THE EFFECT OF COVER CROPS ON SUPPRESSION OF NEMATODES ON PEANUTS AND COTTON IN ALABAMA Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. _____________________________ Sandeep Reddy Marla Certificate of Approval: __________________________ ____________________________ Kathy S. Lawrence Robin N. Huettel, Chair Associate Professor Professor Plant Pathology Plant Pathology __________________________ ______________________________ Jorge Mosjidis Joe F. Pittman Professor Interim Dean Agronomy and Soils Graduate School THE EFFECT OF COVER CROPS ON SUPPRESSION OF NEMATODES ON PEANUTS AND COTTON IN ALABAMA Sandeep Reddy Marla A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Master of Science Auburn, Alabama December 19, 2008 iii THE EFFECT OF COVER CROPS ON SUPPRESSION OF NEMATODES ON PEANUTS AND COTTON IN ALABAMA Sandeep Reddy Marla Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iv VITA Sandeep Reddy Marla, son of Pulla Reddy Marla and Lalitha Marla was born on October 21, 1981, in Khammam, Andhra Pradesh, India. He has one younger sister Swapna Marla. Mr. Sandeep Marla graduated in Bachelor of Agricultural Sciences from Acharya N G Ranga Agricultural University, Rajendra Nagar, Hyderabad, India, in 2005. He joined the graduate school at Auburn University, Alabama to pursue a Master?s program in the Department of Entomology and Plant Pathology in January 2006. v THESIS ABSTRACT THE EFFECT OF COVER CROPS ON SUPPRESSION OF NEMATODES ON PEANUTS AND COTTON IN ALABAMA Sandeep Reddy Marla Master of Science, December 19, 2008 (B.S., Acharya N.G. Ranga Agricultural University, 2005) 71 Typed pages Directed by Robin N. Huettel Cover crops were evaluated in the greenhouse and in field locations to determine their host status and nematode suppressive effect on root-knot nematodes, Meloidogyne incognita and M. arenaria and the reniform nematode, Rotylenchulus reniformis. The winter grain cover crop cultivars included commercially available cultivars of wheat ?Triticum aestivum? ?Pioneer 26R12?, ?AGS 2000?, ?Coker 9152?, ?Panola?; oats ?Avena sativa? ?Georgia Mitchell? and ?Bob?; rye ?Secale cereale? ?Elbon? and ?Abruzzi?. This research also evaluated the host status and nematode suppressive effect of Crotolaria juncea populations. The treatments included the C. juncea populations; PI 207657, PI 314239, PI 322377, PI 391567 and PI 426626 collected in different countries and the commercially available cultivar ?Tropic Sun?. vi Field evaluations of winter grain cover crop cultivars described previously were conducted at WREC in Headland, AL and in a grower?s field in Huxford, AL. There were no significant differences (P ? 0.05) between cover crop cultivars on nematodes. This was most probably due to severe drought and uneven rainfall during both the cropping years. However, the greenhouse studies indicated that ?Elbon? rye; oats ?Bob? and ?Georgia Mitchell? supported low populations of M. incognita. While ?Bob? oats and the rye ?Elbon? and ?Abruzzi? supported significantly (P ? 0.05) lower R. reniformis populations. C. juncea populations were able to significantly suppress (P ? 0.05) M. incognita and R. reniformis in the greenhouse tests. C. juncea roots stained with McCormick Schilling? red food color were found to contain all juvenile stages, low numbers of mature females of M. incognita with egg masses and 1-2 female reniform nematodes per 10 gm of roots, indicating that these nematodes were able to infest and reproduce on C. juncea populations. Freeze-dried root exudates tested against both M. incognita and R. reniformis demonstrated that concentrated exudates could kill both nematodes whereas the water control had no effect. Field trial at EVSRC in Shorter, AL indicated that there were no significant differences observed on Meloidogyne spp. suppression among the C. juncea populations, might be due to severe drought and extreme high temperatures. The knowledge obtained from this study suggests that some winter cover crop cultivars and C. juncea populations may be poor hosts and suitable for crop rotation in a region with specific nematode histories, thus minimizing usage of synthetic nematicides and yield losses. However, further research studies should focus on extensive long-term field studies under controlled irrigation conditions. vii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Robin N. Huettel for her constant support, for sharing her vast knowledge of Plant Pathology and Nematology, and for her guidance throughout my research at Auburn University. I am grateful to her for the critical review of the manuscript and for improving my research skills. I would like to thank Dr. Jorge Mosjidis for his assistance with performing the Crotalaria juncea evaluations. I thank Dr. Kathy S. Lawrence for sharing her knowledge of Nematology with me. I would like to thank my entire committee for their patience, comments and suggestions during my research, and their critical review of the manuscript. I would also like to thank Kathy Burch for support in my field research. I wish to dedicate this work to my parents and younger sister whose love and encouragement provided me the strength to achieve my goals. I would like to thank my friends Vasavi Chilamantula and Hanumantha Rao K. S. V. for their support and encouragement. viii Style manual or journal used Journal of Nematology Computer software used Microsoft Word 2007, Microsoft Excel 2007 ix TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xii I. LITERATURE REVIEW???????????????????????? 1 II. EFFECT OF WINTER GRAIN COVER CROP CULTIVARS ON MELOIDOGYNE SPP. AND ROTYLENCHULUS RENIFORMIS SUPPRESSION .................................... 11 Introduction .......................................................................................................... 11 Materials and Methods .......................................................................................... 12 Results???. ...................................................................................................... 17 Discussion?????????????????????????? ..... 20 III. EVALUATION OF CROTALARIA JUNCEA POPULATIONS TO MANAGE PLANT PARASITIC NEMATODES?????????????????? ....... 29 Introduction?. ..................................................................................................... 29 Materials and Methods .......................................................................................... 30 Results???. ...................................................................................................... 36 Discussion?? ..................................................................................................... 39 IV. SUMMARY ................................................................................................................ 51 LITERATURE CITED ..................................................................................................... 54 x LIST OF TABLES II. EFFECT OF WINTER GRAIN COVER CROP CULTIVARS ON MELOIDOGYNE SPP. AND ROTYLENCHULUS RENIFORMIS SUPPRESSION 1. Effect of winter grain cover crop cultivars on populations of peanut root-knot nematode, Meloidogyne arenaria at Wiregrass Research and Extension Center, Headland, AL, during the cropping cycle of year 2005-06.?? ....................................................... 23 2. Effect of winter grain cover crop cultivars on populations of peanut root-knot nematode, Meloidogyne arenaria at Wiregrass Research and Extension Center, Headland, AL, during the cropping cycle of year 2006-07??. ??. .............................................. 24 3. Effect of winter grain cover crop cultivars on populations of reniform nematode, Rotylenchulus reniformis at Huxford, AL, during the cropping cycle of year 2005-06. .. 25 4. Effect of winter grain cover crop cultivars on populations of reniform nematode, Rotylenchulus reniformis at Huxford, AL, during the cropping cycle of year 2006-07. .. 26 5. Effect of winter grain crop cultivars on the populations of root-knot nematode, Meloidogyne incognita in the greenhouse at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL????????. ............................... 27 6. Effect of winter grain crop cultivars on populations of reniform nematode, Rotylenchulus reniformis in the greenhouse at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL????????????? ............. 28 xi LIST OF TABLES III. EVALUATION OF CROTALARIA JUNCEA POPULATIONS TO MANAGE PLANT PARASITIC NEMATODES 1. Effect of Crotalaria juncea populations on southern root-knot nematode, Meloidogyne incognita under controlled conditions at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL. ..................................................................... 42 2. Effect of Crotalaria juncea populations on reniform nematode, Rotylenchulus reniformis under controlled conditions at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL. ..................................................................... 43 3. Evaluation of the nematicidal activity of Crotalaria juncea populations root exudates on root-knot nematode, Meloidogyne incognita. .............................................................. 44 4. Evaluation of the nematicidal activity of Crotalaria juncea populations root exudates on reniform nematode, Rotylenchulus reniformis. ............................................................ 45 5. Effect of Crotalaria juncea populations planted with tomato on root-knot nematode, Meloidogyne incognita under controlled conditions at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL.? ............................................... 46 6. Effect of Crotalaria juncea populations planted with cotton on reniform nematode, Rotylenchulus reniformis suppression under controlled conditions at the Plant Science Research Center, located on campus of Auburn University, Auburn, AL.... .................... 47 7. Effect of Crotalaria juncea populations on root-knot nematode, Meloidogyne spp. under field conditions at E V Smith Research Center, Shorter, AL????????.. 48 xii LIST OF FIGURES III. EVALUATION OF CROTALARIA JUNCEA POPULATIONS TO MANAGE PLANT PARASITIC NEMATODES 1. Third-stage juvenile (J3) of root-knot nematode, Meloidogyne incognita present inside the Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels). .................................................... 49 2. Adult female of root-knot nematode, Meloidogyne incognita present inside the Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels) ................................................................. 49 3. Female reniform nematode, Rotylenchulus reniformis present semi-endoparasitically on Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? Eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix 4500 camera (4 mega pixels).????????? ............... 50 4. Nematode eggs inside an adult female of reniform nematode, Rotylenchulus reniformis present semi-endoparasitically on Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels) ...... 