Available on-line at http://www.bjbabe.ro Banats Journal of Biotechnology 2012, III(6),

OTIORRHYNCHUS LIGUSTICI L. (COLEOPTERA: ) I. COMPARATIVE STUDY OF THE RESISTANCE OF ALFALFA VARIETIES

DOI: 10.7904/2068 – 4738 – III(6) – 68

Ivelina NIKOLOVA, Nataliya GEORGIEVA

Institute of Forage Crops, Pleven, Bulgaria E–mail: [email protected]

Abstract: From studies carried out during the period 2006–2009 in the experimental field of the IFC–Pleven with 8 alfalfa varieties included in the Official variety catalog of Bulgaria (2005): Europe (France), Prista 2 (Obraztsov Chiflik, Rouse), Prista 3 (Obraztsov Chiflik, Rouse), Prista 4 (Exemplary Chiflik, Rouse), Obnova 10 (EFC, Pleven), Pleven 6 (EFC, Pleven), Dara (EFC, Pleven) and Multifoliate (Obraztsov Chiflik, Rousse) it was found that in the year of the establishment of the stand there was partial damage from Otiorrhynchus ligustici. It was found that surface of caused damage was a major sign to determination of the degree of infestation from the pest as it increased from second to fourth year on average from 5.6 to 42.1 cm2. Varieties of Multifoliate, Europe and Prista 2 were the least preferred and with low values of surface of damage, number and length of the gnawed furrows. These varieties can be used as sources of germplasm resistant to Otiorrhynchus ligustici.

Keywords: alfalfa, varieties, damage, Otiorrhynchus ligustici

Introduction hormones SKENE, 1975 and plant stability, Otiorrhynchus ligustici L. is resulting in lodging GRAY and TOLLEFSON, 1987. widespread across Europe. The main method for control against The species is a polyphag and that pest is the chemical one, but it is causes serious damades not only on expensive and not always effective alfalfa, but also red clover, sainfoin, sugar WILLSON et al., 1976. With the development of beet, pea, vetch, vine LIKVENTOV, 1949; TRIBEL, organic farming as an alternative of 1969; ČAMPRAG, 1973; PISAREK, 2001; ČAMPRAG, 2005; chemical plant protection increasingly SHEBL et al., 2009. importance and priority in the researches The high population density disturbs of many researchers hold the strongly the density of alfalfa stands development of environmentally friendly (occurs thinning) which impose their early methods and means for control against plowing. Otiorrhynchus ligustici–biological control, Strongly attacked stands can be resistant varieties, bioinsecticides and destroyed for one–two years and at a others POPOVA, 1968; GRIGOROV, 1972; LEATH and moderate infestation the permanence of HOWER, 1993, NEUMANN, 2007; NEUMANN and SHIELDS, alfalfa reduces to two–three years PENG, 2008; SCHROEDER et al., 1995. 1994; SHIELDS et al., 2009. The object of the present study was The potential problems to host to to make a comparative study of alfalfa plants arising from root attack by varieties in regard to the degree of include: drought stress to the plant infestation (attack) of Otiorrhynchus caused by pruning of the root system ligustici L. with purpose finding of stable ANDERSEN, 1987; HOU et al., 1997, loss of plasma for alfalfa selection. reproductive output DUTCHER et. al., 1979 and an increase in the plant’s susceptibility to Material and methods infection by soil–borne pathogens JIN et al., The studies was carried out during 1992; CAESAR, 2003. Further, root–feeding by 2006–2009 period in the experimental insects may directly reduce food reserves field of IFC–Pleven with 8 alfalfa varieties such as carbohydrates DINTENFASS and BROWN, included in the Official variety catalog of 1988, synthesis of numerous growth Bulgaria (2005): Europe (France), Prista 2 68 Banat’s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Contact: web: http://www.bjbabe.ro, e-mail: bjb@usab–tm.ro

