and eventually destroys the roots causing vine death (Omer et al., 2002). The injured grapevines are usually attacked by second- ary soil-borne pathogens at the insect feeding site (Omer and Granett, 2000). Granett et al. (1998) and Lotter et al. (1999) revealed that fungal infections associated with pyl- loxera galls are pathogenic and cause root death. In addi- tion, fungal invasion of the roots at grape phylloxera feed- ing sites cause severe infections in grapevines. Fusarium is a large genus of filamentous fungi, and most Fusarium species are harmless saprobes and relatively abundant members of the soil microbial community (Domsch et at., 1980; Nwanma et al.,1993). This ecological habitat of the fungus implies that Fusarium could be a useful resource of extra cellular enzymes. The fungal isolate F. solani SY7 was the best xylanase producer among tested isolates (Arabi et al., 2011; Bakri et al., 2012). Vine varieties sus- ceptible to grape phylloxera were also highly susceptible to F. oxysporum (Omer et al., 1995; 1999). Granett et al. The relationship between grape phylloxera and Fusarium root infection I. Idris,(1) M.I.E. Arabi Department of Biotechnology, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria. Key words: Fusarium ssp., grape phylloxera, grape root. Abstract: Phyloxera seems to have key role in the fungal pathogen infection ratio while the fungal spread reduces the abil- ity of pheloxera to reproduce. Intact roots of four-month-old grape plants were inoculated with phylloxera eggs in pres- ence or absence of fungal pathogens. Fusarium solani SY7 infection was detected in all plant parts when grapevine roots were infested with phylloxera. The spread ratio of Fusarium solani SY7 increased from 74 to 100% of the infested plants with phylloxera. On the other hand, the phylloxera on F. solani SY7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly decreased 49% and 31% respectively. The total plant biomass decreased to 29% in the presence of both F. solani SY 7 and phylloxera as compared to 9 and 17% in the presence of F. solani SY7 or phylloxera, respectively. This study sheds light on the correlation between fungi, phylloxera and grapevine and could help in the application of integrated pest management (IPM) programs against grape phylloxera. Adv. Hort. Sci., 2014 28(1): 14-19 (1) Corresponding author: ascientific@aec.org.sy Received for publication 26 March 2014 Accepted for publication 29 April 2014 15 (1998) also demonstrated that the two root types with V. vinifera parentage, Carignane and AXR#1, which are most susceptible to phylloxera feeding, are also susceptible to infection by F. oxysporum pathogen. The present study therefore aims to investigate whether the presence or absence of phylloxera could enhance F. solani SY7 fungal infection in the local Balady V. vinifera variety vine roots and the consequences of the interaction between the grape phylloxera and F. solan SY7 on the vine. In this context, the interaction between root infection and the three Fusarium species root and root- feeding grape phylloxera were first investigated under controlled condi- tions, then the effects of fungal infection with and without the presence of phylloxera on grape vine vigor (plant bio- mass, roots, branches and leaves, internodes weights) were determined. The process included identification of the Fu- sarium species that invade the roots from grape phylloxera feeding sites. 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 according to the procedures mentioned by De Benedic- tis and Granett (1993). Fresh and healthy pieces of local grape variety (Makee et al., 2008) “Balady” roots, 4-7 mm in diameter and 5-7 cm long, were taken and washed with tap water. Each piece was wrapped with moist cot- ton wool at one end, and then 10 to 15 phylloxera eggs were placed on each piece. The infested root pieces were placed on a wet filter paper disk inside plastic Petri dish- es (12 cm diameter and about 1 cm deep, three to four root pieces per dish). For ventilation purposes, the Pe- tri dish lid was modified with a 1-1.5 cm cloth–screened hole. Dish edges were sealed with parafilm, kept in plas- tic boxes with tightly fitting lids and incubated at 25°C in the dark with 75% relative humidity. The root pieces were replaced when they desiccated rotted or the phyl- loxera became crowded. Potted inoculation procedure Before inoculation, three-day-old eggs (n =100) were removed from the colony and placed in 1.5 ml plastic tubes for surface sterilization. One ml of formaldehyde was add- ed to the eggs and the tubes were gently shaken for 10 min. The sterilizing solution was removed from the mix and the eggs were extracted and placed on a sterile filter paper and dried for 5 min. They