346

16 Invasive Gall () in Eucalypt and Its Management

A.S. Vastrad and S.H. Ramanagouda

1. Introduction Eucalypt species are an important source of short fibre pulp for the production of high-quality paper. The trees having rapid growth rate and short rotation times, can be grown in coppiced production and are extraordinarily well suited for large-scale plantation in diverse parts of the world. As the area of plantation under eucalypts has increased worldwide, so has the number of utilizing them as host. Many of these insects now pose biosecurity threats to eucalypts in regions where they are grown as exotics. Large-scale plantings of eucalypts for a variety of purposes have occurred throughout Asia, from India to Indonesia, Thailand, Malaysia, Philippines, Vietnam and China. Asia has had very few introductions of Australian insects, but large numbers of endemic insects utilize eucalypts as hosts (Sen-Sarma and Thakur, 1983). This appears to be a common theme throughout Southeast Asia where eucalypts have been grown. Invasive , Leptocybe invasa La Salle and Fisher is the only of Australian origin to have been introduced in to South and Southeast Asia and to have caused significant damage with introductions occurring between 2002 and 2007. First reported from Middle East during 2000, the gall wasp wreaked havoc on eucalypt plantations throughout the world (Aytar, 2003; Mutitu, 2003; Mendel et al., 2004; Nyeko, 2005; Neser et al., 2007; Costa et al., 2008; Gaskill et al., 2009; Dhahri et al., 2010; Karunaratne et al., 2010; Aquino et al., 2011). An unconfirmed news item reported its first occurrence in India from Karnataka during 2001 (EF, 2007). However, its definitive invasion was first reported during 2004 from Tamil Nadu which subsequently spread to the neighboring states of Andhra Pradesh, Karnataka and Kerala (Jacob et al., 2007), Maharashtra, Goa and Gujarat (Kumar et al., 2007), Madhya Pradesh (ICFRE, 2007). Since 2009, it has spread to northern India and is causing heavy loss to nursery and plantations in Punjab, Haryana, Uttarakhand and Uttar Pradesh (FRI, Dehradun). Severe incidence of the pest both in nurseries and plantations in Invasive gall wasp (Leptocybe invasa) in eucalypt... 347

Orissa and on new shoots of grown up plants in Jammu and Kashmir has been observed during 2011 (Fig. 1). The pest causes galls on midribs, petioles and stems of new shoots of eucalypt. Heavy infestation leads to deformed leaves, shoots and reduction in growth. Infested seedlings become unfit for planting (Fig. 2). In Karnataka, the gall wasp was reported to be on an attacking spree and damaged 2.5 M eucalypt saplings in the

Fig 1. Distribution of L. invasa in India. 348 A.S. Vastrad and S.H. Ramanagouda

Fig. 2. Gall wasp damage in nursery (above) and plantation (below). nurseries of two major wood based industries (West Coast Paper Mills and Harihara Polyfibres) (EF, 2007). Three lakh grown up trees were severely affected by L. invasa in Punjab. Since its wide spread outbreak during 2007, work carried out at the University of Agricultural Sciences, Dharwad on various aspects of invasive gall wasp is presented below.

2. Biology Biology of the eucalypt gall wasp, L. invasa was studied during winter and summer of 2009-10 (Fig. 3 a to f). During summer season, the symptoms of tissue disruption (Stage I) were first evident within 10 days while it took 10-15 days during winter and occupied 8.66 and 12.0 days during the respective season. The characteristic bump shaped green colored, II stage galls lasted for 45.0 and 57.4 days during summer and winter season, respectively. Stage III galls characterized by glossy pink color lasted for 29.6 and 30.2 days during summer and winter season. While the stage IV galls characterized by dull pink color occupied 20.7 days during summer and 18.8 days during winter. Stage V galls were noticed three to six days after stage IV during summer and four to six days during winter season. Total life cycle of the pest occupied 100-115 (mean 109 days) days during summer and 100-143 (mean 123.6 days) days during winter (Table 1). Adult longevity was 4.3 days without food while those fed Invasive gall wasp (Leptocybe invasa) in eucalypt... 349

a. Oviposition on young leaf d. Gall development

b. Oozing after oviposition e. Adult emergence

c. Tissue disruption f. Emergence hole Fig. 3. Sequence of gall development (a-f). 350 A.S. Vastrad and S.H. Ramanagouda

Table 1. Biology of gall wasp, L. invasa on E. tereticornis Developmental Range (d) Mean ± SD (d) stage Winter 2009 Summer 2010 Winter 2009 Summer 2010 Stage I 10-15 8-9 13.00±1.87 8.66±0.47 Stage II 34-89 40-46 54.75±5.49 45.00±3.74 Stage III 31-40 29-32 31.00±3.80 29.66±2.05 Stage IV 18-30 19-22 19.50±4.15 20.66±1.88 Stage V 3-9 4-6 5.00±0.70 5.00±1.41 Total 100-143 100-115 123.6±15.37 109.00±6.48 Source: Ramanagouda et al. (2010).

on 10 per cent honey solution lived for 5.7 days. These results are in agreement with Mendel et al. (2004) who reported that the mean developmental time from egg to adult emergence as 132.6 days at room temperature. According to Hesami et al. (2005), the developmental period of L. invasa was 126.2 and 138.3 days under laboratory and field conditions, respectively.

3. Seasonal Incidence During survey 30 cm shoot from 10 infested eucalypt plants were collected and the observations on different gall stage were recorded separately from top, middle and bottom portion of the sample (10 cm each) monthly. Sample was kept separately (top, middle and bottom) for pest and emergence in a pin holed polythene bags. Adult emergence of the pest and was recorded daily till the cessation of adult emergence. Different parasitoids emerging from these samples were identified by Dr. T.C. Narendran, Trust for Insect , University of Calicut, Kerala. Mean and standard deviation was calculated and per cent parasitization was worked out by using the following formula (Kim et al., 2008).