(Obraztsov Chiflik, Ruse), Prista 3 data were statistically processed with the (Obraztsov Chiflik, Ruse), Prista 4 programme product Statgraph. (Obraztsov Chiflik, Ruse), Obnova 10 (IFC, Pleven), Pleven 6 (IFC, Pleven), Results and discussion Dara (IFC, Pleven) and Multifoliate Otiorrhynchus ligustici developed (Obraztsov Chiflik, Ruse). one generation per year and wintered as The trial was conducted on slightly imago and larva at depth of 30–40 сm. leached chernozem, non irrigated Overwintered imagos gnawed conditions and natural population density leaves, buds and top parts of plants. of Otiorrhynchus ligustici (from 0.2 to 0.6 These damages had no economic 2 imago/m for 2007–2009 period). importance. The main damage was The sowing was carried out by the caused by the larvae which gnawed deep block method at row spacing 11.5 cm and longitudinal furrows on alfalfa roots and sowing rate 2.5 kg/da, in 4 replications. hindered its growth and development. In The alfalfa was harvested only for the year of the establishment of the stand forage. The degree of damage from (2006) it is not found damages on the root Otiorrhynchus ligustici was established by system of plants from Otiorrhynchus taking soil monoliths 20х200х40 cm ligustici. Single furrows are established (width/length/depth). It was reported the only in autumn regrowth in varieties of following indicators: number, length and Prista 2 and Multifoliate. In the study width of the gnawed furrows/root, period the percentage of damaged plants diameter of the root collar. The area of by pest increased on average from 23.9% damage was calculated based on length in 2007 to 79.8% in 2009 (Table 1). and width of gnawed furrows. The obtain Table 1. Degree of infestation by Otiorrhynchus ligustici L. at different vareties of alfalfa % plants with damaged root system Varieties 2007 2008 2009 Average 1 2 3 4 1 2 3 4 1 2 3 4 2007–2009 Europe 10.0 30.0 30.6 23.5 31.0 68.0 71.0 56.7 73.0 80.4 88.0 80.5 53.6 Prista 2 11.9 21.2 24.8 19.3 35.0 71.0 76.0 60.7 76.0 77.3 79.2 77.5 52.5 Prista 3 4.1 37.8 38.6 26.8 40.0 71.0 73.0 61.3 74.0 83.2 86.8 81.3 56.5 Prista 4 3.8 25.4 37.6 22.2 66.0 74.0 76.0 72.0 77.6 78.8 94.3 83.6 59.3 Obnova 10 4.4 30.4 35.4 23.4 42.0 70.0 71.0 61.0 71.7 84.9 86.1 80.9 55.1 Pleven 6 7.0 33.1 35.7 25.3 37.0 72.0 72.0 60.3 72.6 79.1 83.3 78.3 54.6 Dara 5.3 38.4 41.5 28.4 58.0 69.0 74.0 67.0 78.2 81.2 88.0 82.5 59.3 Multifoliate 6.2 29.5 29.9 21.9 30.0 52.0 54.0 45.3 68.2 75.0 77.8 73.7 47.0 Average 6.6 30.7 34.3 23.9 42.4 68.4 70.9 60.5 73.9 80.0 85.4 79.8 54.7 Legend: 1– spring regrowth; 2–summer regrowth; 3–autumn regrowth; 4–average

During the year the values of that The results from the comparative parameter increased from the spring analysis of the studied alfalfa varieties at regrowth to autumn one as the most different regrowths showed that the essential increase to the previous percentage of damaged roots by the larva regrowth was observed in summer of Otiorrhynchus ligustici was similar and regrowth–on average by 18.7% (by 24.1% took values close to the average of the in 2007 and by 26.0% in 2008). trial. An exception was observed in 2008 In the last year of study the when the value of the considered damaged plants by Otiorrhynchus ligustici parameter in Prista 4 exceeded the in the spring regrowth exceeded 70% and average value for the trial by 11.5%, and in the next regrowths their number in Multifoliate the values was by 15.2% increased less in compare with the lower. The considered parameter was not regrowths in 2007 and 2008. enough precision to determine the degree of infestation by Otiorrhynchus ligustici. It 69 Available on-line at http://www.bjbabe.ro Banats Journal of Biotechnology 2012, III(6),