50 1 I. LITERATURE REVIEW Plant-parasitic nematodes Nematodes are microscopic, unsegmented invertebrate roundworms with bilateral symmetry (Veech, 1984). Nematodes are ubiquitous and regarded as an important part of every ecosystem. Most of the nematodes are beneficial because of their free-living and saprophytic nature and play a major role in decomposition of organic matter and nutrient recycling. Only 10% of the total known nematodes are considered to be plant-parasites (Schumann and D?Arcy, 2006). These parasitic nematodes are obligate parasites and always require a host plant for their survival. Most of the plant-parasitic nematodes belong to order Tylenchida while a few belong to order Dorylaimida (Agrios, 1997). Plant-parasitic nematodes are identified by the presence of a stylet which is a specialized feeding structure used for penetrating the plant tissues. They can feed externally as ectoparasites or develop as endoparasites within the roots. Plant-parasitic nematodes life cycle starts with the egg and develops through four juvenile stages with the last molt to an adult (Schumann and D?Arcy, 2006). Plant-parasitic nematodes have a very wide host range and are found to infest a wide variety of agronomic crops. Plant-parasitic nematodes are identified as one of the major limiting plant parasites for all the cash crops throughout the world. The Society of Nematology and 2 other organizations estimates worldwide crop losses due to nematodes at $100 billion per annum (Schumann and D?Arcy, 2006). In the United States, plant-parasitic nematodes cause an estimated $10 billion of crop losses each year (Mani et al., 2005). Two major crops infested by nematodes are peanuts and cotton. Severe economic losses in peanuts and cotton results from infestations of plant-parasitic nematodes, such as the root-knot nematode, Meloidogyne spp. and the reniform nematode, Rotylenchulus reniformis (Rodriguez-Kabana et al., 1991). These nematodes reduce crop productivity by their direct action and association in pathogenic disease complexes. Meloidogyne spp. account for yield losses up to 12% in peanuts (Handoo, 1998) and R. reniformis causes around 9 % loss in cotton (Blassingame, 2007). These parasitic nematodes cause severe yield losses when infested during early stages of crop growth. Peanuts and root-knot nematode Peanuts (Arachis hypogaea L.) belong to family Fabaceae and have their origin from Peru (South America). Peanuts are one of the most important oil seed crops in the world. Peanut is a self-pollinated, erect or prostate, sparsely hairy, annual herbaceous legume and geocarpic as it produces underground pods. Peanuts play a pivotal role in the crop economy of the United States and in the Southeastern United States. The U.S. peanut production accounted to 3.37 billion pounds in the year 2006 (NASS, USDA, 2006), a substantial decrease from the 2005 production of 4.86 billion pounds. In Alabama, the peanut production accounted to 331 million pounds 3 (NASS, USDA, 2006). The harvested area decreased from 1.62 to 1.21 million acres from the year 2005 to 2006 in the nation (NASS, USDA, 2006). The reason for the gradual decrease in peanut yields is not clear but may be due to adverse climatic conditions, severe pest problems and a commodity market shift. Approximately 60% of the peanut production in the United States occurs in Georgia, Florida and Alabama (Fletcher, 2002). Damage caused by the Meloidogyne spp. is one of the most serious constraints for peanut production in these states. Meloidogyne spp. nematodes are present throughout the world and found to attack more than 2000 plant species (Agrios, 1997). Meloidogyne spp. are sedentary endoparasites. They establish feeding sites inside the roots known as giant cells and continue their life cycle within the roots. The life cycle of a Meloidogyne spp. nematode is completed in 3-4 weeks under favorable conditions. In the Southeastern United States, the peanut root-knot nematode, Meloidogyne arenaria, is one of the major Meloidogyne spp. that causes severe yield losses in peanuts (Rodriguez-Kabana et al., 1991). Meloidogyne spp. can occasionally damage the entire crop when the infestation is wide spread. The symptoms produced by Meloidogyne spp. include formation of galls on roots, pegs and pods (Porter et al., 1984). In case of severe infestations, plants are stunted in growth and light green in color, resembling nutrient deficiencies. Nematode damage can be identified by the presence of small spots with a dark center on peanut pods (Porter et al., 1984). Above ground symptoms includes yellowing of foliage, midday wilting and stunted growth in patches over the entire field. The losses incurred by these nematodes 4 can be effectively reduced by use of synthetic nematicides, fallowing and crop rotation with non-host crops. Cotton and reniform nematode Cotton (Gossypium spp.) is the most important fiber crop of the world. Cotton is a perennial plant with indeterminate growth habitat but is cultivated as an annual crop. There are four domesticated species of cotton: Gossypium arboretum L., Gossypium herbaceum L., Gossypium barbadense L., and Gossypium hirsutum L. (Lee, 1984). Cotton can be grown effectively around the world between the latitudes of 470 north and 320 south (Lee, 1984). The growing period for cotton is from six to eight months depending on climatical conditions and the weather patterns. Cotton is generally planted between April and early June and generally harvested from September to October. Cotton is primarily grown for fiber and the seeds are an important source of oil. The United States is the second-largest producer and the largest exporter of cotton in the world (Fry, 2001). The U.S. cotton production in the year 2006 accounted to 20.5 million bales (NASS, USDA, 2006) while cotton production in 2005 is 23.2 million bales (NASS, USDA, 2005). Cotton production in the Southeastern United States is hindered by insects and plant-parasitic nematodes (Koenning et al., 2004). The major nematode infesting cotton in the Southeastern states is the reniform nematode, Rotylenchulus reniformis, accounts 5 for around 9% of the yield losses on this crop (Blassingame, 2007). In surveys of Alabama cotton fields conducted by Gazaway and McLean (2003), 47% of the fields contained R. reniformis. The symptoms produced by R. reniformis infestation are not conspicuous, and require root and soil analysis to determine the nematode?s presence (Gaur and Perry, 1991). Symptoms produced by R. reniformis include uneven growth of plants, severe stunting, yellowing of foliage and premature death of the plants (Lawrence and McLean, 2001). Rotylenchulus reniformis is a sedentary, semi-endoparasitic nematode that infests a wide range of agronomic crops. The life cycle consists of an egg, four juveniles and an adult stage, and is completed within 24-29 days from egg to egg (Guar and Perry, 1991). Adult males do not feed on the roots. The vermiform females penetrate roots to establish a feeding site in the stele (Gaur and Perry, 1991). Winter cover crops Cover crops are planted between cycles of the main cash crop or intercropped with cash crops to improve soil fertility, soil structure, water infiltration, and reduce soil erosion (Hooks et al., 1998). They have the potential to suppress pathogens, weeds and nematode pests. According to Barker and Koenning (1998) crop rotation with cover crops provided diversity in time and space and is often considered as a preferred means to manage plant-parasitic nematodes. A significant reduction of R. reniformis nematodes has been observed when cotton is rotated with winter grain crops (Jones et al., 2006). In 6 addition to controlling parasitic nematodes, specific cereal crops have also decreased weeds more efficiently at early stages of crop growth (Wang et al., 2004a). Taking into consideration of all the above mentioned beneficial qualities, winter cover crops may serve as an alternative to chemical nematicides. Meloidogyne spp. and R. reniformis are major plant nematode pathogens causing severe yield losses in the Southeastern United States. The present method of controlling these nematodes is use of synthetic nematicides; however, it is not cost-effective and can be harmful to environment and humans. Crop rotation with cover crops can offer a supplement to this current nematode management strategy (McSorley, 1999). Crittenden (1961) has reported many commercial wheat cultivars to be hosts of M. incognita and M. javanica. Recent studies with winter cover crops demonstrated some winter grain crops decreased numbers of M. incognita better than the leguminous cover crops (Wang et al., 2004a). Field evaluations described that winter grain cover crops maintained low populations of M. incognita throughout the winter season but nematode densities were found to increase after planting a susceptible host (Wang et al., 2004a). Greenhouse studies for evaluating winter cover crop cultivar host status has observed reduction of R. reniformis populations on ?Gulf? ryegrass (Lolium multiflorum), ?Wren?s Abruzzi? rye (Secale cereale), ?Soil Saver? blackoats (Avena strigosa), ? AU Homer? lupin (Lupinus albus), and ?Coker 9663? wheat (Triticum aestivum), suggesting that these crops were non-host crops (Jones et al., 2006). In field experiments, Wang et al., (2004a) observed that rye and oats were poor hosts of M. incognita and reduced nematode populations more effectively than fallow. 7 Crop rotation of cotton with rye supported the least number of M. incognita eggs and there was very low root-galling in cotton during the next cropping season (Timper et al., 2006). McSorley (1994) described rye to be partially suppressive to M. arenaria; however, there was no decrease in the nematode populations. A regression model of M. arenaria indicated slight decline of populations over the initial populations (Pi), indicating that rye can be used as a winter cover crop or rotation crop (McSorley, 1994). Planting rye as cover crop can also lower the risk of increasing populations of M. incognita when compared to use of clovers and vetches (Timper et al., 2006). According to Zasada et al., (2005) rye tissue degradation products contained chemicals DIBOA (2, 4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one) and DIMBOA (2,4-dihydroxy-7-methoxy- (2H)-1,4-benzoxazin-3(4H)-one). These chemicals resulted in a mortality rate of 73% and 71% for M. incognita. However, the nematode suppressive effect lasted through a single cropping season and nematode densities increased again after planting susceptible crops. Specific cover crops are found to suppress plant-parasitic nematodes but the mechanism of suppression is not clearly known. Reduced numbers of plant-parasitic nematode abundance in cover crops may be due to poor host status, production of allelochemicals or enhancement of nematode-antagonistic flora and fauna (Wang et al., 2002). Sunn hemp Crotalaria juncea L. (sunn hemp) used as a cover crop in crop rotation has been demonstrated to have many beneficial qualities. It is an effective legume cover crop that 8 adds nitrogen and organic matter to the soil and enhances soil fertility (Wang et al., 2004b). Crotalaria juncea has the potential to grow and cover the soil surface rapidly while protecting the soil surface from erosion (Mansoer et al., 1997). In addition to controlling weeds and soil erosion, C. juncea also produced a biomass of 7.6 Mg/ha after 14WAP with an average N content of 144 kg/ha (Balkcom and Reeves, 2005). Crotalaria juncea can be grown as a green manure crop or intercropped with the cash crops. Crotalaria juncea has shown high degree of resistance to several Meloidogyne spp. (Wang et al., 2004b) and increase free-living nematode populations in pineapple cultivation (Wang et al., 2003). Besides increasing beneficial nematode populations, C. juncea can increase nitrogen especially in organic production systems (McSorley, 1999). This combination of nematode and nitrogen management could be especially useful in sustainable and organic production systems where neither nematicides nor synthetic nitrogen fertilizers could be used (McSorley, 1999). In West Africa, Crotalaria spp. such as C. podocarpa, C. senegalensis and C. sphaericarpa are used as nematostatic green manure crops (Jourand et al., 2004). Crotalaria juncea used for green manure production can also be used in crop rotation to decrease nematode population levels and propagate arbuscular mychorrhizal (AM) fungi for subsequent crops (Germani and Plenchette, 2004). Crotalaria juncea can also be cultivated as green manure crop and ploughed into the soil in organic farming systems. Several research studies have found that this leguminous cover crop has considerable potential for use by farmers in developing countries to control nematode populations in low-value cropping systems (Jourand et al., 2004). 