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Studies of epidemiology of maize streak virus and itsCicadulina leafhopper vectors in Nigeria
Mbey-yame, Asanzi Christopher, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zceb Rd. Ann Arbor, MI 48106
STUDIES OF EPIDEMIOLOGY
OF MAIZE STREAK VIRUS
AND ITS CICADULINA LEAFHOPPER VECTORS
IN NIGERIA
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of the Ohio State University
by
Asanzi Christopher Mbey-yame
Ir. M.S.
*******
The Ohio State University
1991
Dissertation Committee Approved by
Dr. L.R. Nault
Dr. D .T. Gordon
Dr. D.J. Horn
Dr. D.L. Denlinger Department of Entomology This dissertation is dedicated to my family
ii ACKNOWLEDGEMENTS
I am greateful to all those who provided me with their assistance and expertise throughout the realization of this work. Dr. Lowell R.
Nault, my advisor, for his guidance, advice and constructive suggestions which improved the quality of this work. Dr. D.T. Gordon for his deep commitment in this Research and innovative suggestions in the elaboration of this document. Dr. Nilsa A. Bosque-Perez for her enthusiastic supervision of this Research at the International Institute of Tropical Agriculture, support and useful suggestions. I owe a lot of gratitude to Drs. David Horn and David Denlinger for their support and helpful comments.
I express sincere thanks to Dr, Larry Madden, Mr. Bert Bishop and
Mr. Peter Walker for their assistance and advice on statistical problems. I express my sincere appreciation to Dr. H. Gasser for his patience and understanding, to Dr. G. Thottappilly for his contributions in some aspects of this work and to Dr. I. W. Buddenhagen for useful discussions at the early phase of this research.
I am Indebted to IITA Maize Research and Training Program staff for their support during my entire stay in Ibadan. I thank the
Government of Zaire for giving me this life time opportunity and the
USAID for providing me with financial support.
I am grateful to friends and collegues Kasongo Ngindu and Muyolo
ill Gilumbu for their help in the preparation of this document and to Dr.
Mabaya Nyabul and Muwala Bolmakob for their moral support.
Finally, I thank my wife Musitu Jannet, my children Esperance,
Mireille, Philip and Mamie-Ange, my sister Jolie and ray nephew Alson for their patience, sacrifices and above all their loveand, constant support throughout this long ordeal.
iv VITA
December 12, 1947 ...... Born - Mikingi, Kwilu
1975 ...... Ir. , Faculty of Agronomy National University of Zaire (UNAZA)
1975-1979 ...... Research Assistant, National Maize Research Program, Department of Agriculture
1982 ...... M.Sc., Entomology, University of Nebraska, Lincoln
1982-1986 ...... Entomologist, National Maize Reseach Program, Department of Agriculture
1988-1990 ...... Research fellow at the International Institute of Tropical Agriculture, Ibadan, Nigeria
Major Field: Entomology
v TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
VITA ...... iv
LIST OF T A B L E S ...... vii
LIST OF FIGURES ...... xi
INTRODUCTION ...... 1
CHAPTER ...... PAGE
I. STUDIES ON MAIZE STREAK VIRUS AND CICADULINA LEAFHOPPERS IN NIGERIA ...... 7
Introduction ...... 7 Materials and Methods ...... '...... 9 R e s u l t s ...... 15 D i s c u s s i o n ...... 47
II. DISPERSAL OF CICADULINA STOREYI IN MAIZE FIELD AND ITS BEHAVIOR IN RELATION TO MAIZE VARIETY, MAIZE GROWTH STAGE AND PRESENCE OF MAIZE STREAK VIRUS ...... 54
Introduction ...... 54 Materials and Methods ...... 56 R e s u l t s ...... 62 D i s c u s s i o n ...... 79
III. CICADULINA BIOLOGY AND ITS TRANSMISSION OF MAIZE STREAK V I R U S ...... 87
Introduction ...... 87 Materials and Methods ...... 89 R e s u l t s ...... 92 D i s c u s s i o n ...... 102
vi VI. TRANSMISSION OF MAIZE STREAK VIRUS ISOLATES BY CICADULIA STOREYI ...... Ill
Introduction ...... Ill Materials and Methods ...... 112 R e s u l t s ...... 118 D i s c u s s i o n ...... 130
V. MAIZE STREAK VIRUS DISEASE PROGRESSION CURVE AND PATTERN OF SPREAD UNDER FIELD CONDITIONS IN A TROPICAL RAIN FOREST LOCATION ...... 135
Introduction ...... 135 Materials and Methods ...... 136 R e s u l t s ...... 139 Discussion ...... 150
EPILOGUE ...... 153
LIST OF R E F E R E N C E S ...... 157
vii LIST OF TABLES
TABLE ...... PAGE
1. Maize streak virus (MSV) incidence and severity in October 1989 planting at Ikenne ...... 24
2. Maize streak virus (MSV) incindence and severity in July 1989 planting at Mokwa ...... 25
3. Maize streak virus (MSV) incidence and severity in August 1989 planting at Mokwa ...... 26
4. Maize streak virus (MSV) incidence and severity in September 1989 planting at M o k w a ...... 27
5. Maize streak virus (MSV) incidence and severity in June 1989 planting at Funtua ...... 29