were then kept in a Petri dish, sealed with parafilm to prevent contamination and the escape of the phylloxera crawlers. Egg sterilization was carried out under sterile conditions (Makee et al., 2003). Fungal isolates The fungal isolates were obtained from the plant pa- thology laboratory of the Atomic Energy Commission of Syria (Arabi et al., 2011) (Table 1). Host plant root samples infested with Fusarium were collected from dif- ferent locations in Syria. Roots were sterilized in 5% so- dium hypochlorite (NaOCl) for 5 min. After three wash- ings with sterile distilled water, roots were dipped in 70% Ethanol for 1 min and then washed once with dis- tilled water. Roots were cut into small slides under ster- ile conditions and transferred to Petri dishes containing potato dextrose agar (38g/L) (PDA, DIFCO, DETROIT, MI, USA) (Alazem, 2007). Thirteen mg/1 Kanamycin sulphyate were added after autoclaving and 10 days in- cubation at 23 ± 1°C in the dark to allow mycelia growth. All isolates were identified morphologically, according to Nelson et al. (1993). Emphasis was placed on select- ing isolates that induced differential reactions on specific genotype, pathogencity and in vitro xylanase activity (Alazem, 2007; Arabi et al., 2011; Bakri et al., 2012). The above mentioned parameters lead to select of three monosporic isolates F. culnorum SY3, F. solani SY7 and F. equisesti SY24. The Fusarium isolates used in this study, their location, year of collection and xylanase production are listed in Table 1 (Arabi et al., 2011). The cultures were maintained on silica gel at 4°C until needed. Eighty “Balady” grape stem pieces were dipped in a solution of 2000 ppm IAA (Indol Butric Acid) for 2 min, then planted in plastic pots contacting sterile moistened soil. Finally, they transferred to 10-L plastics pots, after four months. The experiment was conducted in greenhouse, using a randomized complete block design with four rep- licates of four plants for each of the following treatments in each treatment. 1 - Infection with phylloxera: roots of vines were infected with phylloxera eggs (50 eggs/root). 2 - Infection with Fusarium spp: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (F. culnorum SY3, F. solani SY7, F. equiseti SY24) for 15 s, then dried for 30 min. 3 - Infection with Fusarium spp and phylloxera: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (F. culnorum SY3 , F. solani SY7, F. equiseti SY24) for 15 s, dried for 30 min, and then infected randomly by phylloxera eggs. 4 - Plant control (free of Fusarium spp. and phylloxera): plants were transplanted into 10-L pots. Each experi- mental unit consisted of two plants. Pots were filled with sterile soil. The pots were all placed in a green- house at 25 ± 1°C ( day) and 23±1°C (night) with 16-h Table 1 - Fusarium isolates, location, year of collection and extra cel- lular xylanase production in solid state fermentation after five days of inoculation at 30°C Isolate Location of Syria Year of collection Xylanase (U/G) F. culnorum SY3 North-West 2005 163.69 F. solani SY7 Middle-Region 2003 908.2 F. equiseti SY24 North-East 2005 122.43 16 daylight and 85-95% relative humidity. Plants were ir- rigated with water as needed. A year later, the following parameters were measured: plant biomass, root weight, root number, internode weight. Five root pieces of each tested plant were sampled. Micro- scopic inspection was performed to determine the number of tuberosities and feeding nymphs for each tested plant. Fungal inspection Plant samples (roots, leaves and branches) were col- lected from each tested plant, surface sterilized for 3 min in 5% NaOCl, and rinsed twice in distilled water. Six disks to each roots, leaves and branches were transferred to Petri dishes containing PDA with 13 mg/L Kanamycin sulphy- ate added after autoclaving and incubated for 4 weeks at 23 + 1°C in the dark to allow mycelia growth. Mycelia edges of F. culnorum SY3, F. solani SY7, and F. equiseti SY24 were identified morphologically as describe by Nel- son et al. (1993). The infection percentage of fungal ratio was calculated using the formula R = F e / Ft x 100, where R = the percentage of fungal ratio, F e = number of disks that contain the fungi from roots, leaves and branches, and Ft = number of the total disks with or without fungi from roots, leaves and branches. Statistical analysis was performed using the STATIS- TIC program version 6 (Statsoft, Inc. 2003) at 5% level (P = 0.05). Means were subjected to analysis of variance tested for significance using Tukey HSD test. 3. Results Incidence of Fusarium spp infection in different parts of plant with or without phylloxera The results demonstrated that fungal infection ratio in the whole plant was 74, 1.7 and 