No. of parasitoid adults emerged Per cent parasitization = —————————————————— X 100 Total no. of adults (gall + parasitoids)

3.1. Kulwalli Plantation (2008-09) Studies on seasonal incidence indicated that the pest and its parasitoids were active throughout the year. Gall incidence (top, middle and bottom) indicated an equal number of galls of second, third, fourth and fifth stage (7.8, 7.2, 7.8 and 7.3, respectively) while the number of first stage galls was lowest (4.3). This is understandable since the total surface area available for oviposition is considerably Invasive gall wasp (Leptocybe invasa) in eucalypt... 351 less compared to those which are either unsuitable for oviposition or were occupied by galls of later stages. Irrespective of the gall stages, total number of galls ranged from a lowest of 19.2 (November 2008) to highest of 44.1 (June 2009). During remaining months total number of galls ranged from 29.3 (January 2009) to 40.3 (December 2008). Galls of all stages were recorded throughout the year (Fig. 4). However, according to Mendel et al. (2004), after over wintering as third or fourth stage from October to March, though new growth appeared in February, wasps started emerging and resumed oviposition only in April. Adult emergence was noticed throughout the year. Three peak periods of adult emergence was noticed during December 2008 (133), April 2009 (111) and June 2009 (88) separated by about 120 days. Adult emergence during remaining months ranged from 30 (September 2008) to 95 (January 2009). Though only females emerged from the galls and started laying eggs indicating thelytokous reproduction, a small number of males were regularly encountered which could be easily identified by the absence of characteristic ovipositor and distinctly hairy antennae. During the survey to document the seasonal incidence and natural enemies of the pest, several hymenopteran parasitoids emerged from the infested eucalypts samples collected during October 2008. These include gala Walker and Aprostocetus sp. (), Megastigmus sp. () and Parallelaptera sp (Mymaridae) (Vastrad et al., 2010). Among the different parasitoids, Megastigmus sp. was the most dominant (90.7 %) followed by Aprostocetus sp. (6.5 %) and A. gala (2.72 %). Among several hundred parasitoids collected, one specimen each belonged to Telenomus sp. and Parallelaptera sp. Combined parasitization ranged from 49 per cent to 74 per cent on severely infested early stage galls (2nd and 3rd). However, no parasitoids emerged from the fresh galls (1st stage), low or moderately Number

Fig. 4. Gall incidence, adult emergence and per cent parasitization (Kulwalli, 2008-09). 352 A.S. Vastrad and S.H. Ramanagouda infested coppice and nursery seedlings. Megastigmus sp. was later described as M. dharwadicus Narendran and Vastrad (Narendran et al., 2010).

3.2. Daddikamalapur Plantation (2009-10) Gall incidence indicated that the samples contained more of first stage galls (11.8) followed by third, fourth and fifth stage. Emergence of M. dharwadicus was noticed throughout the year except November and April and accounted for a maximum of 30.7 per cent and minimum of 6.5 per cent parasitization and emergence of A. gala was maximum (26) and minimum (01) and accounted for a maximum of 15.9 per cent and minimum of 0.6 per cent parasitization. Adult emergence was noticed throughout the year and was maximum during July (348) and minimum (63) during March and increased thereafter (Fig. 5). Only females of L. invasa were recorded during the seasonal incidence studies, however, a small number of males were also recorded (<1%) throughout the year. Sex ratio of the parasitoids recorded during the study was ~1:2 (male: female). Parasitoids were active throughout the year except during November. Parasitization was more during December to March and lowest during April. M. dharwadicus was the dominant among the two parasitoids encountered. L. invasa is considered to be a pest of young seedlings and coppice preferring tender leaves and shoots for oviposition. Hence the first stage galls were more on top 10 cm of the sample. Since top portion consisted of only first stage galls no adult emergence was noticed. Middle portion of the sample on the other hand contains more of third stage galls which have reached their maximum size commensurate with the maturation of the leaf and shoot. Highest adult emergence was recorded from middle portion of the sample which incidentally also recorded highest parasitization among different samples. The bottom portion of the sample containing fully expanded leaves Number

Month Fig. 5. Gall incidence, adult emergence and per cent parasitization (Daddikamalapur, 2009-10). Invasive gall wasp (Leptocybe invasa) in eucalypt... 353 and matured twigs contained more of fourth and fifth stage galls while the first stage galls were totally absent and second stage galls were occasionally encountered. Per cent parasitization was more in middle portion throughout the year. Parasitization by M. dharwadicus and Aprostocetus gala was commonly noticed on middle and bottom portion consisting of II, III and IV stage galls. Commensurating with the preference for oviposition on the tender parts of the plant, the number of early stage galls was more on the top portion of the sample than late gall stages. Conversely, first stage galls were least in middle portion of the sample which harbored highest number of second stage galls followed by other stages. First stage galls were conspicuously absent on the bottom portion of the sample characterized by tissue hardening which does not support oviposition.

4. Management of Eucalyptus Gall Wasp Currently no specific management strategies against L. invasa exist. Some of the adhoc measures include, viz., periodic monitoring of infested nurseries and plantation, mechanical removal, avoiding use of susceptible clones. When pest incidence is low, selective pruning or plucking of leaves or shoots, application of systemic insecticide such as dimethoate or oxydemeton methyl (2 ml l-1) or imidacloprid (1ml l-1) at fortnightly intervals and strict quarantine have been suggested by (IFGTB, EPPO). However, the use of insecticides and botanicals to manage pest has not been very encouraging (Javaregowda et al., 2010). Several options available to develop integrated pest management strategies are described below (Fig. 6 a to d).

4.1. Sticky Traps Pest detection with traps has been the first line of defense against exotic pests. Colored sticky traps are used in pest management for monitoring, early detection and possibly for control by mass trapping. In all the situations the color that maximizes trap catches is most appropriate. A strongly attractive color might draw gall wasp from greater distances than other colors.

4.1.1. Trap colour Among the different colored sticky traps evaluated, yellow was found effective by trapping highest number of wasps (Table 2). Held and Boyd (2008) reported that yellow trap was most effective against Gynaikothrips uzieli Zimmermann. Among the yellow and blue sticky traps tested against the adults of Anastrepha spp. and Ceratitis capitata yellow trap was most effective (Rodrigues and Paulo, 2002). Chen et al. (2004) found blue sticky cards to be most effective in trapping and hoverfly. Protasov et al. (2007) reported that green sticky plates were effective in trapping muskelli (Ashmead). The efficacy of yellow sticky traps in attracting various insects both under protected cultivation as well as open field has been reported by various authors (Durairaj 354 A.S. Vastrad and S.H. Ramanagouda

Trap color Fig. 6. Strategies to manage gall wasp (a-d). Yellow Table 2. Evaluation of different coloured sticky traps against Eucalyptus gall wasp Red

Blue

Green

White

Sem ± CD@ 5%

Figures in the parentheses are transformed values. Invasive gall wasp (Leptocybe invasa) in eucalypt... 355

et al., 2006; Ramegowda et al., 2007; Affandi et al., 2008). Thus, Yellow sticky traps can be used both for monitoring of the pest in general as well as mass trapping of the wasps to reduce the infestation both in nursery and under protected cultivation.

