THESES OF DOCTORAL DISSERTATION Tamás Németh KESZTHELY 2012
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THESES OF DOCTORAL DISSERTATION UNIVERSITY OF PANNONIA GEORGIKON FACULTY Institute of Plant Protection Department of Applied Zoology DOCTORAL SCHOOL OF ANIMAL- AND AGRICULTURAL ENVIRONMENTAL SCIENCES Head of school: Prof. Angéla Anda D.Sc. Supervisors: Dr. habil. Miklós Nádasy C.Sc. Dr. Zsolt Marczali Ph.D. STUDY ON THE ECOLOGY AS WELL AS ON THE CONTROL OF WESTERN CORN ROOTWORM (DIABROTICA VIRGIFERA VIRGIFERA LECONTE, 1868) Written by: Tamás Németh Keszthely 2012 3
TABLE OF CONTENTS 1. PRELIMINARIES OF THE RESEARCH...5 2. RESEARCH OBJECTIVES...6 3. MATERIALS AND METHODS...7 3.1. Preparation of soil containing known numbers of WCR eggs in homogenous distribution for further greenhouse and laboratory tests 7 3.2. Study on the ecological interaction between mites and WCR adults in isolated stocks...7 3.3. Effect of cold treatments on the mortality of WCR embryos...8 3.4. Tests of maize hybrids tolerance against larval damage of WCR...8 3.4.1. Pot trial using original and with WCR eggs artificially treated soils...8 3.4.2. Comparative field study of maize hybrids...9 3.4.3. Relation between the total cyclic hydroxamic acid (chx) content of maize roots and tolerance against WCR larval damage...10 4. RESULTS...11 4.1. Preparation of soil containing known numbers of WCR eggs in homogenous distribution for further greenhouse and laboratory tests...11 4.2. Study on the ecological interaction between mites and WCR adults in isolated stocks...11 4.3. Effect of cold treatments on the mortality of WCR embryos...12 4.4. Tests of maize hybrids tolerance against larval damage of WCR...13 4.4.1. Pot trial using original and with WCR eggs artificially treated soils...13 4.4.2. Comparative field study of maize hybrids...13 4.4.3. Relation between the total cyclic hydroxamic acid (chx) content of maize roots and tolerance against WCR larval damage...14 5. NOVEL SCIENTIFIC RESULTS...15 6. SCIENTIFIC PUBLICATIONS IN CONNECTION WITH THE SUBJECT OF DISSERTATION...17 7. OTHER PUBLICATIONS...20 4
1. PRELIMINARIES OF THE RESEARCH Western Corn Rootworm (WCR) (Diabrotica virgifera virgifera LeConte, 1868) is one of the most noxious maize pests in America and Europe. It is indigenous in North America and has been introduced into Europe in 1992. Three years later, in the summer of 1995 the first specimens were caught in Hungary. WCR has a great potential for damage, its control causes significant expenditures. It has one generation per year and the eggs (embryos within) can diapause in the soil. An essential tool of its control is the utilization of agrotechnical possibilities. Keeping its damage under the damage threshold and stabilizing the number of individuals may be solved by using pesticides but it entails an expressive load on the environment as well. It is important to reveal new, alternative control methods that agree with the practice of sustainable agriculture. 5
2. RESEARCH OBJECTIVES Before starting the researches, our aim was to gain soil containing WCR eggs in known numbers and in homogenous distribution. In the course of collecting WCR adults, mites visible to the naked eye have been noticed on the surface of their body. Our aim was to determine the mite species and to study the relationship between them and the WCR adults. One of our additional objectives was to study the biology of the pest. Within the framework of these investigations, among many other factors affecting embryo mortality, the effect of Hungarian winter soil temperature on the embryos was studied. It is essential to reveal alternative protection methods that agree with the practice of sustainable agriculture. In the course of our investigations, our aim was the comparison of untreated maize hybrids to seek possibly resistant or tolerant ones and in case of fortunate seeking to reveal the background of resistance or tolerance. 6