0% of F. solani SY7, F. equiseti SY24 and F. culmorum SY3, respectively. Further- more, the ratio of infection with F. solani SY7 in treatment 4 increased 26% to reach 100% (Table 2). Effect of different treatments on plant The plant biomass (Fig. 1, I) decreased significantly in the presence of phylloxera and Fusarium solani SY7 (9 and 17%, respectively). This effect is more evidenced in the presence of fungi and phylloxera 29% (F=2; df=1, 3; p<0.05). Vine roots infection with Fusarium spp. had no effect on root weight (Fig. 1, II) compared with the con- trol, while infection with phylloxera increased 36% sig- nificantly comparing to control (F=2; df=1, 3; p<0.05). Branch and leaf weight (Fig. 1, III) decreased signif- icantly (F=2; df=1, 3; p<0.05 ) compared to the control in the presence of phylloxera and F. solani SY7 (53% and 31%, respectively). This effect is more evidenced in the presence of phylloxera with infection of F. solani SY7 58%. The average internode weight (Fig.1, IV) in treated plants with both phylloxera and fungi increased consider- ably (50%) as compared to the control. However, no obvi- ous correlation between phylloxera and fungi concerning the internode weights was shown in the experiment. In addition, the root number of vines exposed to the fungus was notably decreased but to a lesser degree compared to plants infested with phylloxera or both fungi and phyllox- era (F=3.6; df=1, 3; p<0.05) (Fig. 2). Effects of F. solani SY7 infection on nymph and tuberosity numbers The numbers of nymphs and tuberosities in vines infest- ed with phylloxera decreased significantly (F=85.3; df=1.1; p<0.05), (F20=; df=1.1; p<0.05) in comparison with vines infested with both phylloxera and fungi. In addition, nymphs and tuberosities were reduced by 49 and 31%, respectively, compared to the control plants (Table 3). 4. Discussion and Conclusions Phylloxera seems to have key a role in the fungal pathogen infection ratio and, in contrast, the fungal spread reduces the ability of pheloxera to reproduce. The high in- fection ratio of F. solani SY7 (74%) may attribute to its ability to spread in the phloem parenchyma through a spe- cial mechanism which allows the fungi to infect the roots (Omer et al., 1999). Phylloxera can serve as a vector and transport fungal propagates from infected to healthy roots (Omer et al., 2000). Therefore, our data suggest that the injury caused by phylloxera may give benefit to F. solani Table 2 - Incidence of Fusarium spp. infection in different parts of plant with or without phylloxera Treatment F. solani SY7 % F. equiseti SY24 % F. culnorum SY3 % Roots Branches Leaves Roots Branches Leaves Roots Branches Leaves Phylloxera 0 0 0 0 0 0 0 0 0 Fusarium mix 72 77 75 0 0 0 0 0 0 Phylloxera + Fusarium mix 100 100 100 5 0 0 0 0 0 Control 0 0 0 0 0 0 0 0 0 Data represents the infection percentage of Fusarium spp. ratio in roots, branches and leaves in each treatment. 17 SY7 to spread throughout the entire plant 100%. Howev- er, the other two fungi (F. culmorum SY24 and F. equiseti SY3) species may not possess this mechanism to spread in the phloem parenchyma and eventually they are not able to infect the vine despite the presence of phylloxera which may be attributed either to the lack of proper growth con- dition in vine or to the presence of immune response in the plant which prevents the growing of the later fungi species (Omer et al., 1999; Fossen, 2002). To confirm the effect of F. solani SY7 and phylloxera on vines, we established an experiment to evaluate five pa- rameters (the weights of plant biomass, root internodes, roots, branches and leaves and root numbers in presence or absence of phylloxera compared to control plants). From these biological parameters, we can estimate the relation- ship between F. solani SY7 and phylloxera. Omer et al. Table 3 - Effects of F. solani SY7 infection on numbers of nymphs and tuberosities Treatments Nymphs number (SE) Tuberosities number (SE) Phylloxera 25 ± 1 a 4.9 ± 0.9 a F. solani SY7 0 0 Phylloxera + F. solani SY7 12.9 ± 0.9 b 3.4 ± 0.5 b Control 0 0 Data represents nymphs number mean in tested roots of 16 plants in each treatment. Data were subjected to ANOVA analysis and the dif- ferences between means were tested for significance using Tukey HSD test. Means followed by different letters (columns) are significantly dif- ferent at P <0.05. B A B B 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi R oo ts w ei gh ts gr am Treatments II A B C D 0 20 40 60 80 100 control phylloxera fungi phylloxera + fungi P la n ts b io m a ss g ra m Treatments I B A A A 0 5 10 15 20 25 control phylloxera fungi phylloxera+fungi In te rn o d es w ei g h ts g ra m Treatments IV A C B C 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi B ra n ch es a n d le av es w ei g h ts g ra m Treatments III Fig. 1 - Effect of different treatments on plant biomass (I), root (II), branches + leaves weights (III) and internode weights (IV). A B B B 0 5 10 15 20 25 30 35 40 45 50 control phylloxera fungi phylloxera+fungi R o o ts n u m b er s Treatments Fig . 