4.1.2. Sticky materials The use of host odour and chemical attractants has been reported to improve the efficiency of traps. In all the treatments addition of eucalypt oil numerically enhanced the trap catches (Table 3). Insect gum with eucalypt oil trapped maximum number of wasps (192/trap) and was equally effective without oil (169.3/trap), while petroleum jelly with oil (122.3/trap) was the second best treatment. Automobile grease (with or without eucalypts oil) trapped lowest number of wasps (54.3 to 56.3). Results indicated the superiority of insect gum with eucalypts oil in attracting gall wasp. Insect gum was most effective in trapping the wasps while eucalypts oil enhanced the trap catches in petroleum jelly and grease. Influence of host odour in attracting phytophagous insects has been reviewed by Visser (1986). While, insect gum has

Table 3. Efficacy of sticky materials in trapping Eucalyptus gall wasp Treatment Number of gall wasp/trap on different days 7th 14th 21st 28th Pooled T1 Insect gum + eucalypts oil 28.50 123.50 21.75 18.25 192.00 (5.43) (11.16) (4.77) (4.39) (13.89) T2 Insect gum 27.25 77.00 36.75 28.25 169.25 (5.32) (8.33) (6.14) (5.41) (13.05) T3 Petroleum jelly + eucalypts oil 8.25 83.00 16.50 14.50 122.25 (3.04) (9.17) (4.18) (3.94) (11.1) T4 Petroleum jelly 4.25 58.25 17.25 6.00 85.75 (2.29) (7.7) (4.27) (2.65) (9.31) T5 Grease + eucalypts oil 4.50 30.00 13.00 8.75 56.25 (2.35) (5.57) (3.74) (3.12) (7.57) T6 Grease without oil 7.50 22.50 16.00 8.25 54.25 (2.91) (4.85) (4.12) (3.04) (7.43) SEm ± 0.49 0.64 0.49 0.22 0.54 CD @ 5% 1.48 1.94 1.50 0.66 1.64 Figures in the parentheses are transformed values.

been extensively used for trapping (Rodrigues and Paulo, 2002), alternatives such as vinyl chloride (Saito et al., 1988), 5 per cent polybutane (Rushtapakornchai et al., 1989) and grease (Protosav et al., 2007) have also been successfully tried.

4.1.3. Trap shape Literature throws light on use of different types of sticky traps for managing different pest species both under field and protected cultivation. Flat trap with glue on both sides (1,260 cm2) trapped significantly higher number of wasps followed by other 356 A.S. Vastrad and S.H. Ramanagouda

traps, the surface areas of which ranged from 415 (cylindrical trap) to 1,073 cm2 (sphere trap) indicating direct relationship between the surface area and number of trap catches (Table 4).

4.2. Chemical Control in Nursery Different methods of insecticide application, viz., seed treatment, cutting dip and foliar spraying were evaluated.

4.2.1. Seed treatment The seeds (cv. Mysore gum) were soaked for 15 minutes in three dosages of the test insecticides which were replicated thrice. Seeds soaked in water alone formed the control. Later, the treated seeds were shade dried and sown in polythene bags. After emergence of seedlings, observations were recorded on pest infestation (oviposition damage) at weekly intervals and converted to per cent infestation. The data was suitably transformed and analyzed using two factor Randomized Block Design. Neonicotinoids, viz., thiacloprid, imidacloprid and acetamiprid were equally effective in preventing the infestation up to 32 days, whereas azadirachtin was least effective (Table 5). At 39 and 46 days after emergence the infestation levels though increased drastically the test chemicals were still superior over untreated control (92.79%). Higher dosage of neonicotinoids did not affect the establishment of seedlings. Kavitha Kumari (2009) reported acetamiprid to be the best insecticides followed by imidacloprid and thiamethoxam.

Table 4. Evaluation of different shaped yellow sticky traps in nursery Shape Surface Number of adult wasps trapped on difference days area (cm2) 7th 14th 21st 28th Pooled Rectangular cube 947.0 158.40 5.20 22.60 15.20 67.33 (12.58) (2.49) (4.85) (3.97) (827) Sphere 1073.0 233.20 8.00 27.20 16.80 92.27 (15.58) (3.00) (5.30) (4.15) (9.66) Cylindrical 414.9 106.80 1.60 10.20 15.8 52.93 (10.35) (1.61) (3.28) (4.03) (7.34) Flat both sided 1260.0 454.40 3.80 8.40 32.4 176.53 (21.23) (2.19) (3.05) (5.72) (13.32) Flat single sided 630.0 176.20 6.00 24.00 27.4 75.07 (13.29) (2.65) (4.98) (5.21) (8.72) SEm ± - 0.78 0.19 0.21 0.40 047 CD @ 5% - 2.34 0.59 0.64 1.20 1.41 Figures in the parentheses are transformed values. Invasive gall wasp (Leptocybe invasa) in eucalypt... 357

45.01 47.51 55.97 67.92 92.79 (41.98) (43.48) (48.70) (56.01) (78.81) 48.97 32.83 31.11 43.20 61.82 84.84 (46.19) (46.19) (34.73) (34.73) (33.76) (33.76) (40.93) (40.93) (52.96) (52.96) (68.59) 46 DAT 46 DAT 100 100 62.17 42.44 54.44 54.90 68.51 (56.50) (40.56) (47.58) (48.36) (56.05) (89.96) CD@ 5% CD@ (58.69) (58.69) (50.64) (50.64) (49.11) (49.11) (56.82) (56.82) (59.01) (59.01) (77.88) 59.76 56.98 69.80 73.42 93.51

heses are arc sine transformed values. 69.49 37.95 38.96 41.61 51.74 78.88 (37.83) (38.27) (39.93) (46.03) (65.79) 26.58 25.44 32.15 36.16 77.28 39.52 (30.93) (30.93) (29.87) (29.87) (34.07) (34.07) (36.93) (36.93) (61.56) (38.67) 39 DAT CD@ 5% SEm+ CD@ gall wasp 35.92 44.74 44.82 53.28 86.59 53.07 (36.76) (41.95) (41.96) (46.86) (68.77) (47.26) (45.79) (45.79) (42.98) (42.98) (43.75) (43.75) (54.30) (54.30) (67.05) (50.77) Eucalyptus Per cent ovipositiondamage/seedling 51.34 46.69 47.87 65.77 83.84 59.10 24.19 24.19 24.86 24.86 26.85 26.85 42.19 42.19 73.77 (29.03) (29.03) (29.29) (29.29) (30.41) (30.41) (40.42) (40.42) (59.48) 14.48 16.21 21.14 32.09 79.51 32.68 (22.32) (23.13) (25.85) (34.46) (63.20) (33.79) 32 DAT 32 DAT on level of infestation by on level of infestation by CD@ 5% SEm+ 5% CD@ 23.12 25.75 30.23 42.16 74.34 39.12 (28.56) (30.27) (32.86) (40.46) (59.92) (38.41) 2.19 6.24 2.11 2.11 6.03 1.67 4.76 1.63 4.67 1.29 3.74 3.66 NS 2.89 NS 6.24 2.19 4.83 1.69 NS 3.79 SEm+ C1 C2 C3 Mean C1 C2 C3 Mean C1 C2 C3 Mean Mean C1 C2 C3 Mean C1 C2 C3 Mean C1 C2 C3 34.97 34.97 32.62 32.62 29.17 29.17 52.31 52.31 67.46 43.30 (36.21) (36.21) (34.49) (34.49) (32.51) (32.51) (46.33) (46.33) (55.33) (40.97)