determined what part of plants had of damage increased almost eightfold in damaged root system but it did not reflect compare with 2007 when the surface of the degree of damage. damage was slightly expressed and The main sign for determination of because of low population density of pest the degree of larva infestation by (Table 2). Significantly a higher value was Otiorrhynchus ligustici on root system of observed in 2008 as the larva damage plants was the surface of caused increased nearly fourfold compared to the damage. The values of studied parameter previous year. increased consistently over the years on The largest surface and twofold average from 5.6 to 42.1 cm2 as during increase of that parameter was found in the last year of alfalfa growing the surface the last year from the growing of stands. Table 2. The surface of damage on root system by Otiorrhynchus ligustici L. in different varieties of alfalfa Surface of damage /10 roots, cm2 Average Varieties 2007 2008 2009 2007– 1 2 3 4 1 2 3 4 1 2 3 4 2009 Europe 3.08abc 5.25ab 6.30b 4.88 7.09b 24.31bc 24.40b 18.60 32.66c 34.22b 38.42a 35.10 19.53 Prista 2 2.80ab 5.95bc 6.50bc 5.08 7.41b 22.01b 27.48cd 18.97 31.66b 33.72b 42.30b 35.89 19.98 Prista 3 3.48c 7.75f 8.70d 6.64 11.52c 24.30bc 28.79ef 21.54 33.60d 46.47e 61.28de 47.12 25.10 Prista 4 3.20bc 7.35ef 9.50e 6.68 16.01e 24.85bc 29.78fg 23.55 35.62f 45.21d 59.04d 46.62 25.62 Obnova10 3.40bc 7.00de 7.10c 5.83 7.18b 23.42b 26.69c 19.10 34.56e 44.31d 66.85f 48.57 24.50 Pleven 6 2.80ab 6.60cd 7.00bc 5.47 7.72b 29.93d 30.59g 22.75 36.50f 37.86c 41.90b 38.75 22.32 Dara 2.47a 6.65cde 8.40d 5.84 12.75d 26.90c 28.23de 22.63 33.28cd 47.92f 56.53c 45.91 24.79 Multifoliate 3.18bc 5.20a 5.50a 4.63 5.64a 13.49a 19.91a 13.01 24.00a 31.25a 61.83e 39.03 18.89 Average 3.05 6.47 7.38 5.63 9.42 23.65 26.98 20.02 32.74 40.12 53.52 42.12 22.59

* Values with different letter are differed significantly at LSD0.05 Legend: 1– spring regrowth; 2–summer regrowth; 3–autumn regrowth; 4–average During the years of study the increased in less degree–average by populations of О. ligustici were in relation 20.5% to summer regrowth. among themselves and were in relation to The degree of infestation on root the age of alfalfa determined from the system of studied varieties varied in biology of culture. О. ligustici wintered as certain limits over the years. In the adult and larva in the stands and with the second year of alflfa growing (2007) with aging of the stands its numbers increased the least surface of damage as in the respectively the surface of damage. different regrowth, and on average for the The gnaws on root system of plants year was distinguished Multifoliate (4.63 in the spring regrowth were by cm2) followed by Europe (4.88 cm2) and overwintered larvae of О. ligustici which Prista 2 (5.08 cm2) respectively by 17.8, activated in the spring and feeded to the 13.4 and 9.7% lower values from the moment of turn into a pupa (the second average for the year. An analogical half of May and first half of June). tendency was observed in 2008 as the In next regrowths the damages decrease in Europe and Prista 2 was caused by the larvae were in significantly respectively by 7.1 and 5.3%. higher degree and they due to the Variety of Multifoliate distinguished individuals from new generation. significantly from the rest varieties with The surface of damage increased in the lowest degree of damage–13.01 cm2 ascending order from the spring regrowth at average value from 20.02 cm2 and by to the autumn one on average by 130.6%. 35.0% lower value from the average one The most active was the feed for the year. Some change in the activity of larvae in the summer regrowth sequence of these varieties was observed when the surface of damage increased on in the last year of growing which had no average by 95.2% in compare with the significant influence on the final results. spring regrowth. At the autumn regrowth The least affected was Europe the value of analyzed parameter (35.10 cm2), followed by Prista 2 (35.89