3. MATERIALS AND METHODS 3.1. Preparation of soil containing known numbers of WCR eggs in homogenous distribution for further greenhouse and laboratory tests Before the further greenhouse and laboratory tests, a soil containing known numbers of eggs in homogenous distribution was needed. For that purpose, 500-500 adults were placed into 20 litres plastic containers in 1:1 sexual ratio. The containers were kept in a climate chamber on 26 C and L/D 16/8 photoperiod. The number of eggs per unit soil was determined with egg washing method. 3.2. Study on the ecological interaction between mites and WCR adults in isolated stocks During rearing of isolated populations of WCR, foretic mites were occurred in some containers. Specialists were requested to determine the mites. The collected specimens were mounted by Dr. Géza Ripka and later identified by Dr. Árpád Szabó as Proctolaelaps striatus (Westerboer, 1963), a new species for the Hungarian fauna. Effect of the number of mites on the lifespan of adults and fecundity of females was investigated. Observations were made 7
to reveal the type of ecological relationship between the species. 3.3. Effect of cold treatments on the mortality of WCR embryos Soil containing known numbers of eggs was used for modelling the effects of Hungarian winter temperatures on WCR eggs. For that purpose, 1356 pieces of eggs were mixed homogenously in 1.1 kg soil cubes and kept at -2 o C for 8 days and at -4 o C for 4 days. From the difference between the number of hatched larvae and mixed eggs per soil cubes conclusions were drawn to the mortality on the effect of treatments. Relation between the duration and temperature of cold treatments moreover the embryo mortality was also studied. 3.4. Tests of maize hybrids tolerance against larval damage of WCR 3.4.1. Pot trial using original and with WCR eggs artificially treated soils The tests started with samples collected from field and continued with artificially treated soils filled into experimental pots. 3-3 grains were sowed in each 20 litres pot, which were 8
later rarefied. The hybrids were compared according to the extent of larval damage using modified IOWA (Hills-Peters) corn root rating scale. Later we turned to the node injury (Oleson-Tollefson) scale, which contained a 0.00 value, to make clear the minor damages as well. 3.4.2. Comparative field study of maize hybrids Comparative field studies were carried out in three consecutive years (2007-2009). Several monoculture fields were selected in the late winter season. All possible sites were tested with egg washing method. Such field was chosen which contained numerous WCR eggs in a relatively homogenous distribution. The hybrids were compared regard for larval damage and plant lodging. The modified IOWA (Hills-Peters) corn root rating scale was used for evaluation of larval damage. The plant lodging was evaluated according to the presence of the swan-neck symptom and all other plants were considered to be lodged which did not show the typical symptom but enclosed less than 60 of angle with the soil. 9
3.4.3. Relation between the total cyclic hydroxamic acid (chx) content of maize roots and tolerance against WCR larval damage In the course of gleaning information in the scientific literature, the role of hydroxamic acids in insect resistance of maize hybrids has aroused our interest. Thus, based on the results of our earlier studies, six maize hybrids were chosen of which chx content in their 8 days stage was analysed by Dr. Péter Makleit. The maize plants were raised in climate chamber using nutrient solution. The corns were germinated under controlled abiotic circumstances, within moist filter paper. Maize plant were cultivated in a climate chamber using nutrient solution.. The chx content of roots was measured according to the method of Long et al. (1974). That method is based on the photometric measurement of the iron complex content of cyclic hydroxamic acids. 10