C1 – 2 ml or g, C2 – 3 ml or g and C3 – 4 ml or g. DAT – Days after treatment: NS Non-significant. Figures in the parent C1 – 2 ml or g, C2 3 g and C3 4 g. DAT Treatment Acetamiprid 20 SP Imidacloprid 600 FS Thiacloprid SL 21.7% SL 0.03% Azadirachtin Control Mean means comparing For of (T) Insecticides (D) Dosage X D) (T Interaction Table 5. Effect of seed treatment 5. Effect of seed treatment Table

358 A.S. Vastrad and S.H. Ramanagouda

4.2.2. Cutting dip Cuttings used for the clonal multiplication were dipped for 15 min. in three dosages of the test insecticides with three replications. Bavistin (2g l-1) was added to all the treatments. Bavistin (2 g l-1) alone formed the control. Treated cuttings planted in root trainers kept in mist chamber for rooting were first transferred to net house after 30 days and finally to the open nursery. Observations were recorded on establishment of cuttings and infestation (oviposition damage) at fortnightly intervals. The data were suitably transformed and analyzed using two factor Randomized Block Design. The establishment of cuttings was low (~55) during the study period due to late season planting. Dipping the cuttings used for clonal multiplication in neonicotinoids, viz., thiamethoxam imidacloprid, acetamiprid, and thiacloprid did not affect the establishment of cuttings (Table 6). All the insecticides were effective in reducing gall wasp infestation compared to untreated control (Table 7). Establishments of cuttings were as good as in untreated control. Profenophos, an organophosphorous compound even at the lowest dosage severely affected the establishment of the cuttings (< 10 % survival). Dipping the cuttings even at higher concentrations of neonicotinoids did not show any phytotoxic symptoms. Neonicotinoids were found to be safe for establishment of cuttings and profenophos to be highly phytotoxic. Similarly chlorpyriphos and dimethoate were also highly phytotoxic even at the lowest dosage and severely affected the establishment of cuttings (Kavitha Kumari, 2009). Vastrad et al. (2011) reported that acetamiprid at 0.4 g l-1 was the best treatment which provided protection up to 35 days followed by imidacloprid and thiamethoxam and did not show any phytotoxic symptoms. The increased dosage of 4 g or ml l-1 extended the protection of treated cuttings only up to 45 days.

4.2.3. Spraying Seedlings from the earlier experiment showing best results (thiacloprid, imidacloprid and acetamiprid) were used for evaluation of insecticidal sprays. The seedlings were kept below the canopy of the infested plants to ensure infestation by the gall wasps in the second greenhouse. Spraying was taken up on seedlings at weekly intervals depending on the number of galls recorded. Observations were recorded on oviposition damage and gall development at weekly interval. The data was suitably transformed and the means were separated by DMRT (p=0.05). Sprays were given only when the number of galls/oviposition exceeded 10/seedlings. Four sprays of thiacloprid and imidacloprid and eight sprays of other chemicals were given. Among different insecticides tested under nursery conditions, thiacloprid was most effective followed by imidacloprid, acetamiprid and thiamethoxam in reducing the oviposition damage, gall infestation and maximum per cent establishment of cuttings (Table 8). However, spraying is not a viable option due to repeated applications required to reduce gall incidence. Javaregowda et al. (2010) reported Invasive gall wasp (Leptocybe invasa) in eucalypt... 359

Mean Mean 1.34 1.34 20.83 20.83 11.39 11.39 16.11 17.83 53.06 (3.37) (3.37) (26.67) (26.67) (16.58) (16.58) (23.55) (23.52) (46.74) 0.05 0.05 4.17 4.17 23.33 23.33 10.00 10.00 33.33 45.83 19.45 (0.80) (0.80) (28.77) (28.77) (11.64) (11.64) (17.84) (35.13) (42.58) (22.73) 0.85 0.85 1.67 1.67 5.17 14.17 14.17 15.03 15.03 10.00 10.00 58.33 (3.30) (6.19) (21.43) (18.57) (17.84) (12.85) (49.78) C1 C2 C3 30.33 30.33 25.00 25.00 28.33 28.33 28.33 15.00 55.00 (6.41) (6.41) (29.81) (29.81) (28.43) (28.43) (31.91) (31.91) (32.00) (22.58) (47.86) 30.33 30.33 Mean Mean

1.95 1.95 20.83 20.83 56.11 56.11 11.95 11.95 15.83 20.06 (8.02) (8.02) (27.15) (27.15) (48.49) (48.49) (20.21) (20.21) (23.44) (26.59) 0.01 0.01 5.00 5.00 23.33 55.00 21.94 15.00 33.33 (0.40) (0.40) (28.87) (47.85) (24.68) (12.92) (22.78) (35.25) 0.84 0.84 1.67 1.67 9.17 5.17 14.17 14.17 58.33 58.33 14.89 (5.25) (5.25) (7.42) (7.42) (22.10) (22.10) (49.70) (49.70) (19.22) (17.62) (17.62) (13.13) C1 C2 C3C1 C2 5.00 5.00 25.00 25.00 29.17 29.17 23.33 21.67 55.00 26.52 Per cent establishment of establishment cent Per cuttings (12.92) (12.92) (29.99) (29.99) (32.67) (32.67) (28.87) (21.73) (47.86) (30.00)

Mean Mean 1.67 1.67 13.06 13.06 13.33 13.33 15.94 23.67 53.06 (4.34) (4.34) (20.19) (20.19) (17.84) (17.84) (21.96) (27.34) (46.74) 0.05 0.05 5.00 5.00 5.84 15.83 15.83 14.59 15.00 15.00 45.83 (0.40) (0.40) (23.04) (23.04) (18.85) (22.48) (22.48) (42.58) (10.74) (10.74) (13.90) 0.84 0.84 6.67 6.67 4.17 4.17 9.16 15 DAT 15 DAT 30 DAT 45 DAT 16.80 16.80 21.67 21.67 58.33 (3.30) (3.30) (9.74) (9.74) (14.47) (14.47) (20.13) (16.57) (16.57) (26.96) (49.78) 1.94 5.50 2.55 2.55 1.76 5.50 7.33 1.94 5.00 3.05 8.65 12.69 4.43 9.53 3.36 SEm± CD@ 5% SEm± CD@ 5% SEm± CD@ 5% 5% SEm± CD@ 5%SEm± CD@ SEm± CD@ C1 C2 C3 16.67 16.67 28.94 30.83 30.83 (9.31) (9.31) (23.08) (23.08) (30.21) (33.05) (33.05) (26.82) (41.15) (47.86)

Profenophos 50 EC 50 EC Profenophos 4.17 Dosage (D) (D) 1.81 1.24 3.89 5.18 1.37 3.53 For comparing of means Insecticides (T) Dosage Interaction (TXD) Thiamethoxam 25 Thiamethoxam WG Mean Treatment 17.8 Imidacloprid SL 20 SP Acetamiprid 23.67 SL 21.7 Thiacloprid 43.33 55.00 Control DAT – Days after Treatment. Figures in the parentheses are arc sine transformed values. Treatment. – Days after DAT Table 6. Effect of insecticide dip on establishment cuttings Table