70 Banat’s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Contact: web: http://www.bjbabe.ro, e-mail: bjb@usab–tm.ro

cm2), Pleven 6 (38.75 cm2) and Obnova 10 during the third year of stand Multifoliate (39.03 cm2). growing when the surface of furrows had On average for 2007–2009 periods values under the average ones for the the least preferred and with low values of year as in the fourth year the parameter surface of damage were Multifoliate, was with the highest value (on average Europe and Prista 2 which were by 16.4, 48.57 cm2). 13.6 and 11.5% under the average value The possible reason for that was for the study period. the biological development of the variety The mathematical processing of related to weakly express reducing of the results showed that in variety of stand in 2008 compared to 2009 when it Multifoliate there was a significant lower was significantly stronger and created difference of the studied parameter to all favourable conditions for feeding of the rest varieties in the autumn regrowth larvae. It was found that the strong of 2007 as in the regrowths of 2008 and thinning of stands was related with high 2009. In the spring regrowth of 2007 in degree of infestation on root system by Multifoliate there was significance to Dara Otiorrhynchus ligustici. and in the summer regrowth–in all the rest Significantly with the largest surface varieties with exception of Europe. of damage in more regrowths during the The varieties of Europe and Prista 2 years distinguished Prista 4, Prista 3 and were with significantly lower values to the Dara to the other studied varieties. An same in the other varieties in most important parameter complementary the regrowths during the years as especially surface of damage was the number of demonstrative was the last year of study. gnawed furrows on the root system (Table Strongly damaged by О. ligustici 3). In different years it increased on were Prista 4, Prista 3, Dara and Obnova average from 4.5 to 28.2 number/10 roots 10 which had leading positions in the and during the study period it increased separate regrowths and years with large sixfold (Table 2). surface of damage whose values The most expressed increase from increased respectively from 6.68, 6.64, the spring regrowth to the autumn one 5.84 and 5.83 cm2 on average for 2007 to was observed in 2007 (nearly eightfold) 46.62, 47.12, 45.91 and 48.57cm2 on followed by twofold increase in 2008 and average for 2009. onefold in 2009. The degree of increase The varieties exceeded the average of the density of furrows decreased as value for the study period respectively by simultaneously the degree of increase of 13.4, 11.1, 9.8 and 8.5%. The variation in their length rose with the development of the degree of damage was observed in stands during different years. Table 3. Density of furrows on the root system in alfalfa varieties Number furrows/10 roots Varieties 2007 2008 2009 Average 1 2 3 4 1 2 3 4 1 2 3 4 2007–2009 Europe 1.7c 4.4ab 5.7ab 3.9 6.2a 17.2d 18.4b 13.9 23.2bc 29.0cd 29.2b 27.1 15.0 Prista 2 1.0b 3.6a 4.6a 3.1 5.8a 15.9bc 19.1bc 13.6 22.6b 25.6b 28.1a 25.4 14.0 Prista 3 0.4a 7.1e 7.6cde 5.0 9.3c 16.4cd 21.4e 15.7 24.3d 29.7d 33.3d 29.1 16.6 Prista 4 0.6ab 3.6a 8.7e 4.3 14.6f 19.0f 20.4d 18.0 26.1e 31.8e 34.9e 30.9 17.7 Obnova 10 0.9ab 6.2cd 6.5bc 4.5 11.3e 18.2e 19.7cd 16.4 27.1f 30.9e 39.2g 32.4 17.8 Pleven 6 1.0ab 5.3bc 7.8de 4.7 8.3b 15.2b 20.0d 14.5 23.6cd 25.7b 29.3b 26.2 15.1 Dara 0.9ab 6.7e 8.0de 5.2 10.3d 15.6bc 21.8e 15.9 26.1e 28.1c 36.8f 30.3 17.1 Multifoliate 1.1bc 6.2cd 7.6cd 5.0 7.8b 11.2a 13.7a 10.9 19.5a 22.5a 31.2c 24.4 13.4 Average 0.9 5.4 7.1 4.5 9.2 16.1 19.3 14.9 24.1 27.9 32.8 28.2 15.9 * Values with different letter are differed significantly at LSD0.05 Legend: 1– spring regrowth; 2–summer regrowth; 3–autumn regrowth; 4–average The values of analysed parameter 2 and Europe respectively by 15.7, 11.9 corresponded with the surface of and 5.7% under the average value. High damage. With the lowest density of values was established in varieties of furrows on average for 2007–2009 Obnova 10, Prista 4, Dara and Prista 3 periods distinguished Multifoliate, Prista