9 Crotalaria juncea plant residues incorporated into soil have been described to contain antagonistic activity against some plant-parasitic nematodes (Rodriguez-Kabana and Kloepper, 1998). Previous studies describe C. juncea as a poor host to many important plant-parasitic nematodes, including M. incognita, M. javanica, M. arenaria, R. reniformis and Pratylenchus brachyurus (Wang et al., 2004b). Germani and Plenchette (2004) indicated that plant extracts of C. juncea apparently inhibited egg hatching and were found to be lethal to second-stage juveniles (J2) of M. incognita. The aqueous crude extracts from C. juncea shoot and root paralyzed J2 of M. incognita, M. javanica and M. mayaguensis (Jourand et al., 2004). McSorley (1999) indicated C. juncea are highly resistant to nematodes but not immune. Significant differences are observed in invasion and developmental rates of M. incognita and M. javanica on C. juncea and on susceptible control. McSorley et al., (1994) described C. juncea as a trap crop that reduced population densities of several Meloidogyne spp., while enhancing the yield of subsequent nematode susceptible vegetable crop. However, these favorable effects last only through a single cropping season. Crotalaria juncea is found to be a poor host to R. reniformis, allowing the nematode to penetrate the roots but restricting their development and reproduction (Wang et al., 2003). Nematode invasion rates on C. juncea are very low when compared to the susceptible control (tomato). The juvenile J2 which invaded the tomato plants developed into adults, while those on C. juncea rarely developed beyond the third-stage juvenile J3, thus confirming C. juncea to be nonhosts or poor hosts (Germani and Plenchette, 2004). Previous studies have reported the ability of C. juncea to enhance the activity of 10 nematode-antagonistic microorganisms (Rodriguez-Kabana and Kloepper, 1998) and population densities of free-living nematodes (Wang et al., 2002). Crotalaria juncea amendments when incorporated into soils with low organic matter increased numbers of beneficial nematodes. Research studies of Wang et al., (2004b) with C. juncea hay as organic fertilizer is found to increase shoot and root weight of squash and were able to enhance the activity of some nematode-antagonistic fungi in soils. Moreover, C. juncea also enhanced free-living nematodes which play a major role in nutrient cycling (Wang et al., 2004b). Plant extracts of C. juncea apparently inhibited hatching of eggs and are lethal to second-stage juveniles of M. incognita and R. reniformis (Rich and Rahi, 1995). Chemical analysis of root and leaf exudates from Crotalaria spp. described the presence of allelopathic compounds such as monocrotaline and pyrrolizidine alkaloids (Rich and Rahi, 1995). These compounds produced are toxic to plant-parasitic nematodes and inhibited the formation of Meloidogyne spp. galls (Araya and Caswell-Chen, 1994). The mechanism by which C. juncea limits the plant-parasitic nematodes is not clearly known but this may due to the production of nematostatic or nematicidal compounds contained in the root system or in the aerial vegetative parts (Germani and Plenchette, 2004). 11 II. EFFECT OF WINTER GRAIN CROP CULTIVARS ON MELOIDOGYNE SPP. AND ROTYLENCHULUS RENIFORMIS SUPPRESSION INTRODUCTION In Southeastern United States, most of the peanuts (Arachis hypogaea L.) and cotton (Gossypium hirsutum) are monocultured, creating a favorable environment for enhancing plant-parasitic nematode densities (Rodriguez-Kabana et al., 1991). These pests cause severe yield losses when they infest plants at early stages of the crop growth. The most effective strategy for controlling plant-parasitic nematodes is chemical-based management. However, these synthetic nematicides may potentially have adverse affects on human health and environment. Crop rotation with cover crops is often considered as the most practical means of controlling plant-parasitic nematodes (Barker and Koenning, 1998). Knowledge of the host status of cover crops plays a vital role in successful usage of these crops since many crops are susceptible to plant-parasitic nematodes. There are contradictory reports in the literature on susceptibility of cover crops. Some literatures suggest that winter cover crops decrease plant-parasitic nematodes while other suggests that they may be non-hosts (Jones et al., 2006). For instance, many commercial wheat (Triticum aestivum) cultivars were hosts of Meloidogyne incognita and Meloidogyne javanica (Crittenden, 1961; 12 Opperman et al., 1988) and resulted severe infestations in the next cropping season. Wang et al., (2004a) described rye (Secale cereale) and oats (Avena sativa) to be poor- hosts of M. incognita. Previous studies showed that lower plant-parasitic nematode densities in cover crops may be due to poor-host status, production of allelochemicals or enhancement of nematode-antagonistic flora and fauna (Wang et al., 2002). This research was conducted to determine the effect of commercially available winter grain cover crop cultivars on root-knot nematode, Meloidogyne spp. and the reniform nematode, Rotylenchulus reniformis suppression. MATERIALS AND METHODS Field experiments were conducted at two locations, Wiregrass Research and Extension Center (WREC), Headland, AL, and in a grower?s field near Huxford, AL. The field evaluations were conducted for two cropping cycles (2005-06 and 2006-07). The following winter grain cover crops, wheat (Triticum aestivum) cultivars ?AGS 2000?, ?Coker 9152?, ?Pioneer 26R24?; oats (Avena sativa) cultivars ?Bob? and ?Georgia Mitchell?; rye (Secale cereale) cultivars ?Elbon? and ?Abruzzi? were used for evaluations. During the second cropping year (2006-07), in addition to the above cultivars, wheat (Triticum aestivum) cultivars ?Pioneer 26R61?, ?Pioneer 26R12? and ?Panola? were also evaluated for nematode suppression. Fallow was used as control for all the field studies. These research plots were planted in November 2005 with the winter grain cover crops and sampled monthly. In April, the cover crops were harvested and yield data was 13 collected. After harvesting of cover crops at WREC, peanuts (Arachis hypogaea) were planted. In Huxford, AL, cotton (Gossypium hirsutum cv. DP 555 BG/RR) was planted in the same plot. Nematode extraction and quantification Each plot was sampled monthly in a zigzag pattern using a soil probe. Five samples of soil per treatment were taken per plot. Soil samples were stored in a one- gallon plastic bag, at 10 oC until the nematodes were extracted. Each soil sample was mixed thoroughly and a 100 cm3 aliquot of soil was taken from each sample to extract the nematodes. Nematodes were extracted using gravity screening and centrifugal flotation method (Jenkins, 1964). Aliquots of soil (100 cm3) taken from each sample was mixed with water in a container. The sediments were allowed to settle to the bottom. The solution was passed through a series of nested sieves (60-?m on top, 350-?m in middle and 500-?m at the base). The material on the 500-?m sieve was washed into a 50-ml plastic tube and centrifuged for 4 min at 2400 rpm. The supernatant was decanted and 1M sucrose solution was added to the plastic tubes and centrifuged for 2 min at 1200 rpm. The sucrose supernatant containing the nematodes was decanted into a 500-?m sieve, rinsed with water and collected into separate test tubes. Nematodes were identified to genus level and counted on grid plates on a Nikon T-100? inverted microscope at 10x magnification. 14 Field evaluations Experiment I was conducted at WREC, Headland, AL to evaluate the effect of winter grain cover crops on peanut root-knot nematode, Meloidogyne arenaria suppression. Each plot was 10 m in length and 4 m in width. The soil type was Dothan sandy loam. This experiment was conducted for two years from November 2005 to October 2007. The treatments included winter grain cover crop cultivars of wheat, oats and rye described previously and one fallow (control). This experiment was arranged in a completely randomized block design, replicated four times. The winter grain cover crop treatments were row planted in November 2005 and 2006 using a John Deere? tractor mounted plot planter with 15 cm spacing between the rows in individual plots, harvested after maturity, and replanted with peanuts in June 2006 and 2007. Peanuts were harvested at maturity in October 2006 and November 2007 and the yield was recorded separately for each plot. The yield of winter grain crop cultivars and peanuts was recorded for both these years. Soil sampling was done at monthly intervals during the cover crop life cycle and at pre-season (June), mid-season (August) and harvesting (November) during the peanut cropping cycle. Nematodes were extracted and quantified as described above. Experiment II was conducted in a grower?s field near Huxford, AL to evaluate the effect of winter grain cover crops on reniform nematode, Rotylenchulus reniformis. The soil type was Ruston very fine sandy loam. Each plot size measured 10 m in length and 4 m in width. The experiment arrangement was similar to the above experiment. This experiment was repeated for two years. The same winter grain cover crops used in experiment I were planted in November 2005 and 2006, harvested and shoot weights of 15 winter grain crops was recorded. Cotton was planted in the same plots with 90 cm spacing between the rows during June 2006 and 2007, and was harvested in October 2006 and November 2007. After harvesting, yield of cotton was recorded. Soil samples were collected as described previously and nematodes were extracted from soil samples. Soil sampling was done at monthly intervals during the winter cover crop life cycle, at pre-season (June), mid-season (August), and at the time of harvesting (November) during the main cash crop cycle. Nematodes were extracted, and quantified to genus level. Greenhouse evaluations Greenhouse evaluations were conducted at the Plant Science Research Center, located on the campus of Auburn University, Auburn, AL. Two experiments were conducted to evaluate the host status of winter grain crops for M. incognita and R. reniformis. Isolates of M. incognita and R. reniformis nematodes were maintained in the greenhouse on tomato (Lycopersicon esculentum cv. Rutgers) and cotton (Gossypium hirsutum cv. DP 555 BG/RR), respectively. The winter grain crop treatments used were wheat (Triticum aestivum) cultivars ?AGS 2000?, ?Pioneer 26R12?; oats (Avena sativa) cultivars ?Bob? and ?Georgia Mitchell?; rye (Secale cereale) cultivars ?Elbon? and ?Abruzzi?. Tomato and cotton were the controls for M. incognita and R. reniformis evaluations respectively. The soil used was a mixture of autoclaved loamy sand soil field soil (72.5%, 25%, 2.5%, S-S-C, pH 6.4) and sand in the ratio of 3:1 in 500 cm3 polystyrene cups. Both the experiments were arranged in a completely randomized block design on raised benches, replicated ten times. Both the M. incognita and R. reniformis experiments were repeated for the second time. Winter grain crop seeds were hand-sown 16 into the cups, allowed to germinate and grow. One week after germination of winter grain crops nematode eggs were infested as described below. On the day of nematode infestation, nematode eggs from the host plants were extracted using hypochlorite method by agitating roots in 0.6% sodium hypochlorite solution (Hussey and Barker, 1973). Eggs were collected by washing through a 350-?m and 500-?m sieve. Nematode eggs were rinsed with water and the solution containing nematode eggs was collected. The number of eggs for innoculum was standardized by adding water to the solution. Every individual cup was infested with ca. 4000 M. incognita eggs or ca. 2000 R. reniformis eggs near the root zone by making small holes at the base of the plant. After 50 days, the roots were washed gently with water; the shoot weight and root weight of the winter grain crops was recorded. Nematode eggs present on the winter grain crop roots were extracted as described above and counted. Nematode eggs present per gram of root were determined using the formula (Number of eggs/gm of root = Total number of eggs present on plant / Total root weight of the plant) and the data were analyzed as described in data analysis. Data analysis The field data and the greenhouse data of nematode populations were log transformed and analyzed separately for each experiment. The transformed values were submitted to analysis of variance using Generalized Linear Model in Statistical Analytical System software (SAS institute, Inc., Cary, NC). Probability of F-value was used to determine the significant effects of winter grain cover crop treatments (P ? 0.05). 17 Treatment means were separated by Fisher?s protected Least Significant Difference (LSD) and the effect of winter grain cover crop treatments on M. incognita and R. reniformis nematodes was compared. RESULTS Field evaluations In the field studies during 2005-06 at WREC, there was no significant difference (P ? 0.05) observed in the nematode suppression among the different winter grain crop cultivars evaluated (Table 1). Even though there were no significant differences on nematode populations among winter grain cover crops, the numerically decreasing order of winter grain crop susceptibility to M. arenaria nematode demonstrated was ?Bob? oats, ?AGS 2000? wheat, ?Elbon? rye, ?Georgia Mitchell? rye, ?Pioneer 26R24? wheat, Fallow, ?Coker 9152? wheat and ?Abruzzi? rye. During this cropping cycle, M. arenaria counts remained low throughout the winter cropping season and were lowest at harvest of winter grain cover crops. Nematode densities were found to gradually decrease from sowing to harvesting of winter grain cover crops (Table 1). Nematode populations were found to resurge after planting of peanuts. Highest numbers of M. arenaria were present after harvesting of peanuts. During the second cropping season of 2006-07 at WREC, the winter grain cover crops had no significant effect on nematode suppression (P ? 0.05) (Table 2). The 18 decreasing order of numerically relative host susceptibility was ?Coker 9152? wheat, ?Elbon? rye, ?Pioneer 26R61? wheat, Fallow, ?AGS 2000? wheat, ?Georgia Mitchell? oats, ?Bob? rye, ?Abruzzi? rye, and ?Panola? wheat. The overall nematode populations were highest during the harvesting of winter grain cover crops (April), followed by nematode counts during harvesting of peanuts in October (Table 2). There was no significant difference between the cover crop yields and the peanut yields during the two cropping seasons. In Experiment II during 2005-06 Huxford, AL, the winter grain cover crop cultivars supported low R. reniformis nematode populations. No significant difference (P ? 0.05) was observed among the winter grain cover crop cultivars on R. reniformis populations (Table 3). Nematode counts were varied between different sampling months. Highest nematode counts were recorded at mid-season sampling of cotton followed by pre-plant sampling of cover crops (Table 3). Nematode counts were comparatively low throughout the winter grain cover cropping cycle. In the cropping cycle of 2006-07 at Huxford, AL, there were no significant differences (P ? 0.05) on nematode susceptibility between the different winter grain cover crop cultivars evaluated (Table 4). The numerically decreasing order of winter grain cover crop susceptibility for R. reniformis nematode was ?Georgia Mitchell? oats, ?Bob? oats, ?Abruzzi? rye, ?Panola? wheat, ?Pioneer 26R61? wheat, ?Coker 9152? wheat, ?Elbon? rye, Fallow, and ?AGS 2000? wheat. The nematode densities were highest during initial stages of cover crop growth ?January?, and remained high throughout the rest of the winter grain cover crop cycle. Nematode densities were very low at pre-planting of 19 cover crop ?November?. There was no significant relationship between the winter cover crop yields and the cotton yields during both cropping years. Greenhouse evaluations Winter grain crop cultivars had a significant effect (P ? 0.05) on the rate of M. incognita nematode reproduction (Table 5). The nematode reproduction among the treatments ranged from 3.516 [Log (x+1) of number of M. incognita per gram of root weight) on ?Rutgers? tomato to 1.854 ?Bob? oats. Among the winter grain crops evaluated, wheat cultivars supported highest number of nematodes, followed by rye, and oats supported the lowest number of M. incognita nematodes (Table 5). Wheat supported numerically high amounts of M. incognita nematodes than oats and rye cultivars; however, the reproduction of M. incognita nematodes on wheat was less than the tomato control. The nematode susceptibility of winter grain crop cultivars in decreasing order was Tomato, ?AGS 2000? wheat, ?Pioneer 26R12? wheat, ?Abruzzi? rye, ?Elbon? rye, ?Georgia Mitchell? oats, and ?Bob? oats (Table 5). The greenhouse evaluations of R. reniformis nematodes, there was a significant relationship (P ? 0.05) on R. reniformis nematode reproduction among the winter grain crop cultivars and the control (Table 6). Cotton supported numerically highest number of R. reniformis nematodes. Among the winter grain crops, ?Pioneer 26R12? wheat supported numerically highest number of R. reniformis nematodes and ?Abruzzi? rye supported the lowest number of R. reniformis nematodes (Table 6). The decreasing order of relative host status of winter grain crops to R. reniformis nematode demonstrated from 20 this experiment was Cotton, ?Pioneer 26R12? wheat, ?Georgia Mitchell? oats, ?AGS 2000? wheat, ?Bob? oats, ?Elbon? rye, and ?Abruzzi? rye (Table 6). DISCUSSION Field evaluations In experiment I at WREC, the reason for gradual decrease in nematode populations among different sampling intervals was due to adverse climatic conditions. There was a severe drought during the winter grain cover crop cycle, resulted in poor crop growth and no significant differences between the winter grain crop yields and nematode suppression, as observed in the greenhouse evaluations. Highest numbers of M. arenaria nematode populations were present at time of harvesting of peanuts, which indicated that under field conditions the winter grain cover crops had no residual effects on M. arenaria populations. However, this was previously observed by Wang et al., (2004a). During the first cropping year 2005-06 at WREC, Wheat cultivar ?Pioneer 26R24? had no effect on nematode suppressive effect, therefore this cultivar was removed from the trials and other wheat cultivars described above were included during the second cropping year. In 2006-07 at WREC, ?Panola? wheat and ?Abruzzi? rye supported very low M. arenaria reproduction, this may be due to uneven rainfall during the winter grain cover 21 crop cycle. Meloidogyne arenaria nematode densities were high during winter grain cover crop harvesting; which may have been the result of increased rainfall before harvesting of winter cover crops. Experiment II conducted at Huxford, AL, during 2006-07 demonstrated contrasting results from that of the first cropping year. In 2005-06 cover crop cycle, R. reniformis nematode densities were low on winter grain cover crop cultivars, whereas R. reniformis densities were high during the 2006-07 winter grain cover crop cycle. The reason for this contrasting result was due to uneven rainfall received during cover crop cycle. The greenhouse results suggest that rye cultivars and oats may be used as winter cover crops in crop rotation to manage R. reniformis nematodes. However, due to adverse climatic conditions these field studies were inconclusive. Greenhouse evaluations Among the different winter grain crop cultivars evaluated in the greenhouse, wheat cultivars ?AGS 2000? and ?Pioneer 26R12? served as hosts for M. incognita. ?Abruzzi? rye also supported higher number of M. incognita nematodes, similar to wheat cultivars. While cultivars ?Elbon?, ?Bob? and ?Georgia Mitchell? were found to be poor hosts, supporting very low populations of M. incognita. This experiment demonstrated that wheat cultivars of ?Pioneer 26R12?, ?AGS 2000?, and rye ?Abruzzi? were more susceptible to M. incognita nematode, suggesting that these crops should not be used as cover crop in crop rotation sequences to manage M. incognita nematodes. Based on the difference in the levels of reproduction of M. incognita nematodes on winter grain crops, 22 oats cultivars ?Georgia Mitchell? and ?Bob?, and ?Elbon? rye gave the better results and could be recommended in crop rotation with peanuts to control M. incognita nematodes. These greenhouse results were similar to those reported by Wang et al., (2004a). Winter grain crop cultivars supported low reproduction of R. reniformis nematode. There was a significant difference between the winter grain crop cultivars and the control. Among the winter grain crop cultivars, wheat cultivars ?Pioneer 26R12? and ?AGS 2000? were most susceptible to R. reniformis, indicating these cultivars were good hosts for R. reniformis. Oats cultivar ?Georgia Mitchell? supported high R. reniformis populations, suggesting this cultivar should not be used in crop rotation. However, the oats cultivar ?Bob? supported low nematode reproduction between the different winter grain crops evaluated. Therefore, cultivars selection is important in crop rotation. Rotylenchulus reniformis reproductions were very low on rye cultivars ?Abruzzi? and ?Elbon?, suggesting these cultivars to be poor-hosts and can be used in a crop rotation sequence with cotton for managing R. reniformis nematodes. 