360 A.S. Vastrad and S.H. Ramanagouda

8.69 6.95 33.13 33.13 10.55 10.55 53.68 53.68 Mean Mean (15.65) (15.65) (12.75) (12.75) (34.73) (34.73) (17.19) (17.19) (47.10) (47.10) 2.50 4.25 5.00 20.83 20.83 15.28 15.28 53.33 53.33 (7.47) (7.47) (11.79) (26.98) (12.91) (21.21) (46.90)

8.58 8.58 1.67 1.67 5.83 5.83 28.58 18.09 55.00 (4.57) (4.57) (16.90) (16.90) (32.24) (32.24) (11.57) (11.57) (22.63) (22.63) (47.86) (47.86) C1 C2C1 C3 15.00 15.00 15.00 20.83 29.55 29.55 52.73 (22.58) (22.58) (21.89) (21.89) (44.98) (44.98) (27.08) (27.08) (32.62) (32.62) (46.55) (46.55)

5.36 5.36 3.08 3.08 3.33 3.33 14.01 14.01 32.50 32.50 (5.86) (5.86) (6.28) (6.28) Mean (11.70) (11.70) (19.43) (19.43) (34.41) (34.41) Per cent infestation of cuttings of infestation cent Per 0.83 0.83 0.92 0.92 8.33 8.33 8.70 8.70 gall wasp 0.005 0.005 41.66 41.66 (3.30) (3.30) (3.45) (3.45) (0.40) (0.40) (13.86) (13.86) (40.08) (40.08) (12.22) (12.22) Eucalyptus 6.08 6.08 9.67 9.67 0.005 0.005 13.58 13.58 0.005 0.005 35.00 35.00 (0.40) (0.40) (0.40) (0.40) 30 DAT 30 DAT DAT 45 (14.22) (14.22) (17.88) (17.88) (36.11) (36.11) (13.81) (13.81) C1 C2 C3 9.16 9.16 8.33 20.12 20.12 10.00 10.00 20.83 20.83 13.32 13.32 (17.58) (17.58) (13.75) (26.54) (26.54) (18.04) (18.04) (27.02) (27.02) (20.59) (20.59)

Treatment Imidacloprid SL 17.8 Acetamiprid 20 SP Thiamethoxam Thiamethoxam 25 WG Thiacloprid Thiacloprid SL 21.7 Control Mean Mean Forcomparing of means (T) Insecticides (D) Dosage (TXD) SEm± Interaction 2.32 CD@ 5% 4.02 1.93 SEm± 6.63 11.48 5.51 CD@ 5% 1.69 2.93 1.40 4.82 8.36 4.01 DAT- Days after treatment. Figures in the parentheses are arc sine transformed values. DAT- Table 7. Effect of cutting dip on infestation by 7. Effect Table

Invasive gall wasp (Leptocybe invasa) in eucalypt... 361 Figures in the parentheses are transformed values. Table 8. Effect of insecticidal spray on gall incidence Table 362 A.S. Vastrad and S.H. Ramanagouda that foliar spray of imidacloprid 17.8 SL and spot application of carbofuran 10 G at root zone and were effective in reducing the number of fresh galls. Vastrad et al. (2011) reported that thiacloprid 21.7 SL (1 ml l-1) was most effective followed by imidacloprid 17.8 SL (0.3 ml l-1), acetamiprid 20 SP (0.2 g l-1) and thiamethoxam 25 WG (0.2 g l-1) in reducing adult emergence in nursery.

4.3. Biological Control with Native Parasitoids Classical biological control has been a preferred approach for management of alien insects. Though management of invasive pest can be ideally attempted through classical biological control, it works well if introduced during early part of invasion as it gives the best results. However, concerns have been raised about the risks of classical biological control (Howarth, 1991; Samways, 1997). Further, many exotic natural enemies have been released without considering the use of native species (van Lenteren et al., 2006). In the light of increasing evidence of non-target host use and resultant threat to native associated with it, the classical biological control needs to be weighed carefully (Louda et al., 2003). The literature is replete with many examples of native parasitoids exploiting the exotic hosts (Aebi et al., 2006; Cooper and Rieske, 2007). It is reported that the recently described M. dharwadicus is an efficient parasitoid of L. invasa in India (Vastrad et al., 2010). Contrary to this view, none of the Megastigmus spp. from Brazil, Israel, Thailand and Turkey appears to be an efficient natural enemy of Eucalyptus gall wasp (Protasov et al., 2008; Doganlar et al., 2013; Sangtongpraow et al., 2013). However, recent studies have shown the potential utility of native parasitoids to manage the invasive Eucalyptus gall wasp in green houses (Kulkarni et al., 2010). Therefore, an effort was made to utilize the native parasitiods for the biological control of invasive Eucalyptus gall wasp. In release and recovery studies, along with parasitoids adults of L. invasa were also released to maintain high gall density necessary for parasitization (Fig. 7). Hence the level of parasitization recorded in both the greenhouses where the release and recovery studies were made was less than expected during the initial period. Megastigmus species are generally phytophagous feeding on the seeds of Gymnosperms and Angiosperms and some develop inside fig and are believed to be gall formers on eucalypts. They also develop as parasites in the galls of and other insects (Narendran et al., 2007). An experiment was conducted to rule out the possibility of the M. dharwadicus and A. gala as gall inducer. No gall development was noticed even after two months of exposure to the parasitoids thus ruling out the possibility of M. dharwadicus and A. gala as gall inducers.

4.3.1. Release and recovery of parasitoids in greenhouse After recording the emergence of parasitoids from eucalypt samples collected during survey and seasonal incidence studies, they were collected with the help of aspirator Invasive gall wasp (Leptocybe invasa) in eucalypt... 363

a. L. invasa (female, F) b. L. invasa (male, M)

c. M. dharwadicus (F) d. M. dharwadicus (M)

e. A. gala (F) f. A. gala (M)

Fig. 7. Eucalyptus gall wasp (a and b) and its parasitoids (c to f). 364 A.S. Vastrad and S.H. Ramanagouda

Table 9. Release of parasitoids in greenhouse Month Greenhouse I Greenhouse II M. dharwadicus A. gala Total M. dharwadicus A. gala Total 2009 July 24 00 24 - - - August 42 49 91 - - - September 20 20 40 - - - October 28 01 29 - - - November 91 70 161 10 19 29 December 33 26 59 38 26 64