4. RESULTS 4.1. Preparation of soil containing known numbers of WCR eggs in homogenous distribution for further greenhouse and laboratory tests Based on our results, it has been found that the number of WCR eggs per pot is cardinally influenced by the physiological conditions of collected adults. These soils can only be used in further studies when their egg content is previously determined using modified egg washing method or eggs in known numbers are added to clear soils. Taking 90 pieces of eggs into 20 litres plastic containers 1.73 average value of node injury was observed. Comparing our results to the former laboratory test results of Bayer et al. (2002) it has been found, that a WCR female can lay an average of 773.6±308 pieces of eggs in July while an average of 459.2±300.8 pieces of eggs in August. 4.2. Study on the ecological interaction between mites and WCR adults in isolated stocks The lifetime of adults carrying mites on their body has been remarkably (about 25%) shortened. As regards to the fecundity, no differences were detected between females carrying mites and females free from them. The shortened lifetime was probably triggered by the stress caused by the 11
mass attendance of mites, which however did not decrease the number of the laid eggs. Based on our studies, the ecological relation between the two species is the phoresis. 4.3. Effect of cold treatments on the mortality of WCR embryos It has been established, that under Hungarian climatic conditions not the temperature of cold treatments but the duration of the cold effect influences the mortality. The longer the duration of the cold effect influencing the eggs, the higher mortality can be observed; moreover, the cold treatments can narrow the duration of hatching as well. A one-day long colt treatment with -2ºC temperature caused 0.795% increase in the mortality. The treatment with -4ºC temperature did not cause any further increase in the mortality. Thus, the Hungarian weather conditions cannot highly influence the successfulness of WCR eggs overwintering. The heat stress could increase the mortality of less viable individuals to the highest degree. 12
4.4. Tests of maize hybrids tolerance against larval damage of WCR 4.4.1. Pot trial using original and with WCR eggs artificially treated soils Two hybrids Zamora and SUM2162 were compared using the soil collected from the field. While the sensitive Zamora showed 1.8 average value of node injury, the SUM2162 showed not more than 1.35. However, the difference was not significant. In the course of our studies with WCR eggs artificially treated soils, nine different maize hybrids were tested. The SUM2162 hybrid, developed by the Südwestsaat GbR with conventional improvement methods, showed significantly lower root damage in comparison with other hybrids. Some chewing damages on its root system were observed only sporadically. The hybrid has proved a superior ability to resist larval damage, because in case of a 1.73 average value of node injury damage level it showed only 0.12 so it was classified tolerant. 4.4.2. Comparative field study of maize hybrids In our field studies performed in 2007 PR38A24 and PR37D25 hybrids showed significantly less extent of root damage than the most sensitive PR37D79 hybrid. The 13
difference remains under the experimental average in 2008. Differences among hybrids similar to those of found in pot trials were not proved in our field studies in 2008 and 2009, we had to rely on our empirical observations only. Based on these facts, PR38A24 and PR37D25 hybrids were considered less sensitive. 4.4.3. Relation between the total cyclic hydroxamic acid (chx) content of maize roots and tolerance against WCR larval damage Total cyclic hydroxamic acid content of three non-tolerant maize hybrids (on the average 101.51 mg/kg fresh weight) and the tolerant SUM2162 hybrid (on the average 70.50 mg/kg fresh weight) was measured. Based on the data published earlier in the scientific literature, the tolerant SUM2162 hybrid should have been contain an outstanding amount of chx in our studies. However, such differences were not observed. On the contrary, the chx content of sensitive hybrids were trend like always and in some cases significantly higher than that of the tolerant hybrid The total hydroxamic acid content in itself cannot be responsible for the tolerance against larval damage of WCR. 14