71 Available on-line at http://www.bjbabe.ro Banats Journal of Biotechnology 2012, III(6),

which exceeded the average value of the the same parameters in 2008 and 2009 trial by 11.9, 11.3, 7.5 and 4.4%. (respectively r=+0.837 and r=+0.866). These varieties distinguished with In anlysing of the results related to significant higher values to the values in the diameter of root collar in regard to the the rest varieties during nearly all infestation by O. ligustisi it was regrowths over the years as the most established that the plants with larger demonstrative in this respect was 2008 diameter of root collar were more and 2009. In the autumn regrowth of preferred from the pest and respectively 2009 was established statistical with larger surface of damage. significance of the differs among the It was existed moderate positive different varieties with exception of correlation between surface of damage Europe and Pleven 6. It was found and diameter of root collar (r=+0.400) in correlation with low positive value 2007, low positive one–in 2008 between the surface of damage and (r=+0.109) and strong positive one during number of furrows (r=+0.302) in 2007 the last year of alfalfa growing and strong positive correlation between (r=+0.853)–Table 4. Table 4. Dependence between diameter of the root collar and density of damage in alfalfa varieties 2007 2008 2009 Varieties 1 2 1 2 1 2 Europe 3.71 4.88 5.93 18.60 7.29 35.10 Prista 2 4.24 5.08 6.10 18.97 7.17 35.89 Prista 3 4.05 6.64 6.41 21.54 7.82 47.12 Prista 4 4.54 6.68 6.35 23.55 8.13 46.62 Obnova 10 4.81 5.83 6.58 19.10 8.36 48.57 Pleven 6 4.48 5.47 6.23 22.75 7.34 38.75 Dara 4.00 5.84 6.20 22.63 7.49 45.91 Multifoliate 4.03 4.63 6.28 13.01 7.55 39.03 r 0.400 0.109 0.853 1–diameter of root collar, cm; 2–surface of damage/10 roots, cm2

According to HAKL et al., 2007 strong The value of the parameter in the pronounced differences in the morphology autumn regrowth increased in significantly of the root system among alfalfa varieties lower degree–on average by 15.9% and particularly diameter of tap root was compared to the summer regrowth. established during the last year of the There was difference in the length development of stands. In the fourth year of the furrows between different varieties. were the most demonstrative and strong Less length was observed in Europe, pronounced the preferences of O. ligustisi Prista 2 and Multifoliate respectively by to some alfalfa varieties as a result of that 13.5, 9.2 and 9.2% under the average the interdependence among the studied value for the period. That trend was parameters was the greatest. observed in different regrowths and years Another parameter studied in the and corresponded to the surface of present investigation was the length of the damage. In these varieties was observed gnawed furrows (Table 5). significantly lower differences to the other The increase of the degreeof varieties in more regrowts over the years damage over the years was related to (especially demonstrative was the last prolongation of the furrows as average for year–2009). In the rest of varieties the the study period the values of the length of furrows exceeded the average parameter increased fourfold from 6.0 to for the trial from 2.8 to 9.9%. 21.8 cm/10 roots. In the result of the most It was established a moderate active feed activity of the larvae of O. positive dependence between the length ligustisi in the summer regrowth was of furrow ant the surface of damage in observed and the most pronounced rise 2007 and 2008 (respectively r=+0.679 of the length of furrows–on average by and r=+0.472) and strong positive 94.7% to the spring regrowth. dependence in 2009 (r=+0.953). 72 Banat’s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Contact: web: http://www.bjbabe.ro, e-mail: bjb@usab–tm.ro