2010 January 18 04 22 19 13 32 February 177 39 216 93 44 137 March 128 07 135 1664 92 1756 April 00 00 00 48 19 67 May 00 00 00 107 28 135 Total 561 216 777 1979 241 2220

and released in green house at monthly interval (Table 9). Forty days after release, 10 random samples were collected at monthly interval and kept for adult emergence (pest and parasitoids) and per cent parasitization was worked out as mentioned in section 2. In control, no parasitoids were released. In the first greenhouse, a total of 777 parasitoids were released from July 2009 to March 2010, whereas in the second greenhouse, 2,220 parasitoids were released from November 2009 to May 2010. A total of 1,131 M. dharwadicus were recovered from 10 randomly collected samples from both greenhouses. Highest recovery (268) of M. dharwadicus and per cent para- sitization (57.6 %) was noticed during May, 2010. During June, 2010, 314 individuals of M. dharwadicus were recovered accounting for 36.1 per cent parasitization. Recovery of A. gala from both the greenhouse was 343 and parasitization ranged from 5.4 (October) to 15.4 per cent (March). Though maximum parasitoids (1979 M. dharwadicus and 241 A. gala) were released in the second greenhouse, no parasitoids were recovered during April and May 2010. This was due to the spraying experiments conducted during March and April 2010. Though no parasitoids were released during April 2010 in the first green- house, recovery of M. dharwadicus was maximum during May (268) and June (314) accounting for 57.6 and 36.1 per cent parasitization. In the control plot, where parasi- toids were not recorded till April, 42 individuals of M. dharwadicus were recovered accounting for 8.5 per cent parasitization. The migration of the released parasitoids from the second greenhouse where insecticide trial was conducted may have been the cause for the parasitization recorded in the control plot (Table 10). Invasive gall wasp (Leptocybe invasa) in eucalypt... 365 Per cent Per parasitization ------M A M A M Total A emerged emerged - L Per cent parasitization Total no. of adult emerged emerged 874 25 00 2.2700 874 25 0.00 2.78 Per cent parasitization Per of adults no. Total A. gala. A- dharwadicus;

emerged emerged M.

L M A M A Total L M A M A Total Total M A A M L M A Total L M A 555 314 00 36.13 0.00 36.13 36.13 36.13 5550.00 314 00 Total no. of adults Total no. M- L. invasa; Total 2,681 1,161 202 30.21 7.00 33.70 2,357 309 141 11.59 5.66 16.03 1,725 42 00 2.37 0.00 2.37 0.00 00 2.37 42 1,725 11.59 5.66 16.03 141 309 2,357 Total 30.21 202 7.00 33.70 1,161 2,681 Month Month 2009 Greenhouse I Greenhouse II Control September 159 - 45 - 28.57 - - 0.00 September5.40 25.53 22.05 - 105 36 06 0.00 0.00 - 00 - 00 22.05 October129 - - - November - 487 137 31 21.95 - 5.94 25.64 - December 401 144 - - 40 26.42 316136 9.07 - 00 31.91 85 0.0029.62 0.00 0.0013.18 48 21.19 2010 - - 112 00 0.00 0.00 0.00 269 January 76 - 40 251 22.89 30.12 61 12.14 26 19.55 125 9.38 00 0.00 25.73 0.00 0.00 - February225 00 0.00 0.00 0.00 332 89 53 260208 21.14 00 29.95 76 0.0030.29 March 13.76 0.00 0.0012.45 37 22.61 176 April 52 32 204148 22.80 00 32.30 62 0.0031.08 15.38 0.00 0.0012.82 30 23.30 May - - June 197 268 00 - 57.63 297 0.00 57.63 00 00 0.00 - 0.00 455 0.00 42 00 8.45 8.45 0.00 - - 155 00 00 0.00 0.00 187 0.00 00 0.00 0.00 0.00 L- Table 10. Recovery of parasitoids from greenhouses 10. Recovery of parasitoids from Table

366 A.S. Vastrad and S.H. Ramanagouda

Mendel et al. (2007) reported the release and recovery of chamaeleon in eucalypts for the management of another gall wasp, where C. chamaeleon is an efficient biological control agent in lowering the population density by parasitizing second and third instar gall wasp. A total of 210 S. kryseri adults and 670 Q. mendeli adults were liberated in three sites in the coastal plain and the first parasitoids were recovered four months after release and a total of 99 individuals of S. kryseri and 36 individuals of Q. mendeli were recovered from all three sites and found that they successfully parasitized approximately 2.2 and 2.5 gall units per days, respectively (Kim et al., 2008). Male and female M. dharwadicus lived for 2.33 and three days, respectively without food, whereas with food they survived for 2.7 and 3.7, respectively. The longevity of male and female of S. kryseri with food was 6.4+0.7 and 6.5+0.7 days, respectively. Whereas, Q. mendeli survived for 6.0+0.6 days (Kim et al., 2008). Protasov et al. (2008) reported that female of Megastigmus sp. I survived for 3 days without food and with food survival ranged from 4.5 to five days where as males of Megastigmus sp. I survival lasted for two to three days with and without food. Males and females of A. gala survived for two and three days, respectively without food and with food they lived for 2.3 and 2.7 days, respectively. To study the pattern of parasitoid emergence from different stage galls, 50 galls collected during August, February and May were enclosed in a perforated polythene bag. Since the different gall stages could not be separated due to overlapping occurrence of galls they were designated as II-III, III-IV and IV-V stages. The ratio of each gall stage was 80:20. The adult emergence was recorded daily till the cessation of adults and parasitization was worked out. Investigations on preferred stage for parasitization indicated that the parasitoids emerged from all gall stages except I stage. Maximum numbers of parasitoids were recorded from second-third and third-fourth stage galls (Table 11). During release and recovery studies, 1,470 and 343 individuals of M. dharwadicus and A. gala were, respectively recovered from 10 randomly selected samples. Number of branches per plant and the plant height was more in the greenhouse where parasitoids were released than control. The methodology adopted for the release and recovery studies may also be adopted for mass multiplication of these parasitoids.

4.3.2. Large scale field release of parasitoids for the management of gall wasp 4.3.2.1. Mass multiplication: Heavily galled seedlings supplied by the West Coast Paper Mills nursery were used for the mass multiplication. Parasitoids that emerged from eucalypts samples collected during the routine survey were released on six month old galled seedlings kept in the greenhouse for mass multiplication following Invasive gall wasp (Leptocybe invasa) in eucalypt... 367 Per cent cent Per parasitization emerged emerged No. of adult adult of No.

Per cent cent Per parasitization III-IV IV-V Gall stage stage Gall emerged emerged No. of adult adult of No.