23 Ta ble 1. E ffe ct of w int er gr ain c ov er c rop cul tiva rs on po pul ati ons of pe anut root -knot ne ma tode , M eloi do gy ne ar enar ia a t W ire gra ss R ese arc h a nd Ex tens ion C ent er, H eadl and, A L, dur ing the cr op ping cy cle of ye ar 2005 -06. Cul tiv ar W int er gra in co ve r c rop sa mp lin g Pe an ut sam pli ng __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ No ve mb era Jan ua rya Fe bru ary a Ma rch a Ap rila Ju ne a Au gu sta Oc tob era ___ _______ _______ ________________ _____________ _____________ _____ ___ __ __ __ __ __ ___ __ ___ __ __ __ __ __ ___ __ __ Ab ruz zi 39 .3 13 .5 11 .5 5.5 13 .8 10 22 14 8.3 Fa llo w 42 .5 20 .6 11 .9 12 .3 7.5 15 .9 38 .1 29 6.8 Pio ne er 26 R2 4 49 .9 7.4 11 .1 20 .6 6.3 8.8 45 20 2.5 AG S 2 00 0 17 .3 17 15 .4 19 .9 11 .6 11 .8 49 .9 12 6.9 Elb on 12 .5 17 .3 5 14 .3 18 .8 10 88 .3 68 Bo b 32 .8 9.3 19 .3 24 .3 21 .3 7 34 .8 12 8 Co ke r 9 15 2 12 .4 18 .5 9.6 13 10 .4 14 .8 44 .4 14 8.1 Ge org ia Mi tch ell 23 .8 17 .3 9.8 13 15 10 .3 33 .3 13 7 LS D (P ? 0.0 5) 48 .1 18 .2 13 .4 17 .5 15 .7 16 37 .7 21 6.4 a N um be r o f n em ato de s p res en t in a 10 0 c m3 sa mp le. Co ve r c rop w as pla nte d i n t he m idd le of No ve mb er 20 05 an d w as ha rve ste d i n A pri l 2 00 6. Pe an uts w ere pl an ted in Ju ne an d h arv est ed in O cto be r. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0.0 5). 24 Ta ble 2. E ffe ct of w int er gr ain c ov er c rop cul tiva rs on po pul ati ons of pe anut root -knot ne ma tode , M eloi do gy ne ar enar ia a t W ire gra ss R ese arc h a nd Ex tens ion C ent er, H eadl and, A L, dur ing the cr op ping cy cle of ye ar 2006 -07. Cu ltiv ar W int er gra in co ve r c rop sa mp lin g Pe an ut sam pli ng __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ No ve mb era Jan ua rya Fe bru ary a Ma rch a Ap rila Jun ea Au gu sta Oc tob era ___ _______ _______ ________________ _____________ _____________ _____ ___ __ __ __ __ __ ___ __ __ __ ___ __ __ __ __ __ ___ Co ke r 9 15 2 14 8.2 12 .6 11 .8 78 .8 52 7.8 89 .5 10 9.3 15 4.3 Ge org ia Mi tch ell 13 7 19 .5 35 .5 62 35 3.8 77 51 .3 20 6 Pio ne er 26 R1 2 20 2.5 41 .2 26 41 31 5 12 1.7 51 10 2.8 AG S 2 00 0 12 6.8 15 .5 23 .3 23 .8 16 7.3 10 9.2 13 4.8 23 8 Pio ne er 26 R6 1 44 44 9.5 43 22 5 83 .5 44 .8 19 9.3 Elb on 68 12 .6 10 .8 47 .8 36 0.3 96 10 2.8 25 0.8 Pa no la 11 11 22 29 .3 21 2.3 70 .5 57 .5 57 .5 Fa llo w 29 6.7 21 .2 12 .5 46 .5 63 0.8 96 31 .8 15 4.5 Bo b 12 8 20 .7 16 .5 38 .3 37 9.5 83 .5 83 .5 21 8.8 Ab ruz zi 14 8.2 25 9.5 60 .3 14 7.8 51 .3 64 .3 96 .3 LS D (P ? 0.0 5) 21 6.4 29 25 39 .5 31 5 13 6 76 .5 15 1.3 a N um be r o f n em ato de s p res en t in a 10 0 c m3 sa mp le. Co ve r c rop w as pla nte d i n t he m idd le of No ve mb er 20 06 an d w as ha rve ste d i n A pri l 2 00 7. Pe an uts w ere pl an ted in Ju ne an d h arv est ed in O cto be r. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed LS D (P ? 0 .05 ). 25 Ta ble 3. E ffe ct of w int er gr ain c ov er c rop cul tiva rs on po pul ati ons of re nif orm ne ma tode , R oty lenc hul us r eni for mi s a t Hux for d, A L, dur ing the cr opp ing cy cle of ye ar 20 05 -06. Cu ltiv ar W int er gra in co ve r c rop sa mp lin g Co tto n s am pli ng __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ No ve mb era Jan ua rya Fe bru ary a Ma rch a Ap rila Jun ea Au gu sta Oc tob era ___ _______ _______ ________________ _____________ _____________ _____ ___ __ __ __ __ __ ___ __ __ __ __ __ ___ __ __ __ __ _ AG S 2 00 0 65 3.5 15 7 99 .3 10 8.8 11 5.3 11 5.3 20 85 .5 36 7.7 5 Pio ne er 26 R2 4 65 1.2 15 0 93 .8 85 .5 10 5.3 10 5.3 19 81 .5 35 7.2 5 Fa llo w 53 4.7 23 0 12 1.2 3 5 11 7.8 11 7.8 13 70 .7 59 4.5 Elb on 56 4.5 25 6 12 0.5 10 6 82 .5 82 .5 38 94 .2 36 9.2 5 Bo b 52 7.5 13 6 11 5 12 0 13 5.3 13 5.3 16 60 .5 36 6.5 Ab ruz zi 61 6 16 5 75 .3 87 12 9.5 12 9.5 27 03 .5 28 1 Ge org ia Mi tch ell 50 8.7 83 .5 77 66 10 6.3 10 6.3 24 20 .2 53 8.2 5 Co ke r 9 15 2 48 4 14 9.5 14 5.2 55 .5 13 0.8 13 0.8 25 11 .5 19 3.5 Pa no la 62 7 11 9 69 .8 61 96 96 13 44 .5 41 9.2 5 LS D (P ? 0.0 5) 34 5.9 18 0.9 67 .9 81 .7 60 .7 60 .7 11 64 .9 30 3.7 a N um be r o f n em ato de s p res en t in a 10 0 c m3 sa mp le. Co ve r c rop w as pla nte d i n t he m idd le of No ve mb er 20 06 an d w as ha rve ste d i n A pri l 2 00 7. Co tto n w as pla nte d i n J un e a nd ha rve ste d i n O cto be r. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed LS D tes t (P ? 0.0 5). 26 Ta ble 4. E ffe ct of w int er gr ain c ov er c rop cul tiva rs on po pul ati ons of re nif orm ne ma tode , R oty lenc hul us r eni for mi s a t Hux for d, A L, dur ing the cr opp ing cy cle of ye ar 20 06 -07. Cul tiv ar W int er gra in co ve r c rop sa mp lin g Co tto n s am pli ng __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ No ve mb era Jan ua rya Fe bru ary a Ma rch a Ap rila Jun ea Au gu sta Oc tob era A GS 20 00 10 7.5 33 4.3 26 3.5 44 4 28 9.3 28 9.3 38 6.3 37 7 Pio ne er 26 R2 4 21 0 37 3 26 5.7 40 5.3 48 2.5 48 2.5 53 4 45 4.3 Fa llo w 14 5.3 70 1.5 32 8 33 4.5 37 9.3 37 9.3 34 1 46 7.5 Elb on 17 4.8 37 9.5 33 4.5 53 3.8 37 3 37 3 43 7.7 40 3.3 Bo b 35 2 38 9.8 42 4.3 43 1 73 3.8 73 3.8 58 5.5 47 6 Ab ruz zi 23 8.5 8 62 .3 54 7 38 6 54 0.5 54 0.5 45 0.3 36 5.3 Ge org ia Mi tch ell 30 0.8 79 8 57 9 63 7 54 6.8 54 6.8 30 2.3 42 0.3 Co ke r 9 15 2 28 0.3 66 9 45 6.8 45 0.5 62 4 62 4 18 0 18 6.3 Pa no la 28 2 43 0.8 35 3.8 50 8.3 63 0.5 63 0.5 40 5.3 41 1.8 LS D (P ? 0.0 5) 16 1.3 53 1.3 53 1.4 36 1.5 41 7.8 41 7.8 31 7.4 23 6.3 a N um be r o f n em ato de s p res en t in a 10 0 c m3 sa mp le. Co ve r c rop w as pla nte d i n t he m idd le of No ve mb er 20 06 an d w as ha rve ste d i n A pri l 2 00 7. Co tto n w as pla nte d i n J un e a nd ha rve ste d i n O cto be r. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0.0 5). 27 Ta ble 5. E ffe ct of w int er gr ain c rop cul tiva rs o n t he po pul ati ons of root -knot ne ma tode , M eloi do gy ne inc og nit a i n t he gre enhous e a t the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on c am pus of A ub urn U nive rsi ty, A ub urn, AL . Cul tiv ar Sh oo t w eig ht Ro ot we igh t Eg gs/ gm of ro ot Ne ma tod e e gg sa __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ Ge org ia Mi tch ell 10 .82 10 .04 93 0 2.2 85 7 bc B ob 13 .46 12 .73 67 3 1.8 54 8 c Ab ruz zi 9.0 6 18 .26 13 65 2.6 64 3 b Elb on 9.7 0 23 .65 10 63 2.4 00 5 bc Pio ne er2 6R 12 10 .11 21 .94 16 12 2.8 57 0 b AG S2 00 0 11 .15 19 .57 16 95 2.9 01 4 b To ma to 9.7 7 8.7 1 39 51 3.5 16 2 a LS D (P ? 0.0 5) 0.8 64 a L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0.0 5). 28 Ta ble 6. E ffe ct of w int er gr ain c rop cul tiva rs o n p op ula tions of re nif orm ne ma tode , R oty lenc hul us r eni for mi s i n t he gre enhous e a t the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on c am pus of A ub urn U nive rsi ty, A ub urn, AL . Cul tiv ar Sh oo t w eig ht Ro ot we igh t Eg gs/ gm of ro ot Ne ma tod e e gg sa __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ Ge org ia Mi tch ell 13 .85 9.9 0 50 82 .9 1.5 81 3 bc Bo b 16 .68 11 .64 52 0.3 1.0 53 3 cd e Ab ruz zi 16 .35 28 .81 58 .5 0.5 72 4 e Elb on 20 .22 30 .07 15 6.5 0.9 65 5 de Pio ne er 26 R1 2 19 .12 27 .19 10 63 .4 1.6 12 6 b AG S 2 00 0 19 .93 29 .56 14 17 .4 1.4 56 5 bc d Co tto n 7.2 0 6.3 2 15 13 7.6 2 3.1 33 4 a LS D (P ? 0.0 5) 0.5 43 a L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0.0 5). 29 III. EVALUATION OF CROTALARIA JUNCEA POPULATIONS TO MANAGE PLANT-PARASITIC NEMATODES INTRODUCTION Crotalaria juncea L. (sunn hemp) is a legume crop that has received attention due to its green manure properties, the potential to fix nitrogen and its ability to suppress plant-parasitic nematodes (McSorley et al., 1999). It is also reported to increase number of free-living nematodes and improve nutrient levels in soils with low organic matter content. Considering all these beneficial qualities, C. juncea may be used effectively in organic and sustainable agricultural systems to suppress plant-parasitic nematodes. The major constraint affecting the extensive use of C. juncea in the continental United States is its limited reproduction and non-availability of seeds on large scale for cultivation. Crotalaria juncea cannot reproduce under these climatic conditions. In United States, most of the C. juncea evaluations and breeding programs were limited to Hawaii (Rotar and Joy, 1983). To overcome the problem of seed production, C. juncea populations collected from different countries were evaluated for their ability to produce seeds under the Southeastern U.S. climatic conditions. Along with the seed production evaluations, the nematode suppressive effect of these populations was evaluated. This research was conducted to evaluate the efficiency of different C. juncea populations to suppress the 30 southern root-knot nematode, Meloidogyne incognita and the reniform nematode, Rotylenchulus reniformis. MATERIALS AND METHODS Crotalaria juncea populations collected in different countries were obtained from the National Plant Germplasm System. Seed of these populations were increased under the same environmental conditions and under isolation in 2003 at Tallassee, AL. The populations were: PI 207657 from Srilanka, PI 314239 from Russia, PI 322377 from Brazil, PI 391567 from South Africa and PI 426626 from Pakistan. These C. juncea populations were evaluated for their ability to suppress southern root-knot nematode, M. incognita and reniform nematode, R. reniformis in the greenhouse and in the field. The C. juncea host status was evaluated in the greenhouse at the Plant Science Research Center, Auburn University, AL and in one field experiment at E V Smith Research Center (EVSRC), Shorter, AL during the summer of 2006. Tropic Sun (C. juncea cv. Tropic Sun) was used as the control in the field experiment. Additional populations of Selection FCU-2005 and Selection PBU-2005 were also used in mixed planting experiments and in a field evaluation. Nematode extraction from roots Meloidogyne incognita and R. reniformis populations were maintained on the host plants of tomato (Lycopersicon esculentum cv. Rutgers) and cotton (Gossypium hirsutum 31 cv. DP 555 BG/RR), respectively. Nematode eggs on the roots were extracted using 0.6% sodium hypochlorite solution (Hussey and Barker, 1973). The hypochlorite solution containing nematode eggs was allowed to pass through a series of sieves with 350-?m sieve on top and 500-?m at the base. Nematode eggs were collected on 500-?m sieve and were gently washed with water. The solution containing nematode eggs was standardized and quantified under an (Nikon-T 100?) inverted microscope. Greenhouse evaluations of C. juncea populations Experiments were conducted to evaluate the host status of C. juncea populations for plant-parasitic nematodes. Crotalaria juncea populations were grown in 500 cm3 polystyrene cups filled with autoclaved loamy sand soil field soil (72.5%, 25%, 2.5%, S- S-C, pH 6.4) and fine sand in 3:1 ratio. One C. juncea seed of each treatment was hand- sown in each cup. Tomato (Lycopersicon esculentum cv. Rutgers) and cotton (Gossypium hirsutum cv. DP 555 BG/RR) were used as the controls for M. incognita and R. reniformis nematodes, respectively. The experiment was arranged in a completely randomized block design, replicated eight times and repeated twice. Crotalaria juncea seeds were allowed to germinate and grow for one week. On the day of nematode infestation, nematode eggs were extracted from host plants as described previously. One week after the C. juncea germination, the seedlings were inoculated with ca. 4000 eggs of M. incognita or ca. 2000 eggs of R. reniformis by making small holes near the root zone of the plants by using a Repipett? Jr. Dispenser. After 50 days the C. juncea roots were gently washed, fresh weight of the aerial parts and root weight was 32 recorded. Nematodes present on the roots of C. juncea populations were extracted as described above and the numbers of eggs present were counted. The number of eggs present per gram of root was calculated, and the data were analyzed. Staining of C. juncea roots 10 grams of C. juncea roots of all the populations infested with M. incognita or R. reniformis were stained using McCormick Schilling? red food color (Thies et al., 2002). Crotalaria juncea roots were washed gently with water and blotted dry by using a paper towel. The roots were cut in to 2-cm-pieces and were suspended for 15 sec in a 500-ml beaker containing 10% (v/v) solution of McCormick Schilling? red food color (Thies et al., 2002). The stained roots were rinsed in tap water and blotted dry. Meloidogyne incognita and R. reniformis egg masses present in the roots were observed and counted under a Nikon T-100? inverted microscope (10x). To identify the nematode presence within the root tissues, the stained roots were suspended into acidified glycerin (40 ml glycerin and 5 drops of 5N HCL). The roots were mounted between glass microscopic slides to observe the juvenile stages present in roots and photographs were taken using a Nikon? Coolpix4500 camera (4 mega pixels). For counting the number of M. incognita juveniles present within the roots, the roots were chopped in an Oster? blender for 20 sec by adding water; the chopped suspension was observed under a Nikon? inverted microscope and juvenile stages were counted. 