6. Maize streak virus (MSV) incidence and severity in July 1989 planting at F u n t u a ...... 30
7. Maize streak virus (MSV) incidence and severity in August 1989 planting at F u n t u a ...... 31
8. Maize streak virus (MSV) incidence on maize varieties at 14 and 28 days after inoculation with different numbers of MSV-exposed leafhoppers following a 24 hr inoculation access period (IAP)...... 42
9. Maize streak virus (MSV) severity on maize varieties at 28 and 56 days after inoculation with different numbers of MSV-exposed leafhoppers following a 24 hr inoculation access period (IAP)...... 43
10. Maize streak virus (MSV) incidence on maize varieties at 14 and 28 days after inoculation with different numbers of MSV-exposed leafhoppers at various time periods following a 24 hr inoculation access period (IAP)...... 4 5
11. Maize streak virus (MSV) severity on maize varieties at 28 and 56 days after inoculation with different numbers of MSV-exposed leafhoppers at various time periods following 24 hr inoculation access period (IAP)...... 47
viii 12. Effect of fluorescent dye on survival of C. storeyi...... 63
13. Effect of fluorescent dye on Cicadilina storeyi flight activity and its virus transmission efficiency. .... 64
14. Cicadulina population density in maize field with different growth s t a g e s ...... 75
15. Numbers of Clcadulina storeyi settling on healthy maize varieties...... 76
16. Numbers of Cicadulina storeyi settling on maize varieties infected with maize streak virus (MSV) ...... 76
17. Number of eggs laid by female Cicadulina storeyi on three maize varieties over 7 day oviposition period (free choice test)...... 80
18. Number of eggs laid by female Cicadulina storeyi on three maize varieties over 7 day oviposition period (No choice test) ...... 81
19. Developmental period (egg to adult) of Cicadulina storeyi on maize vaireties...... 82
20. Mean and median developmental period (number of days) of Cicadulina species reared on maize (variety Pool 16) at different temperature ...... 93
21. Survival time (t25, t50 and t75 in weeks) of Cicadulina spp. reared on maize at different temperatures ...... 94
22. Fecundity parameters (Ro, tc and rc) of Cicadulina spp. at different temperatures ...... 96
23. Transmissibility of maize streak virus (MSV) by Cicadulina arachidis and Cicadulina ghaurii given variable acquisition access periods (AAP) ...... 97
24. Transmissibility of maize streak virus by C. arachidis and C. ghaurii given variable inoculation access periods ( I A P ) ...... 99
25. Maize streak virus (MSV) latent period for Cicadulina arachidis after a 2 hr and 8 h r - A A P ...... 100
26. Maize streak virus (MSV) latent period for Cicadulina ghaurii after a 8 hr acquisition access p e r i o d ...... 101
27. Persistence of maize streak virus (MSV) in Cicadulina spp. given a 24 hr acquisition access period . . 103
ix Transmission of maize streak virus (MSV) by four Cicadulina. spp. given variable acquisition access period and a 24 hr inoculation access period and a 24 hr inoculation access period ...... 104
Comparison of maize streak virus (MSV) transmission by males and females of four Cicadulina species ...... 105
Transmission by Cicadulina storeyi of maize streak virus (MSV) given acquisition access periods (AAP) of 6, 12, or 24 hr on three maize varieties infected with MSV and a 24 hr inoculation access period (IAP) ...... 119
Transmission by Cicadulina storeyi of maize streak virus (MSV) given a 96 hr acquisition access period (AAP) on three maize varieties and a 6, 12 and 24 hr inoculation access period (IAP) ...... 120
Transmission of four maize streak virus (MSV) isolates acquired from four grass species given various acquisition access periods (AAP) following a 24 hr inoculation access period (IAP) on maize test plants by Cicadulina spp...... 121
Transmission by Cicadulina spp. of maize streak virus (MSV) isolates acquired from four grass species given a 48 hr acquisition access periods (AAP) followed by various inoculation access period (IAP) on maize test plants ...... 123
Latent period of maize streak virus (MSV) isolates acquired from three grass species by Cicadulina storeyi . . 125
Persistance of maize streak virus (MSV) isolates acquired from four grass species by Cicadulina storeyi . . . 127
Transmission by Cicadulina spp. (group of five leafhoppers per species) of maize streak virus (MSV) given a 72 hr acquisition access period (AAP) on four grass species followed by a 72 hr inoculation acces period (IAP) ...... 128
Absorbance at 405 nm (A^g) of dilution of extracts from maize streak virus (MSV) infected maize varieties as determined by Enzme Linked Immunosorbent Assay using MSV antiserum ...... 129
Ordinary run analysis on a sequence of 100 MSV-infected and healthy maize plants for 85 quadrats . . . 140