Per cent cent Per parasitization A. gala. II-III II-III A- emerged emerged No. of adult adult of No. M. dharwadicus; L L M A M L A Total M A L M M A A Total M A Total 49 48.95 31 24.61 38.75 56 16 39 41 13.84 46.15 0941.05 67 16 07 44.62 46 10.66 28.57 35.9340.7014.58 59 08 41 40 23.37 50.00 1841.00 11 04 21.56 9.09 27.27 108 204 65.38 0.00 65.38 00 112 122 00 52.13 0.00 99 52.13 73 00 42.44 0.00 42.44 ; M- L. invasa L – Period Period of study Aug. 2009 Feb. 2010 May 2010 Mean 74.66 93.66 08 48.27 11.75 52.98 75.66 67.33 09 44.72 12.40 49.42 60 33.33 3.66 30.85 7.89 35.21 Table 11. Preference for different stage galls for parasitization stage galls for different for Preference 11. Table

368 A.S. Vastrad and S.H. Ramanagouda

the methodology of Kulkarni et al. (2010). A total of 2,305 M. dharwadicus and 82 A. gala were released in the green house between July 2010 and February 2011 (Table 12). Seedlings were kept in the green house for a minimum of 45 days before they were distributed in the plantation. Parasitized galled seedlings and the adult parasitoids collected from the greenhouse were used for field release. Before the

Table 12. Parasitoids released in the greenhouse for mass multiplication Month M. dharwadicus A. gala Total 2010 July 845 00 845 August 201 00 201 September 206 00 206 October 624 00 624 November 153 39 192

December 71 26 97

2011 January 07 17 24 February 198 00 198 Total 2,305 82 2,387

field release the extent of parasitization was ascertained from 25 randomly selected seedlings as described by Kim et al. (2008). 4.3.2.2. Field release: The release site belonging to the West Coast Paper Mills, Dandeli consisted of two to six years old clones mostly derived from E. tereticornis spread over an area of 1,000 ha in Kulwalli village (located between 15o 32’ 07.57" and 15o 34’ 06.52" N, 74o 47’ 34.04" and 74o 47’ 50.51 E). A total of 14,000 parasitised galled seedlings were distributed at 20 randomly selected spots between September 2010 and March 2011. In addition, 1,400 M. dharwadicus and 300 A. gala collected from the greenhouse were released in the centre of the plantation during October and November 2010 (Table 13). The impact of field release on gall incidence and per cent parasitization was recorded over a period of nine months. Galled samples were randomly collected from four locations on the day on which field release were made. In each location 30 centimeter apical shoots from ten eucalypts plants were randomly collected. The samples were equally divided into top, middle and bottom portion (10 cm each). Different gall stages were recorded on each sample as described by Mendel et al. (2004). Based on the Invasive gall wasp (Leptocybe invasa) in eucalypt... 369

Table 13. Number of parasitized galled seedling distributed and adult parasitoids released at Kulwalli during 2010-11 Month No. of Adult emergence Parasitization (%) No. of parasitoids parasitized from 25 randomly recorded on galled released galled selected galled seedlings before seedlings seedlings distribution L M A M A Total M A September 22, 2010 500 75 108 38 48.86 17.19 66.05 - - October 7, 2010 12,500 17 100 04 82.64 3.30 85.94 860 250 November 6, 2010 500 19 34 31 40.47 36.90 77.37 540 50 March 7, 2011 500 41 35 65 24.82 46.09 70.91 - - Total 14,000 152 277 138 48.85 24.33 73.19 1,400 300 L- L. invasa; M- M. dharwadicus; A- A. gala.

number of gall stages recorded mean gall incidence was worked out for each spot from 30 samples. Later, the samples were kept in pin holed polythene bags for pest and parasitoid emergence. Adult emergence of the pest and parasitoids was recorded daily till the cessation of adult emergence. During the initial period though the decline in gall incidence was negligible, the par cent parasitization increased from 42.9 to 53.1 per cent. Among the two parasitoids released, M. dharwadicus was the most dominant and mainly responsible for reduction in pest incidence. True impact of parasitoid release was clearly evident three months after third release indicated by substantial reduction in the number of galls (4.6 galls/ 30 cm shoot) coupled with significant increase in per cent parasitisation (95.0%). Faster turnover of parasitoid generation (~ 45 days) compared to the pest (~120 days) seems to have contributed to the overwhelming of the pest by the parasitoids. By the end of June 2011, no fresh oviposition damage and gall incidence was noticed resulting in spectacular control of the pest. Though the native parasitoids were recorded as early as 2008, their impact on gall incidence was not discernible. Despite 42.9 per cent parasitization recorded in the beginning of the study, gall incidence still remained high (16.5 galls/30 cm shoot) three months after the first release. The augmentative biological control through repeated field releases of parasitoids resulted in successful control of the pest (Table 14). Post release evaluation conducted during May 2012 revealed that there was no resurgence of the pest one year after the last field release. This is a rare example of the native parasitoids halting the ravages of an invasion resulting in substantial financial savings on control measures that also avoided the large scale negative environmental impact due to use of insecticides. The present study also highlights the importance of considering the use of native parasitoids before embarking on classical biological control. 370 A.S. Vastrad and S.H. Ramanagouda Per cent cent Per parasitization parasitization M A Total M A (March 2011)(March Fourth release release Fourth Number Number cm sho ot ot sho cm of galls/30

Per cent cent Per parasitization M A Total M A Third releaseThird (November 2010) Number of of Number galls/30 shoot cm

Cumulative A. gala. A- Top portion of the sample sample the of portion Top Middle portion of the sample sample the of portion Middle Bottom portion of the sample of portion Bottom Per cent cent Per parasitization M A Total M A M. dharwadicus; Second release release Second (October 2010) 2010) (October shoot shoot Number of of Number galls/30 cm cm galls/30

Per cent cent Per parasitization parasitization M A Total M A 42.86 0.00 42.8642.860.00 15.40±2.85 0.00 48.27 48.27 16.30±2.51 53.12 0.00 53.12 4.55±3.35 90.09 4.95 95.04 * (September 2010) 2010) (September shoot shoot On theOn of day first release 9.80±1.66 0.000.000.00 13.80±1.32 0.000.00 0.00 9.80±1.66 0.00 0.00 0.00 2.30±1.307.80±1.40 0.00 0.00 0.00 47.61 47.610.00 7.80±1.60 0.00 31.81 31.81 7.80±1.40 47.61 0.00 47.61 3.10±2.15 90.09 4.95 95.04 4.60±1.35 35.00 35.000.00 5.20±1.16 0.00 58.33 58.33 4.60±1.35 65.00 0.00 65.00 1.40±1.01 0.00 0.00 0.00 Number of of Number galls/30 cm cm galls/30 16.50±1.66 * Means of 30 samples from top, middle and bottom. M- Table 14. Gall incidence and per cent parasitization before and after release of parasitoids at Kulwalli during 2010-2011 release and after cent parasitization before 14. Gall incidence and per Table Invasive gall wasp (Leptocybe invasa) in eucalypt... 371