33 Nematicidal activity of freeze-dried C. juncea root exudates To evaluate the nematicidal activity of C. juncea root exudates, the roots of C. juncea plants were submerged in a beaker containing 250 ml distilled water. The beakers were wrapped with an aluminum foil to prevent photolysis of the exudates. The beakers were agitated using an Environ? automatic shaker @ 150 rpm for 12 hours. Crotalaria juncea root exudates collected in the beakers were decanted into brown plastic bottles and stored at 0 oC. These exudates were freeze-dried using a freeze drier. Freeze-dried root exudates were reconstituted by adding 15 ml of distilled water. Nematode isolates maintained on susceptible hosts of tomato and cotton was extracted using 0.6% NaOCl (Hussey and Barker, 1973). Nematode eggs were allowed to hatch by placing in an incubator at 25 oC for one day. Approximately, 25 second-stage juveniles (J2) were handpicked and added to the glass vials containing 5 grams of sand (Halbrendt et al., 2007). The treatments included three different concentrations of root exudates, 50- ?l, 100-?l, 250-?l and the water control. Root exudates were added using a 1000-?l Eppendorf? micropipette, and water was added to make up the volumes in the vials to one ml. This entire experiment was arranged in a complete randomized block design in five replications. After 24 hours, the nematodes were washed into a series of sieves with 350-?m on top and 500-?m at the base. The suspended solution on 500-?m sieve was transferred in to Petri plates and the number of alive J2 stages was counted under Nikon? inverted microscope. The nematodes that were moving and burst open when squished using a needle was considered alive. In contrast, nematodes that were straight in shape, paralyzed and didn?t burst open when squished were considered dead. 34 Mixed planting of C. juncea Crotalaria juncea populations described previously were planted with tomato or cotton in the greenhouse to evaluate their potential to suppress M. incognita or R. reniformis. Autoclaved loamy sand soil field soil (72.5%, 25%, 2.5%, S-S-C, pH 6.4) and sand were mixed in 3:1 ratio and placed in 2000 cm3 plastic pots. Plastic squares cut from a large plastic screen were placed vertically in the center of each pot, dividing it in to equal portions. One seed of a C. juncea population was hand-sown in one half of the pot and one tomato or cotton seed was sown on the other half of the pot. The control pots were planted with tomato or cotton plants on both sides of the screen. The entire experiment was arranged in a completely randomized block design on raised benches in 8 replications and both the experiments were repeated twice. Two weeks after germination of C. juncea plants, ca. 6000 M. incognita or R. reniformis nematodes were infested at the center of the pot near the base of the screen by making small holes. Shoot weight and root weight of C. juncea and tomato or cotton controls was recorded after 50 days. Nematode present on the roots were extracted using hypochlorite method and number of eggs present per gram of root was determined. Field evaluation Field evaluation of C. juncea populations was conducted to evaluate their effect on Meloidogyne spp. at E V Smith Research Center, Shorter, AL. This experiment was conducted during the summer months from June to August 2007. The treatments included the C. juncea populations described above, one fallow and tomato control, replicated four 35 times. Crotalaria juncea populations were planted as a summer crop. Each plot (1 m2) was sampled in a zigzag pattern before planting C. juncea. Five probes of soil was taken, composited into one sample per plot and stored in plastic bags. These samples were maintained at a temperature of 10 oC until nematode extraction. A subsample of soil (100 cm3) was used and nematodes were extracted using gravity screening and centrifugal flotation method and quantified (Jenkins, 1964). Each plot was hand sown with five C. juncea seeds per population by marking with a wooden marker. Tomato was planted as a control in one plot. Weeding was done manually every week for the entire experimental period. All plants and root systems were harvested after 50 days by digging with a shovel. Plants from each plot were placed in separate plastic bags. Fresh weight of leaves and stem and roots was recorded. Soil samples were collected at time of harvesting and nematodes were extracted. Nematodes present on the roots were extracted as described above in nematode extraction from roots. After harvest of C. juncea, all the plots were replanted with two-wk-old tomato seedlings at the same place where the C. juncea plants were planted previously. Tomato plants were harvested manually 50 days after planting and soil samples were collected. Fresh weight and root weights were recorded, and nematodes were extracted from the soil samples and tomato roots. Data analysis The greenhouse data represented the number of eggs present per gram of root weight and the field data reflected the number of nematodes present in each soil sample. 36 Data from both field and greenhouse were log transformed and the transformed data was analyzed using Proc GLM in Statistical Analysis Systems software (SAS institute, Inc., Cary, NC). The significance of effects of C. juncea treatments was determined by the probability of F-value (P less than or equal to 0.05). Treatment means was separated by Fisher?s protected Least Significant Differences, and the suppressive effect of C. juncea populations on M. incognita and R. reniformis nematodes was compared. RESULTS Greenhouse evaluations of C. juncea populations Crotalaria juncea populations supported very low populations of M. incognita nematodes. There was a significant difference in M. incognita populations (P ? 0.05) between all the C. juncea populations and the control. In M. incognita experiment, all the C. juncea populations supported low reproduction and tomato supported the highest reproduction (Table 1). Among the different C. juncea populations evaluated, there were no significant differences between the populations. The numerically relative decreasing order of M. incognita reproduction on C. juncea populations was tomato, PI 207657, PI 314239, PI 322377, PI 391567, and PI 426626 (Table 1). Crotalaria juncea populations also demonstrated a significant difference (P ? 0.05) on suppression of R. reniformis reproduction (Table 2). All the C. juncea populations supported low R. reniformis reproduction while the control cotton supported 37 the highest R. reniformis populations (Table 2). Between the C. juncea populations, PI 322377 supported numerically the lowest R. reniformis reproduction and population PI 314239 supported the highest R. reniformis reproduction. However, there were no significant differences between C. juncea populations on R. reniformis reproduction. The numerically relative decreasing susceptibility of C. juncea for R. reniformis was cotton, PI 314239, PI 391567, PI 426626, PI 207657, and PI 322377. Staining of C. juncea roots All the C. juncea populations were found to contain few M. incognita nematode juveniles (Fig. 1) and adults (Fig. 2) within the root tissues. All the juvenile stages J2, J3 of M. incognita were observed within the roots. In case of R. reniformis nematodes, only 1-2 adult females (Fig. 3 and 4) were present semi-endoparasitically inside the C. juncea root tissues. Nematicidal activity of freeze-dried C. juncea root exudates Crotalaria juncea freeze-dried root exudates were found to kill most of the M. incognita (Table 3) and R. reniformis nematodes (Table 4). There was a significant difference (P ? 0.05) in the nematode mortality rates between the C. juncea root exudates and the water control. The nematode mortality rate was highest at 250-?l concentration. Higher concentrations resulted in higher mortality rates. 38 Mixed planting of C. juncea Mixed planting of C. juncea populations had no significant effect (P ? 0.05) on M. incognita populations (Table 5). However, population?s PI 391567, PI 314239, Selection FCU-05, PI 207657, PI 322377, and Tropic Sun supported numerically lower M. incognita reproduction than the control. Crotalaria juncea populations had no significant effect (P ? 0.05) on R. reniformis suppression (Table 6). Planting of cotton with C. juncea population?s PI 322377, PI 426626, Selection PBU-2005, Tropic Sun and Selection FCU-2005 supported numerically lower R. reniformis nematode densities than the control. Field evaluation Field evaluations provided no significant difference (P ? 0.05) between the C. juncea populations, follow and the tomato control (Table 7). However, the continuous cultivation of tomato followed by tomato supported numerically highest Meloidogyne spp. counts whereas tomato grown in the plots after harvesting of C. juncea was found to support lower nematode populations (Table 7). C. juncea populations PI 322377, Tropic Sun and PI 207657 were found to reduce Meloidogyne spp. counts when compared to the fallow treatment. 39 DISCUSSION Greenhouse evaluations of C. juncea populations All C. juncea populations tested suppressed M. incognita populations more effectively when compared to tomato control. Greenhouse studies revealed that C. juncea populations were effective in reducing the M. incognita nematode densities. The difference in M. incognita reproduction between C. juncea populations may be due to the different genetic constitution of the plants. Crotalaria juncea populations were found to significantly decrease R. reniformis nematodes when compared to cotton control. Among the C. juncea populations, PI 322377 supported numerically the lowest R. reniformis nematode reproduction. In contrast, population PI 314239 supported the highest R. reniformis reproduction. The reason for varying nematode reproduction levels on C. juncea populations may be due to the different genetic constitution of the plants. However, all the C. juncea populations were able to significantly decrease R. reniformis populations. Staining of C. juncea roots Different M. incognita juvenile stages and few adults were present within the root system, indicating there was penetration of M. incognita. This experiment demonstrated that M. incognita juveniles were able to pierce the C. juncea roots, but in limited numbers. 