2. Effects presents of phylloxera and fungi on roots numbers Fig. 2 - Effects presents of phylloxera and fungi on roots numbers I II III IV 18 (1995) demonstrated that greenhouse experiments with ef- fects of fungal infection on grapevine vigor after pruning at week 13 showed that damage was significantly greater in vines infested by phylloxera, F. solani and Pythium ul- timum than the damage in vines infested with phylloxera alone. Total biomass was reduced by 16% in vines infested with phylloxera and by 24% in vines infested by phyllox- era and F. solani (Omer et al., 1995). In our study it was found that the total biomass was reduced by 9% in vines infested with phylloxera and by 29% in vines infested with phylloxera and F. solani SY7, compared to the controls. The differences between F. solani SY7 and F. solani (Omer et al., 1995) may be attributed to the disparity of aggres- siveness of the isolates. Therefore, the infection with phylloxera and fungi together caused a synergetic effect where the plant biomass tend to decrease in presence of phylollxera or fungi. This can be attributed to the dam- age in the vine roots caused by either phylloxera or fungi which cause dysfunction of the root as they should be in the normal conditions. As both phylloxera and fungi affected the roots di- rectly therefore, more work focused on the roots. The root weight and internodes increased significantly by 36 and 63% comparing to the control, in the presence of phyllox- era and fungi respectively. This increase is normal in the presence of phylloxera due to the tuberosities and nodosi- ties formed by the phylloxera. However, fungi alone did not cause changes in the root weight (Omer et al., 1995). Our results demonstrated that the significantly growth of infested vines with phylloxera alone or with a combina- tion of phylloxera and F. solani SY7 may attribute to the rapid growth observed after potting. Moreover phylloxera may require more time necessary to establish feeding sites (Omer et al., 1995). Additionally, a reduction of 50%, 30% respectively was observed in branch and leave weight and in the num- ber of roots in the presence of phylloxera or both phyl- loxera and fungi compared to control, is attributed to roots death.Therefore, F. solani SY7 infection can spread radi- ally causing necrosis in the parenchyma and phloem. Ulti- mately the infection kills the roots and eventually the plant (Omer et al., 1999). Similarly Fossen (2002) found that the virulence isolates in present of V. vinifera on vine roots led to proportion of the root circumference that became necrotic in a 5-week period. Some isolates were highly virulent, causing up to 80% necrosis while other isolates were negligibly virulent (Fossen, 2002). It has been shown in other plant-herbivore-pathogen systems that co-occurrence can, through direct interaction or changes in host’s susceptibility, affect the performance of the pest or the pathogen (Karban et al., 1987; Hatcher., 1995). Omer et al. (2002) reported that the ability of grape phylloxera to exploit grape roots increased in the absence of fungal pathogens and indicated that phylloxera on infected roots developed more slowly, and had substantially reduced survival and reproduction rates. Therefore our results re- vealed the presence of fungi reduced the ability of phylox- era to form tuberosities by 31%. Our data also demonstrated that the total root weight decreased in presence of fungi with the Phylloxera-infested vine roots which were also reflected by a decrease in the number of nymphs. Therefore our assays demonstrated that phylloxera on F. solani SY7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly de- creased by 49% and 31% respectively. The reproduction and feeding activities of phylloxera were significantly de- creased in the presence of fungal infection, consequently this result is in agreement with Omer et al.( 2000). Fur- thermore the ability of F. solani SY7 to spread within the plant parts increased when grapevine roots were infested with phylloxera grape. These results provide interesting piece of information about the relationship between F. solani SY7 pathogens and phylloxera. However these results are to be proved in the field. The present study provides preliminary information that could help in application of integrating pest manage- ment (IPM ) program against grape phylloxera. 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