Point pattern analysis on maize streak virus infected plants in all quadrats ...... 141 x 40. Linear regression statistics of different models describing maize streak virus disease progress curves . 148
xi LIST OF FIGURES
Ecological zones and study sites in Nigeria ..... 10
Maize streak virus (MSV) incidences for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Ikenne . . . 16
Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Mokwa ...... 21
Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Samaru . . . 22
Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Funtua . . . 23
Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Ikenne, 1988-1990 ...... 33
Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Mokwa, 1989 ...... 34
Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Samaru, 1989 ...... 35
Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Futua, 1989 ...... 36
Infective Cicadulina leafhoppers (%) and Cicadulina leafhopper density at Ikenne, 1988-1990 ...... 37
Infective Cicadulina leafhoppers (%) and Cicadulina leafhopper density at Mokwa, 1989 ...... 38
Infective Cicadulina leafhoppers (%) and Cicadulina leafhopper density at Samaru, 1989 ...... 39
Infective Cicadulina leafhoppers (%) and Cicadulina
xii leafhopper density at Funtua during 1989 41
14. Arrangement of yellow sticky traps to capture Cicadulina storeyi (second release) from release point ...... 59
15. Number of Cicadulina storeyi leafhoppers recaptured over time after their release ...... 65
16. Linearized form of exponetial model on number of Cicadulina storeyi leafhopper recaptured over time after their release...... 66
17. Number of Cicadulina storeyi leafhoppers (N) recaptured at different distances over 14 days following release...... 68
18. Number of Cicadulina storeyi leafhoppers recaptured (logten) at various distances from release point for different days after leafhopper release ...... 69
19. Rate of dispersal (ra/day) of Cicadulina storeyi leafhoppers at various distances from release point . . . 74
20. Quadratic effect of log Cicadulina storeyi leafhopper count on maize growth s t a g e ...... 76
21. Maize streak virus (MSV) disease progression curve for the rain forest zone ( O w o ) ...... 143
22. Rate of maize streak virus (MSV) disease increase (dy/dt) for the forest zone (Owo) ...... 144
23. The Logistic fit of maize streak virus disease progress data with k, a parameter for maximum amount of disease ...... 145
24. The Richard fit of maize streak virus disease progress data with m (shape parameter) - 0.5 (upper plot) and m - 3 (lower plot)...... 146
25. The Gompertz fit of maize streak virus disease progress data with k, a parameter for maximum amount of disease...... 147
26. Residual plot (randomly distributed) for Gompertz's fit of maize streak virus disease progress data .... 149 INTRODUCTION
Maize, Zea mays L., was introduced from the Americas to West
Africa some three and a half centuries ago (Fajemisin and Shoyinka,
1976) . It has become a basic staple food and principal crop not only in
West Africa but also in many other parts of sub-Saharan Africa. Maize also is used for animal feed and as a raw material for the manufacture of starch, ethanol and other products.
In the last two decades, maize yield per hectare in the tropical zones of Africa has remained constant (ca. 1 ton/ha) although the overall production of maize increased by 2.5% annually due to an increase in the area planted to this crop (Gelaw, 1986; Connolly, 1987).
High yields of maize, however, are limited by a number of constraints including climate, soil fertility, and lack of improved technology but mostly pests and diseases.
Among the pests and diseases in tropical Africa, maize is mostly attacked by stem borers of the genera Sesamia, Eldana, Busseola and
Chilo (Bosque-Perez and Mareck, 1990; Okoth, 1985; Mulamba and Asanzi,
1986) and Cicadulina species, vectors of maize streak geminivirus (MSV) and maize mottle chlorotic stunt. Maize is seriously affected by other diseases such as Helminthosporiura leaf blight, rusts, stalk and ear rots and systemic foliar diseases (Fajemisin, 1986).
1 2
Maize streak disease, first reported in South Africa in 1901 by
Fuller, is the most damaging and widespread disease of maize throughout the African continent. It is present from South Africa to Egypt in the
North and from Ethiopia to as far west as Sierra Leone (Storey, 1936;
Amraar, 1975; Fajemisin and Shoyinka, 1976; Kim et al. 1981). MSV has also been reported in the neighboring Islands of the Western Indian
Ocean (Bock, 1974; Rose, 1978). A similar virus disease has been observed in graminaceous crops (pearl millet, wheat, etc.) in India
(Seth et al., 1971; 1972a, b, c; Seth and Singh, 1975). However the exact relationship to MSV is not known.