5. NOVEL SCIENTIFIC RESULTS 1. An effective method was developed for homogeneous treatment of experimental pots with known numbers of WCR eggs. Taking 90 pieces of eggs into 20 litres plastic containers 1.73 average value of node injury was observed. 2. It has been proven in isolated stocks of WCR, that the fecundity of females in July may reach an average of 773.6±308 while in August an average of 459.2±300.8 pieces of eggs per female. Our results are the multiples of the data published in the former scientific literature. 3. A mite species Proctolaelaps bickleyi (Bram, 1956) was described for the first time in Hungary and it was established, that living together with the WCR adults the mites may decrease the their lifespan by 25%. Besides, it has been revealed, that the interspecific interaction is the phoresis. 4. Effect of low temperatures on the mortality of WCR embryos was studied on the first occasion in Hungary. It has been found, that a treatment with -2 o C temperature could increase the embryo mortality by 0.795%, while the treatment with -4 o C does not cause any further increase in the mortality. Based on our 15
results, even winters colder than the average in Hungary could not result in the mass destruction of overwintering WCR embryos. 5. It has been established in our pot trials, that maize hybrids sensitive to larval damage showed 1.73 while the SUM2162 hybrid showed only 0.12 average value of node injury. With this, the tolerance of the SUM2162 hybrid against larval damage of WCR has been proven. 6. Total cyclic hydroxamic acid content of three nontolerant maize hybrids (on the average 101.51 mg/kg fresh weight) and the tolerant SUM2162 hybrid (on the average 70.50 mg/kg fresh weight) was measured. In contrast to the data published earlier in the scientific literature it has been found, that the total hydroxamic acid content in itself cannot be responsible for the tolerance against larval damage of WCR. 16
6. SCIENTIFIC PUBLICATIONS IN CONNECTION WITH THE SUBJECT OF DISSERTATION SCIENTIFIC PUBLICATIONS IN HUNGARIAN Oral presentations in Hungarian (abstract) Németh T., Takács J., Nádasy M. (2006): Módszerfejlesztés az amerikai kukoricabogár (Diabrotica virgifera virgifera LeConte, 1868) téli előrejelzéséhez, és annak alkalmazása a precíziós mezőgazdaságban. 52. Növényvédelmi Tudományos Napok, Budapest, 15. Takács J., Nádasy M., Németh T. (2007): Módszertani vizsgálatok az amerikai kukoricabogár (Diabrotica virgifera virgifera) téli felvételezéséhez. 53. Növényvédelmi Tudományos Napok, Budapest, 5. Németh T., Nádasy M. (2008): A kukoricabogár múltja, jelene és jövője Magyarországon. XIV. Ifjúsági Tudományos Fórum, Keszthely, 2008. április 3. Németh T., Nádasy M., Takács J. (2009): Mesterségesen megnövelt kukoricabogár tojásterheltségű talaj előállítása, növényházi és laboratóriumi kísérletekhez. 55. Növényvédelmi Tudományos Napok, Budapest, 2009. február 23, 11. 17
Németh T., Nádasy M., Fábián L., Havancsák A. (2009): Csávázó szerek és talajfertőtlenítő anyagok kukoricabogár lárvák elleni hatékonyságának vizsgálata, 2007-ben és 2008-ban. XIX. Keszthelyi Növényvédelmi Fórum, Keszthely, 2009. február 4-6., 124. Németh T., Nádasy M. (2010): A téli hideg hatása az amerikai kukoricabogár (Diabrotica virgifera virgifera LeConte) tojás mortalitására. XX. Keszthelyi Növényvédelmi Fórum, Keszthely, 2010. január 27-29., 124. Informative articles in Hungarian Németh T., Nádasy M. (2008): A kukoricabogár elleni integrált védekezés. Agronapló XII. (4): 16-17 Németh T., Nádasy M. (2008): Védekezési lehetőségek a kukoricabogár ellen. Őstermelő Gazdálkodók Lapja 2008/5, (október-november): 47-48. 18