4.4. Host Plant Resistance An important strategy to manage pests not amenable for insecticidal control is the exploitation of host plant resistance. With more than 800 species of eucalypts (Myrtaceae) there is ample scope to exploit resistance in host plants to manage the invasive gall wasp. A wide array of chemical substance including inorganic chemicals, primary and intermediary metabolites and secondary substance are known to impart resistance to a wide variety of insect pests. Among the secondary plant metabolites, phenolics are the source of resistance against phytophagous insects and are ubiquitous in plants (Harborne, 1994; Jacob, 2009). Total phenol, reducing sugar and protein were estimated from a total of 48 eucalypt genotypes. Total phenol was inversely related to the gall incidence while no definite relationship between the gall incidence and reducing sugar and protein content was evident. Among 48 eucalypt genotypes screened, 13 were classified as highly susceptible, two were susceptible, 22 were tolerant, two were resistant and nine were immune (Table 15). In general, E. tereticornis and E. camaldulensis were susceptible to Eucalyptus gall wasp. Wide variation in phenol content ranging from 41.7 to 131.0 mg/g among 21 genotypes of E. tereticornis was noticed. Among the 21 genotypes of E. tereticornis, two were immune (C-2N and SRO-16), one was susceptible (C-4), four were highly susceptible (K-14, C-5, KN-13 and C-9) and remaining clones were tolerant. Phenol content in the genotypes of E. camaldulensis ranged from 40.67 (C-411) to 141.3 mg g-1 (C-526). Varied susceptibility of genotypes is attributed to highly cross pollinated nature of eucalypt plants. Therefore, in natural vegetation within the same stand some trees were severely affected while others were totally free from gall wasp damage. E. pellita (BP-6 and P-1) and E. urophylla clones (BC-350, EU-35 and EU-3) though suitable for oviposition exhibited no further gall development. Clone Mu - 8P was immune with highest (133.3 mg g-1) phenol content. Amongst the 10 commercial Eucalyptus hybrids, one was immune, five were tolerant and four were highly susceptible. Lowest gall incidence (0.00 to 1.00) was noticed in all the eucalypt genotypes above two years. Among the six species, four were immune and two were tolerant.

4.4.1. Influence of plant age on biochemical parameters of E. tereticornis Eucalyptus gall wasp is a pest of young coppice and nursery seedlings and its incidence on trees older than two years is rare. The effect of plant age on biochemical constituents was studied. A direct relationship between plant age and phenol content was noticed with an exception at 30 days after new growth. Total phenol content gradually increased over a period of time from 61.0 to 79.0 mg g-1 (Fig. 8). Early bud sprout recorded highest (65.0 mg g-1) reducing sugar which declined gradually and remained more or less constant during subsequent 372 A.S. Vastrad and S.H. Ramanagouda

Table 15. Biochemical parameters of eucalypt genotypes Genotype/ Biochemical parameter (mg g-1) No. of Rating clone galls/10 cm Total Total Reducing terminal phenol protein sugar shoot E. tereticornis ERK-4 82.67±1.52 92.67±1.15 25.00±1.00 2.00 Tolerant C-290 92.00±1.00 53.00±1.00 73.00±1.00 1.50 Tolerant K-16 92.67±0.57 55.00±1.00 81.33±0.57 1.20 Tolerant C-105 92.33±0.57 97.33±0.57 24.00±1.00 1.00 Tolerant K-21 94.00±1.00 101.33±1.52 62.00±1.00 1.00 Tolerant C-2135 96.33±0.57 93.33±0.57 34.33±0.57 1.32 Tolerant C-130 99.33±0.57 95.00±1.00 24.33±0.57 1.30 Tolerant C-288 100.67±1.15 95.00±1.00 68.33±0.57 1.40 Tolerant C-290 102.00±1.00 87.67±0.57 24.00±1.00 2.00 Tolerant C-6 103.33±0.57 85.00±1.00 31.67±0.57 1.20 Tolerant C-2 105.67±0.57 92.33±0.57 22.33±1.52 1.00 Tolerant C-316 105.33±0.57 91.67±1.52 112.67±0.57 1.20 Tolerant C-213 108.00±1.00 94.33±0.57 94.00±1.00 1.30 Tolerant K-11 111.33±1.52 103.33±1.52 81.00±1.00 1.40 Tolerant C-2N 117.67±0.57 85.67±0.57 23.33±0.57 0.00 Immune SRO-16 131.00±1.00 94.33±1.52 41.67±0.57 0.00 Immune E. camaldulensis C-2045 115.00±2.00 87.33±1.52 34.67±1.52 2.00 Tolerant C-526 141.33±1.52 93.33±1.52 48.00±1.00 0.00 Immune E. pellita BP-6 74.33±1.15 85.33±0.57 14.00±1.00 5.00* Resistant P-1 93.00±1.00 86.00±1.00 23.67±1.52 3.00* Resistant Mu-8P 133.33±2.08 97.67±1.15 36.00±1.00 0.00 Immune Hybrids E. camaldulensis 72.67±1.52 79.00±1.00 13.67±1.52 1.00 Tolerant x E. deglupta E. camaldulensis 98.33±0.57 77.33±1.52 24.33±0.57 1.60 Tolerant x E. pellita E. tereticornis 100.67±1.15 101.00±1.00 36.33±1.52 2.20 Tolerant x E. urophylla Contd. on next page… Invasive gall wasp (Leptocybe invasa) in eucalypt... 373

...Contd. from previous page SA – 30 110±1.00 96.67±1.15 52.67±0.57 1.00 Tolerant C – 2306 127.33±1.52 78.33±0.57 33.00±1.00 0.00 Immune E. urograndis 133.00±1.73 115.67±0.57 56.00±1.00 2.20 Tolerant Genotypes (grown up trees >2 years) E. tereticornis 83.67±0.57 97.67±0.57 65.00±1.00 1.00 Tolerant Corymbia 86.67±1.52 62.00±1.00 44.00±1.00 0.00 Immune torelliana C. citriodora 92.00±1.00 85.00±1.00 26.67±0.57 0.00 Immune E. pellita 121.00±1.00 92.67±0.57 45.33±0.57 0.00 Immune E. grandis 122.33±1.15 118.67±0.57 52.67±0.57 1.00 Tolerant E. crebra 122.67±1.52 100.33±0.57 45.67±0.57 0.00 Immune Immune- no oviposition; Resistant- Oviposition observed but no gall development; Tolerant- 1 to 2 galls; Susceptible- 3 to 5 galls; Highly susceptible- >5 galls (10 cm terminal shoot). mg/g

Stage A-Bud sprout; B-15 days after bud sprout; C-30 days after bud sprout; D-45 days after bud sprout; E- 60 days after bud sprout; F-days after bud sprout; G-120 days after bud sprout; H-150 days after bud sprout; I-180 days after bud sprout; J-210 days after bud sprout; K-240 days after bud sprout; L-270 days after bud sprout; M-300 days after bud sprout; N-330 days after bud sprout Fig. 8. Variation in bio-chemical parameters at different growth stages of E. tereticornis. growth stages. However, total protein (65.7 mg g-1) was significantly lower during the bud sprout stage which gradually increased from 65.7 to 78.3 mg g-1 with the advancing age of the plants.

4.4.2. Biochemical parameters from different leaf portions of E. tereticornis L. invasa is considered to