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
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rop
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tiva
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po
pul
ati
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tode
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W
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arc
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Ex
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L, dur
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op
ping
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cle
 of
 ye
ar 2005
-06.
 
 Cul
tiv
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gra
in 
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Pe
an
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Ab
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39
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8.1
  
Ge
org
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(P
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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
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 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
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W
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gra
in 
co
ve
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mp
lin
g 
 
 
 
 
 
Pe
an
ut 
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___
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Co
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8  
   
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on
 
 
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ruz
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(P
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be
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as 
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ng
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sch
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s p
rot
ect
ed
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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
 
 
__
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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
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ar 20
06
-07.
 
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tiv
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int
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gra
in 
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ve
r c
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mp
lin
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tto
n s
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pli
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6.3
 
a  N
um
be
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f n
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de
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t in
 a 
10
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mp
le.
 
 
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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. 
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tto
n w
as 
pla
nte
d i
n J
un
e a
nd
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rve
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d i
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cto
be
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an
s w
ith
in 
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lum
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fol
low
ed
 by
 di
ffe
ren
t le
tte
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gn
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tly
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 Fi
sch
er?
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rot
ect
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 Le
ast
 Si
gn
ific
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t D
iff
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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
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-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
. 
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tiv
ar 
 
Sh
oo
t w
eig
ht 
 
Ro
ot 
we
igh
t 
Eg
gs/
gm
 of
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ot 
Ne
ma
tod
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sa 
 
__
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Ge
org
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Mi
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10
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bc
 
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6 
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 eg
gs 
pre
sen
t p
er 
gra
m 
of 
roo
t w
eig
ht.
 
Me
an
s w
ith
in 
co
lum
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fol
low
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 by
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 Le
ast
 Si
gn
ific
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t D
iff
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nc
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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 
 
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ot 
we
igh
t 
 
Eg
gs/
gm
 of
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ot 
Ne
ma
tod
e e
gg
sa  
__
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__
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Ge
org
ia 
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tch
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13
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0 
 
50
82
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1.5
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3 
bc
 
Bo
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de
 
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tto
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7.6
2  
 
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33
4 
a 
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(P
 ? 
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5) 
0.5
43
 
a  L
og
 (x
+1
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f th
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um
be
r o
f n
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de
 eg
gs 
pre
sen
t p
er 
gra
m 
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t w
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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
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ffe
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t a
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 Fi
sch
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ast
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gn
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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
 
 
 
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47 
 
Ta
ble
 6.     E
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junc
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pul
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 pl
ant
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ith c
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oty
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for
mi
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r c
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led c
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tions
 at
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48 
 
Ta
ble
 7.        E
ffe
ct of
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rot
alar
ia 
junc
ea
 po
pul
ati
ons
 on r
oot
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ma
tode
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eloi
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p. und
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d c
ondi
tions
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ith R
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h C
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ter
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PI 
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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 
  
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