Peer-reviewed articles in Hungarian Németh T., Nádasy M., Takács J. (2009): Az amerikai kukoricabogár (Diabrotica virgifera virgifera LeConte, 1868) téli előrejelzése, és annak felhasználhatósága a precíziós mezőgazdaságban. Növényvédelem 45 (2): 69-72. Németh T., Nádasy M., Takács J. (2009): Nagyszámú kukoricabogár tojást tartalmazó talaj létrehozása, növényházi és laboratóriumi kísérletekhez. Növényvédelem 45 (2): 91-93. Németh T., Nádasy M., Szerecz A. (2010): Hideg kezelések hatása az amerikai kukoricabogár (Diabrotica virgifera virgifera LeConte) tojáskori mortalitására. Növényvédelem 46 (4): 155-160. SCIENTIFIC PUBLICATIONS IN ENGLISH Peer-reviewed articles in English T. Németh, M. Nádasy, Zs. Marczali, J. Takács (2009): Making soil containing numerous eggs of WCR for greenhouse and laboratory experiments. Communications in Agricultural and Applied Biological Sciences, 74. (2): 309-312. 19
Németh T., Makleit P., Nádasy M. (2010): Relation between western corn rootworm tolerance and cyclic hydroxamic acid content of maize roots. Növénytermelés Vol. 59.: 125-128. Szabó Á., Ripka G., Hajdu Zs., Tempfli B., Varga M., Mészáros I., Kutasi Cs., Németh T., Pénzes B. (2013): New data on the Mesostigmatid mite fauna of Hungary (Acari: Mesostigmata). Acta Phytopathologica et Entomologica Hungarica Vol. 48. (in press). 7. OTHER PUBLICATIONS PUBLICATIONS IN HUNGRIAN Oral presentations in Hungarian Takács J., Nádasy M., Pirgi Z., Németh T., Milevoj, L., Trdan, S. (2005): A kukoricabogár (Diabrotica virgifera virgifera LeConte, 1868) téli előrejelzése és annak felhasználhatósága a precíziós növényvédelemben. 10. Tiszántúli Növényvédelmi Fórum, Debrecen, 353-360. Takács J., Nádasy M., Pirgi Z., Németh T. (2006): Mavicell-el a kukoricabogár (Diabrotica virgifera virgifera Le Conte, 1868) ellen Tegyünk pontot a végére! XVI. Keszthelyi Növényvédelmi Fórum, 104-107 20
Cserényi J., Szabó M., Nádasy M., Takács J., Németh T., Polgár Z. (2007): A kukoricabogár (Diabrotica virgifera virgifera LeConte, 1868) elleni védekezés entomopatogén nematodák és baktericid készítmények alkalmazásával. XVII. Keszthelyi Növényvédelmi Fórum, Keszthely, 2008. január 31.-február 2., 76. Németh T., Nádasy M., Marczali Zs., Simon F., Takács J. (2007): A kukoricabogár (Diabrotica virgifera virgifera LeConte, 1868) téli előrejelzése GPS technológia segítségével. Integrált termesztés a kertészeti és szántóföldi kultúrákban XXVIII., Budapest, 39-43. Németh T., Nádasy M., Takács J. (2008): Az EM-1 baktériumtrágya kukoricabogár kártételre gyakorolt hatása 2006-ban és 2007-ben. XVIII. Keszthelyi Növényvédelmi Fórum, Keszthely, 2008. január 30.- február 1., 60. Informative articles in Hungarian Takács J., Pirgi Z., Németh T. (2006): Kukoricabogár - Mi lesz ha idén nem esik? Gyakorlati Agrofórum 17 (4): 31. Nádasy M., Németh T. (2009): Mikrobiológiai oltóanyagok hatása erdészeti csemetekertben a talajlakó kártevők 21
ellen. Őstermelő Gazdálkodók Lapja 2008/6, (2008. december-2009. január): 88-89. Németh T., Nádasy M. (2009): Kukoricabogár lárvakártétellel szembeni ellenállóságának, valamint talajfertőtlenítő anyagok kukoricabogár lárvák elleni hatékonyságának vizsgálata. Őstermelő Gazdálkodók Lapja 2009/1, (2009. február-március): 102-103. Nádasy M., Kadlicskó S., Németh T. (2010): A kukorica integrált védelme a főbb kártevők és kórokozók ellen. Őstermelő Gazdálkodók Lapja 2010/1, (2010. februármárcius): 92-94. SCIENTIFIC PUBLICATIONS IN ENGLISH Peer-reviewed articles in English Németh T., Nádasy M., Marczali Zs., Simon F., Nádasyné I.E., Takács J. (2008): Effect of a soil microbe preparation (EM-1) on the damage of western corn rootworm (Diabrotica virgifera virgifera LeConte). Cereal Res. Comm. Vol. 36. No. 2.: 843-846. Nádasy M., Simon F., Marczali Zs., Nádasyné I.E., Németh T. (2008) Effect of entomopathogenic nematodes on larvae of scarabs and western corn rootworm, 22
Diabrotica virgifera virgifera LeConte. Cereal Res. Comm. Vol. 36. No. 2.: 851-854. Nádasy M., Sáringer Gy., Marczali Zs., Keresztes B., Németh T., Horváth J., Kazinczi G., Takács A. (2008): Important potato pest in Hungary. Global Potato Conference New Delhi India, 148-149. 23