40 Adult females of the R. reniformis nematodes were present semi-endoparasitically inside the roots, indicating that R. reniformis can infest and reproduce on the C. juncea roots. However, the R. reniformis reproduction was very low when compared to the cotton control. Nematicidal activity of freeze-dried C. juncea root exudates Crotalaria juncea freeze-dried root exudates were able to kill the M. incognita and R. reniformis nematodes. The mechanism responsible for nematode mortality is not clearly known but might be due to some nematotoxic compounds released from the roots in to the water. Root exudates may serve as an alternative to chemical nematicides in organic production systems. Further biochemical studies have to be conducted to determine the chemical nature of the compounds present in the C. juncea root exudates. The chemical nature of C. juncea root exudates is not known but these exudates were found to kill the nematodes. Mixed planting of C. juncea In the mixed planting of C. juncea with tomato or cotton, C. juncea populations were able to suppress the nematodes when compared to control. The reason for numerically low nematode densities on tomato or cotton mixed planted with C. juncea might be the result of nematode suppression resulted from root exudates produced by the C. juncea populations. However, there was no significant difference (P ? 0.05) on nematode populations between the control and C. juncea planted with tomato or cotton. Nematodes may have been more affected if the C. juncea was planted earlier than tomato 41 or cotton. Further studies should focus on mixed cropping C. juncea populations with agronomic crops to control plant-parasitic nematodes. Field evaluation Field studies demonstrated that there were no significant differences between the continuous cultivation of tomato and the tomato grown in rotation after C. juncea. However, C. juncea populations were found to decrease small populations of Meloidogyne spp. densities on the tomato grown after C. juncea cultivation. In contrast, continuous cultivation of tomato increased Meloidogyne spp. densities during the summer months and tomato grown in the next cropping season. Extreme temperatures that prevailed during the summer months under field conditions, accompanied by lack of rainfall might be reason for no significant differences as was observed in the greenhouse evaluations. Even though the plots were hand-irrigated, the lack of moisture and high temperatures may have played a role. Therefore, further field studies with C. juncea populations under controlled irrigation conditions should be carried out to determine the potential of C. juncea populations as summer cover crop to manage plant-parasitic nematodes in organic farming and sustainable agriculture. 42 Ta ble 1. E ffe ct of Cr otal ari a j unc ea po pul ati ons on sout he rn root -knot ne ma tode , M eloi do gy ne inc og nit a unde r c ont rol led condi tions at the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on c am pus of A ub urn U nive rsi ty, A ub urn, A L. Cul tiv ar Sh oo t w eig ht Ro ot we igh t Ne ma tod e e gg sa Ne ma tod e e gg sb __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ PI 20 76 57 11 .4 8.7 22 0 1.3 07 0 b PI 31 42 39 15 .2 12 .4 15 1 1.1 65 9 b PI 32 23 77 13 .5 14 76 1.0 27 2 b PI 39 15 67 17 .1 13 .6 10 1 0.7 31 9 b PI 42 66 26 16 .0 11 .8 37 0.5 17 0 b To ma to 10 .5 12 .3 11 17 1 3.7 29 2 a LS D (P ? 0.0 5) 0.8 63 6 a Nu mb er of ne ma tod es pre sen t p er gra m of roo t w eig ht. b L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 43 Ta ble 2. E ffe ct of Cr otal ari a junc ea p op ula tions on reni for m ne ma tode , R oty lenc hul us reni for mi s unde r c ont rol led condi tions at the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on c am pus of A ub urn U nive rsi ty, A ub urn, A L. Cul tiv ar Sh oo t w eig ht Ro ot we igh t Ne ma tod e e gg sa Ne ma tod e e gg sb __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ PI 20 76 57 16 .1 16 .5 26 .1 0.4 85 3 cd PI 31 42 39 16 .1 15 .4 28 2.4 1.1 44 0 b PI 32 23 77 16 .5 20 .2 13 .9 0.1 63 7 d PI 39 15 67 16 .6 16 .9 13 5.2 0.8 91 3 bc PI4 26 62 6 17 .0 17 .4 80 .1 0.6 81 7 bc d Co tto n 9.5 9.5 21 22 .8 3.2 41 6 a LS D (P ? 0.0 5) 0.7 53 9 a Nu mb er of ne ma tod es pre sen t p er gra m of roo t w eig ht. b L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 44 T ab le 3. E va lua tion o f the ne ma tic ida l a cti vit y of C rot alar ia junc ea po pul ati ons root ex uda tes on r oot -knot ne ma tode , Me loi do gy ne inc og nit a. Pop ula tio n Ro ot we igh t Nu mb er of ne ma tod es ali ve in di ffe ren t c on cen tra tio n o f C . ju nc ea roo t e xu da tes __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ 50 -?l a 10 0-? la 25 0-? la __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ _ PI 20 76 57 38 .1 0.1 8 de 0.0 6 c 0 c PI 31 42 39 23 .9 0.6 4 b 0.4 8 b 0.4 b PI 32 23 77 33 .3 0.4 8 bc 0.3 bc 0.2 5 b PI 39 15 67 28 .6 0.3 3 cd 0.1 5 c 0.0 6 c PI 42 66 26 33 .7 0.1 4 e 0.1 2 c 0 c Co ntr ol -- 1.2 4 a 1.2 4 a 1.2 5 a LS D (P ? 0.0 5) 0.1 7 0.2 0.1 6 a L og (x +1 ) o f n um be r o f n em ato de s a liv e i n d iff ere nt co nc en tra tio ns of Cr ota lar ia jun cea roo t-e xu da tes . Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 45 Ta ble 4. E va lua tion of the ne ma tic ida l a cti vit y of C rot alar ia junc ea po pul ati ons root ex uda tes on r eni for m ne ma tod e, Rot yle nc hul us r eni for mi s. Pop ula tio n Ro ot we igh t Nu mb er of ne ma tod es ali ve in di ffe ren t c on cen tra tio n o f C . ju nc ea ro ot ex ud ate s __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ 50 ?l 10 0? l 25 0? l __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ _ PI 20 76 57 38 .1 0.3 7 c 0.1 8 c 0 d PI 31 42 39 23 .9 0.6 8 b 0.4 8 b 0.2 7 bc PI 32 23 77 33 .3 0.6 2 b 0.3 9 bc 0.3 7 b PI 39 15 67 28 .6 0.4 c 0.2 7 bc 0.1 5 cd PI 42 66 26 33 .7 0.2 7 c 0.1 8 c 0 d Co ntr ol -- 1.2 5 a 1.2 7 a 1.2 6 a LS D (P ? 0.0 5) 0.1 9 0.2 8 0.1 5 a L og (x +1 ) o f n um be r o f n em ato de s a liv e i n d iff ere nt co nc en tra tio ns of Cr ota lar ia jun cea ro ot- ex ud ate s. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 46 Ta ble 5. E ffe ct of C rot alar ia junc ea po pul ati ons pl ant ed w ith t om ato on r oot -knot ne ma tode , M eloi do gy ne inc og nit a unde r cont rol led c ondi tions at the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on cam pus of A ub urn U nive rsi ty, A ub urn, A L. C. j un cea po pu lat ion s? To ma to pla nts m ixe d p lan ted w ith C . ju nc ea __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ _ Sh oo t w eig ht Ro ot we igh t Ne ma tod e e gg sa Ne ma tod e e gg sb PI 20 76 57 30 .1 5.9 89 92 3.6 6 PI 31 42 39 34 .7 8.7 99 63 3.7 7 PI 32 23 77 44 .8 7.4 62 11 3.6 3 PI 39 15 67 28 .35 6.0 10 95 3 3.8 8 PI 42 66 26 42 .3 8.1 13 67 5 4.0 9 Tr op ic Su n 20 .4 4.9 79 61 3.4 Se lec tio n F CU -05 30 .7 7.7 11 93 9 3.7 1 Se lec tio n P BU -05 23 .8 5.5 15 07 0 3.9 5 To ma to 38 .8 10 .8 96 22 3.8 9 LS D (P ? 0.0 5) 0.5 9 a Nu mb er of ne ma tod es pre sen t p er gra m of roo t w eig ht. b L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht on to ma to pla nts m ixe d p lan ted w ith C . ju nc ea . Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 47 Ta ble 6. E ffe ct of C rot alar ia junc ea po pul ati ons pl ant ed w ith c ott on on r eni for m ne ma tode , R oty lenc hul us r eni for mi s supp res sion unde r c ont rol led c ondi tions at the Pl ant Sc ienc e R ese arc h C ent er, l oc ate d on cam pus of A ub urn U nive rsi ty, Au bur n, A L. Cul tiv ar Co tto n p lan ts mi xe d p lan ted w ith C . ju nc ea __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ Sh oo t w eig ht Ro ot we igh t Ne ma tod e e gg sa Ne ma tod e e gg sb PI 20 76 57 29 .4 5 19 77 3.1 9 PI 31 42 39 28 .3 4.3 27 74 3.2 2 PI 32 23 77 33 .3 6.4 18 53 3.1 2 PI 39 15 67 32 .2 7.7 27 38 3.3 9 PI 42 66 26 30 .22 7.7 13 75 2.9 9 Tr op ic Su n 42 9.7 15 52 2.8 9 Se lec tio n F CU -05 47 .7 8.3 63 4 2.0 8 Se lec tio n P BU -05 29 .5 4.2 26 58 2.9 2 Co tto n 41 .8 12 .1 20 54 3.1 5 LS D (P ? 0.0 5) 0.7 62 8 a Nu mb er of ne ma tod es pre sen t p er gra m of roo t w eig ht. b L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht on co tto n m ixe d p lan ted w ith C rot ala ria ju nc ea po pu lat ion s. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 48 Ta ble 7. E ffe ct of C rot alar ia junc ea po pul ati ons on r oot -knot ne ma tode , M eloi do gy ne sp p. und er f iel d c ondi tions at E V Sm ith R ese arc h C ent er, Shor ter , A L. Po pu lat ion To ma to pla nte d a fte r h arv est ing of C . ju nc ea __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ _ Sh oo t w eig ht Ro ot we igh t Ne ma tod e e gg sa Ne ma tod e e gg sb ____ ___ _____________ _____________ ______________ __ __ ___ __ __ __ __ __ ___ __ __ __ __ __ __ __ __ __ __ __ __ ___ __ __ __ __ __ ___ __ __ __ __ __ ___ __ __ __ ____ ___ ____ ____ ___ ____ ____ ____ ___ ____ ____ ___ ____ ____ ____ ___ ____ ____ ___ ____ ____ PI 20 76 57 33 6.5 38 .35 11 5.2 2.1 58 5 PI 31 42 39 44 7 47 .97 5 34 1.2 2.5 44 7 PI 32 23 77 30 7.2 5 40 .42 5 50 5.2 2.3 26 7 PI 39 15 67 35 7 47 .3 49 6 2.6 64 6 PI 42 66 26 29 7.2 5 39 .75 46 3.5 2.6 42 5 Tr op ic Su n 35 6.5 42 .72 5 45 0.7 2.2 48 1 FC U- 05 41 8.2 5 51 .57 5 50 8.6 2.6 64 6 PB U- 05 19 5 25 .15 43 7.2 2.6 31 8 Fa llo w 26 3.3 3 37 .03 54 3 2.5 30 4 To ma to 19 3.7 5 29 .55 35 65 .9 3.2 08 7 LS D (P ? 0.0 5) 0.6 96 3 a Nu mb er of ne ma tod es pre sen t p er gra m of roo t w eig ht. b L og (x +1 ) o f th e n um be r o f n em ato de eg gs pre sen t p er gra m of roo t w eig ht. Me an s w ith in co lum ns fol low ed by di ffe ren t le tte rs are si gn ific an tly di ffe ren t a cco rdi ng to Fi sch er? s p rot ect ed Le ast Si gn ific an t D iff ere nc e t est (P ? 0. 05 ). 49 Fig 1. Third-stage juvenile (J3) of root-knot nematode, Meloidogyne incognita present inside the Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels). Fig 2. Adult female of root-knot nematode, Meloidogyne incognita present inside the Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels). 50 Fig 3. Female reniform nematode, Rotylenchulus reniformis present semi-endoparasitically on Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? Eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix 4500 camera (4 mega pixels). Fig 4. Nematode eggs inside an adult female of reniform nematode, Rotylenchulus reniformis present semi- endoparasitically on Crotalaria juncea roots, stained using a McCormick Schilling? red food color, observed under a Nikon? eclipse 80i microscope at 40x magnification, photographed using a Nikon? Coolpix4500 camera (4 mega pixels). 