The length and number of furrows on the degree of damage but between were complementary each other them not always was observed directly parameters which influenced complexly proportional dependence. Table 5. Length of gnawed furrows on root system in alfalfa varieties Length of furrows/10 roots, cm Average Varieties 2007 2008 2009 2007– 1 2 3 4 1 2 3 4 1 2 3 4 2009 Europe 1.6ab 5.1a 6.3a 4.3 11.9b 13.3a 14.1a 13.1 15.8a 19.1a 22.7a 19.2 12.2 Prista 2 0.9a 6.4b 7.4ab 4.9 13.0b 15.3a 17.4b 15.2 17.7abc 18.1a 18.9a 18.2 12.8 Prista 3 2.0bc 7.0bc 9.2bc 6.1 13.7b 15.5ab 16.0ab 15.1 18.0bc 23.3b 29.3b 23.5 14.9 Prista 4 2.6cd 7.9cd 10.0c 6.8 11.2ab 15.5ab 15.0a 13.9 16.9ab 27.6c 31.9b 25.5 15.4 Obnova 10 3.1d 8.6d 8.9bc 6.9 10.0a 14.7a 14.8a 13.2 18.4bc 25.3bc 29.7b 24.5 14.8 Pleven 6 2.2bcd 7.5bcd 10.6c 6.8 13.9b 18.0b 19.5c 17.1 19.5cd 19.6a 19.8a 19.6 14.5 Dara 2.8cd 8.1cd 10.2c 7.0 12.5b 14.7a 17.5bc 14.9 21.0d 24.2b 28.8b 24.7 15.5 Multifoliate 1.3ab 6.3b 8.1abc 5.2 11.5ab 13.5a 16.0ab 13.7 16.4ab 19.1a 23.1a 19.5 12.8 Average 2.1 7.1 8.8 6.0 12.2 15.1 16.3 14.5 18.0 22.0 25.5 21.8 14.1

* Values with different letter are differed significantly at LSD0.05 Legend: 1– spring regrowth; 2–summer regrowth; 3–autumn regrowth; 4–average

Demonstrative in this relation was Europe and Prista 2. The most damaged variety of Prista 4. The length of the varieties were Prista 4, Prista 3 and Dara furrows in that variety in 2008 was under in which the surface of damage, number the average value for the year and and length of furrows were the greatest. corresponded with the highest established With good tolerance to density of furrows. Otiorrhynchus ligustici were the varieties Analogical were the data in 2007 of Europe, Prista 2 and Multifoliate which when less number of gnawed furrows was could be used as sources of resistant compensated by the high values of germplasm in the alfalfa selection. length. Inversely proportional dependence between the two parameters and References relatively large area of damage was found 1. Andersen D. C.; Below–ground herbivory in Obnova 10 in the second year of the in natural communities: a review growing of alfalfa. There was directly emphasizing fossorial , proportional dependence between the Quarterly Reviews of Biology, 1987, length of furrows and their number. 62, pp. 261–286. 2. Caesar A. J.; Synergistic interaction of soilborne plant pathogens and root– Conclusions attacking insects in classical biological In the year of the establishment of control of an exotic rangeland weed, alfalfa stand Otiorrhynchus ligustici L. Biological control, 2003, 28, pp. 144– caused single damages on the plants. 153. During next years of growing the 3. Čamprag D.; Alfalfa snout degree of damaged plants increased on (Otiorrhynchus ligustici L.). Plant average from 23.9% to 79.8%. A main Doctor, 2005, 33 (5), pp. 496–500, sign for determination of the degree of ISSN 0354–6160. infestation from the larvae of 4. Čamprag D.; Štetočine šećerne repe. Otiorrhynchus ligustici on the root system Poljoprivredni fakultet, 1973, Novi Sad. was the surface of the caused damage 5. Dintenfass L. P.; Brown G. C.; Influence which increased from the second to fourth 2 of larval clover root curculio year on average from 5.6 to 42.1 cm . (Coleoptera:Curculionidae) injury on The surface of damage increased in carbohydrate reserves and yield of ascending order from the spring regrowth alfafa, Journal of Economic to the autumn on average by 130.6%. Entomology, 1988, 81, pp. 1803– Least preferred varieties by 1809. Otiorrhynchus ligustici was Multifoliate, 73 Available on-line at http://www.bjbabe.ro Banats Journal of Biotechnology 2012, III(6),

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