51 IV. SUMMARY Cover crops were evaluated in the greenhouse and in field locations to determine their host status and nematode suppressive effect on root-knot nematodes, Meloidogyne incognita and M. arenaria and the reniform nematode, Rotylenchulus reniformis. The winter grain cover crop cultivars included four commercially available cultivars of wheat (Triticum aestivum) ?Pioneer 26R12?, ?AGS 2000?, ?Coker 9152?, ?Panola?; two cultivars of oats (Avena sativa) ?Georgia Mitchell? and ?Bob?; and two cultivars of rye (Secale cereale) ?Elbon? and ?Abruzzi?. This research also evaluated the host status and nematode suppressive effect of Crotolaria juncea (sunn hemp) populations. The treatments included the C. juncea populations; PI 207657 from Srilanka, PI 314239 from Russia, PI 322377 from Brazil, PI 391567 from South Africa and PI 426626 from Pakistan collected in different countries and increased under the same environmental conditions and under isolation in 2003 at Tallassee, AL. Crotalaria juncea commercially available cultivar ?Tropic Sun? was also used for evaluations. Winter grain cover crop cultivars described above were evaluated to determine M. arenaria and R. reniformis suppressiveness and their subsequent effects on peanuts and cotton. Field evaluations were conducted at Wiregrass Research and Extension Center, Headland, AL and in a grower?s field in Huxford, AL to determine the effect of winter grain cover crops on M. arenaria and R. reniformis, respectively. The treatments included 52 commercially available cultivars of wheat, oats and rye described above at both the test locations. There were no significant differences (P ? 0.05) between winter grain cover crop cultivars on nematode suppression. This was most probably due to severe drought and uneven rainfall during both the cropping years at the two test locations. However, the greenhouse studies indicated that the wheat cultivars ?Pioneer 26R12? and ?AGS 2000?and rye cultivar ?Abruzzi? were hosts to M. incognita nematodes, whereas ?Elbon? rye, oats cultivars ?Bob? and ?Georgia Mitchell? supported low populations of M. incognita. The greenhouse studies of winter cover crop cultivars with R. reniformis nematodes demonstrated that the wheat cultivars ?Pioneer 26R12? and ?AGS 2000? and oats cultivar ?Georgia Mitchell? were good hosts, while ?Bob? oats and the rye cultivars ?Elbon? and ?Abruzzi? supported significantly (P ? 0.05) lower R. reniformis nematode populations. This suggests that these oats and rye cultivars are poor hosts and might be used in a crop rotation sequence with agronomic crops to manage these nematodes. Crotalaria juncea populations were able to suppress M. incognita and reniform nematodes in the greenhouse tests. Significant difference (P ? 0.05) was observed in nematode reproduction between C. juncea populations and the control. This indicated that the C. juncea populations could be an efficient summer cover crop to manage M. incognita and R. reniformis nematodes. Roots of C. juncea populations infested with M. incognita and R. reniformis were stained using a McCormick Schilling? red food color (Thies et al., 2002) to determine if either genus could complete its life cycle on the roots. All juvenile stages of M. incognita were found as well as low numbers of mature females with egg masses and 1-2 adult female reniform nematodes were present per 10 gm of 53 roots, indicating that these nematodes were able to infest and reproduce on the roots of C. juncea populations. However, the reproduction on C. juncea was very low when compared to the controls. In the evaluation of the nematicidal activity of C. juncea root exudates, root exudates were collected from each population and freeze-dried. The exudates were reconstituted and tested against both M. incognita and R. reniformis nematodes at several concentrations. All concentrations could kill both nematodes whereas the water control had no effect. The field trial conducted in the summer (2007) at E V Smith Research Center, Shorter, AL indicated that continuous cultivation of tomato followed by tomato increased the nematode densities whereas tomato planted after C. juncea was found to support low Meloidogyne spp. nematode densities. However, there were no significant differences observed on Meloidogyne spp. suppression between the different C. juncea populations evaluated. The knowledge obtained from this study suggests that some winter cover crop cultivars and C. juncea populations may be suitable for crop rotation in a region with specific nematode histories, thus minimizing usage of synthetic nematicides and yield losses. However, further research studies should focus on extensive long-term field studies on cover crops nematode suppressive effect under controlled irrigation conditions. 54 LITERATURE CITED Agrios, G. N. 1997. Plant Pathology 4th Edition. Academic Press, San Diego, CA: 568- 569. Araya, M. and E. P. Caswell-Chen. 1994. Host status of Crotalaria juncea, Sesamum indicum, Dolichos lablab, and Elymus glaucus to Meloidogyne javanica. Journal of Nematology 26: 492?497. Balkcom, K. S. and D. W. Reeves. 2005. Sun hemp utilized as a legume cover crop for corn production. American Society of Agronomy Journal 97: 26-31. Barker, K. R. and S. R. Koenning. 1998. Developing Sustainable Systems for Nematode Management. Annual Review of Phytopathology 36: 165-205. Blassingame, D. 2007. Cotton Disease Loss Estimate. in Proceedings of the Beltwide Cotton Conferences, New Orleans, LA. 9-12 January 2007. National Cotton Council of America, Memphis, TN. Cook, C. G. and L. N. Namken. 1993. Reniform nematode effects on yield and fiber quality of cotton. Proceedings of the Beltwide Cotton Conferences 1: 1727-1729. Crittenden, H. W. 1961. Studies on the host range of Meloidogyne incognita acrita. Plant Disease Reporter 45: 190?191. 55 Fletcher, S. M. 2002. Peanuts: Responding to Opportunities and Challenges from an Intertwined Trade and Domestic Policies. The University of Georgia, National Center for Peanut Competitiveness. Athens, GA. Fry, J. 2001. Industry and Trade Summary Cotton. U. S. Industrial Trade Commission Publication 3391: 3. Gazaway, W. S. and K. S. McLean. 2003. A survey of plant-parasitic nematodes associated with cotton in Alabama. Journal of Cotton Science 7: 7-17. Germani, G. and C. Plenchette. 2004. Potential of Crotalaria spp. as green manure crops for the management of pathogenic nematodes and beneficial mychorrhizal fungi. Plant and Soil 266: 333-342. Guar, H. S. and R. N. Perry. 1991. The Biology and Control of the Plant Parasitic Nematode Rotylenchulus reniformis. Agricultural Zoology Reviews 4: 177-212. Halbrendt, J. M., J. Dean, C. P. Rice, and I. A. Zasada. 2007. Relating the glucosinolate profile of Tropaelum majus cultivars to Xiphinema americanum mortality. Journal of Nematology 36: 91 Handoo, Z. A. 1998. Plant-parasitic nematodes. USDA ARS Publication: http://www.ars.usda.gov/Services/docs.htm?docid=9628. Hooks, C. R. R., H. R. Valenzuela, and J. Defrank. 1998. Incidence of pests and arthropod natural enemies in zucchini grown with living mulches. Agriculture, Ecosystems and Environment 69: 217-231. 56 Hussey, R. S. and K. R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Disease Reporter 57: 1025-1028. Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48: 692. Jones, J. R., K. S. Lawrence, and G. W. Lawrence. 2006. Evaluation of winter cover crops in cotton cropping for management of Rotylenchulus reniformis. Nematropica 36: 53-66. Jourand, P., S. Rapior, M. Fargette, and T. Mateille. 2004. Nematostatic activity of aqueous extracts of West African Crotalaria species. Nematology 6: 765-771. Koenning, S. R., J. A. Wrather, T. L. Kirkpatrick, N. R. Walker, J. L. Starr, and J. D. Mueller. 2004. Plant-Parasitic Nematodes Attacking Cotton in the United States: Old and Emerging Production Challenges. Plant Disease 88: 100-113. Lawrence, G. W. and K. S. McLean. 2001. Reniform nematodes. Pp. 42-44 in D. L. Kirkpatrick and C. S. Rothrock, eds. Compendium of Cotton Diseases second edition. The Disease Series of the American Phytopathological Society. St. Paul, MN. Lee, J. A. 1984. Cotton as a World Crop. Pp. 6-24 in R. J. Kohel and C. F. Lewis, eds. Cotton. American Society of Agronomy, Madison, WI. Mani, A., Z. Handoo, and S. Livingston. 2005. Plant-Parasitic Nematodes Associated with Date Palm Trees (Phoenix Dactylifera L.) in the Sultanate of Oman: Nematropica 35: 135-144. 57 Mansoer, Z., D. W. Reeves, and C. W. Wood. 1997. Suitability of sun hemp as an alternate late-summer legume cover crop. Soil Science Journal 61: 246-253. McSorley, R. 1994. Changes in population densities of Meloidogyne spp. and Paratrichodorus minor on winter rye cover crops. Nematropica: 151-160. McSorley, R. 1999. Host susceptibility of potential cover crops for root-knot nematodes. Supplement to the Journal of Nematology 31: 619-623. McSorley, R., D. W. Dickson, J. A. De Brito, T. E. Hewlett, and J. J. Frederick. 1994. Effects of tropical rotation crops on Meloidiogyne arenaria population densities and vegetable yields in micro plots. Journal of Nematology 26: 175-181. NASS, USDA. 2005. Crop Production, November 2005: http://usda.mannlib.cornell.edu/usda/nass/CropProd//2000s/2005/CropProd-11-10- 2005.pdf. NASS, USDA. 2006. Crop Production, November 2006: http://usda.mannlib.cornell.edu/usda/nass/CropProd//2000s/2006/CropProd-11-09- 2006.pdf. Opperman, C. H., J. R. Rich, and R. A. Dunn. 1988. Reproduction of three root-knot nematodes on winter small grain crops. Plant Disease 72: 869-871. Porter, M. D., D. H. Smith, and R. R. Kabana., eds. 1984. Compendium of peanut diseases. The Disease Series of the American Phytopathological Society. St. Paul, MN. 58 Rich, J. R. and G. S. Rahi. 1995. Suppression of Meloidogyne javanica and M. incognita on tomato with ground seed of castor, Crotalaria, hairy indigo, and wheat. Nematropica 25: 159?164. Rodriguez-Kabana, R. and J. W. Kloepper. 1998. Cropping systems and the enhancement of microbial activities antagonistic to nematodes. Nematropica 28: 144 (abstract). Rodriguez-Kabana, R., D. G. Robertson, L. Wells, C. F. Weaver, and P. S. King. 1991. Cotton as a Rotation Crop for the Management of Meloidogyne arenaria and Sclerotium rolfsii in Peanut. Supplement to Journal of Nematology 23: 652-657. Rotar, P. P. and R. J. Joy. 1983. ?Tropic sun? sunn hemp Crotalaria juncea L. Research Extension Series 036, University of Hawaii, Honolulu. Schumann, G. and C. J. D?Arcy. 2006. Essential Plant Pathology. American Phytopathological Society Press, St. Paul, MN: 67. Thies, J. A., S. B. Merrill, and E. L. Corley. 2002. Red Food Coloring Stain: New, Safer Procedures for Staining Nematodes in Roots and Egg Masses on Root Surfaces. Journal of Nematology 34: 179-181. Timper, P., R. F. Davis, and P. G. Tillman. 2006. Reproduction of Meloidogyne incognita on winter cover crops used in cotton production. Journal of Nematology 38: 83- 89. Veech, J. A. 1984. Cotton Protection Practices in the USA and World. Pp. 311-326 in R. J. Kohel, and C. F. Lewis, eds. American Society of Agronomy, Madison, WI. 59 Wang, K. H., B. S. Sipes, and D. P. Schmitt. 2002. Management of Rotylenchulus reniformis in pineapple, Ananas comosus, by intercycle cover crops. Journal of Nematology 34: 106-114. Wang, K. H., B. S. Sipes, and D. P. Schmitt. 2003. Intercropping cover crops with pineapple for the management of Rotylenchulus reniformis. Journal of Nematology 35: 39-47. Wang, K. H., R. McSorley, and R. N. Gallaher. 2004a. Effect of winter cover crops on nematode population levels in North Florida: Journal of Nematology 36: 517-523. Wang, K. H., R. McSorley, and R. N. Gallaher. 2004b. Effect of crotalaria juncea on squash infected with Meloidogyne incognita. Journal of Nematology 36: 290-296. Zasada, I. A., S. L. F. Meyer, J. M. Halbrendt, and C. Rice. 2005. Activity of hydroxamic acids from Secale cereale against the plant-parasitic nematodes Meloidogyne incognita and Xiphinema americanum. Phytopathology 95: 1116-1121.