MSV is characterized by a geminate particle and genome of circular, single-stranded (ss) DNA of molecular weight of 0.7-0.9 x 10® daltons (Da) (Harrison et al., 1977; Goodman, 1981). MSV possesses a single major coat protein subunit of molecular weight 28,000-34,000 Da
(Bock, 1977; Goodman, 1981). MSV belongs to the leafhopper-transmitted geminivirus subgroup which infect monocotyledonous plants and have a monopartite genome (Mullineaux et al., 1984, Markham et al., 1984a).
Natural hosts of MSV are confined to the Poaceae (Gramineae) including the economic crops Zea mays L., Triticum aestivum L., Sorghum bicolor
(Linn) Moench, Saccharum officinarum L., Permisetum typhoides (Burn.f.)
Stapf and Hubb, Hordetim vulgare L. and Avena sativa L. (Bock, 1974;
Damsteegt, 1983).
The first symptoms of MSV appear on young leaves of infected maize as small spherical chlorotic spots which increase in number and elongate as leaves grow. When the disease is fully expressed, infected leaves are covered with narrow, broken or continuous chlorotic streaks along 3 the veins which may fuse leaving irregular green lines (Storey, 1926;
McClean, 1947; Van Rensburg and Kuhn, 1977; Fajemisin and Shoyinka,
1976), Only new leaves formed after inoculation are infected. Thus, it is possible to estimate the time of infection since the leaves below the inoculated leaf remain uninfected.
MSV symptom severity varies according to maize genotype and the age of plant at the time of infection (Rose, 1976; Kim et al., 1981).
Van Rensburg and Kuhn (1977) reported a logarithmic decline in yield loss with increasing age at the time of infection. Yield reduction of up to 70% has been reported when plants are infected at an early age
(Guthrie, 1978). However, it is difficult to estimate yield loss accurately, particularly under field conditions (Fajemisin et al., 1986;
Van Rensburg, 1981). Yield loss of 71-93% under controlled infection with viruliferous Cicadulina storeyi (syn. C. triangula) (Webb, 1987) has been observed at the International Institute of Tropical Agriculture
(IITA) (Fajemisin et al., 1986).
Many high yielding varieties of maize have been developed and released to the farmers. In particular, during the last decade or so maize rapidly has gained increasing importance in the savanna zone of
West Africa. It partly has replaced millet and sorghum, especially in the Guinea Savanna zone, thus drastically increasing the area under cultivation. Maize also has changed status from a crop grown in a mixed culture with others to a plantation crop grown in pure stands.
Introduction of new maize genotypes, the shift in cultural practices and the increase in acreage of maize under cultivation have led to increased
MSV and Cicadulina occurrences in Africa. Irrigation which allows crops 4 to grow throughout the year also has contributed to conditions for MSV epidemics. This has been the case in Zimbabwe where winter cereal crops that are grown under irrigation remain attractive to Cicadulina. leafhoppers which later invade and inoculate early planted maize with
MSV resulting in severe crop loss (Rose, 1973a). MSV epidemics, which were associated with drought conditions or irregular early rains, were reported in 1966, 1971, 1973, 1983, 1984 and 1986 in Nigeria (Fajemisin and Shoyinka, 1976; Fajemisin et al., 1986; IITA, 1984; 1987). MSV epidemics have also been observed in other African countries including
Burkina Faso, Angola, Ghana, Mozambique, Kenya, Zambia and Zaire (IITA,
1986).
Cicadulina leafhoppers are of considerable economic importance as vectors of MSV rather than as plant feeders (Ruppel, 1965). They are generally regarded as grassland species (Rose, 1978) and have been commonly collected from grass species in the genera Chloris, Brachiaria,
Eleusine, Digitaria, Setaria, Sporobolus, Paspalum, Pennisetum, Cynodon, etc. (Rose, 1978). According to Webb (1987) there are 22 species of
Cicadulina worldwide of which 18 occur in Africa and eight of those are vectors. Cicadulina species also have been reported to occur in the
Middle East, India, Colombia and Australia. The distribution of most species seems to be limited to large regions in Africa (Rose, 1972b) which is presumed to be the center of origin of Cicadulina. Some species of Cicadulina can live undetected in low density populations on seasonal grasses, although high density populations have been sampled
(Rose, 1973a). 5
The biology of C. mbila on maize was first described by Van der
Merwe (1926) who discovered that eggs were inserted linearly along the midribs of the leaf. The egg incubation period varied between 9-12 days depending upon season and temperature. There are five nymphal instars with the nymphal development period averaging 23 days. Following this first investigation, the biology of C. triangula (syn. C. storeyi) , C. mbila, C . parazeae, C. china, and C. bipunctata have been extensively studied (Rose, 1973b; Ammar, 1975, 1977; Dabrowski, 1985). Dabrowski
(1985) reported that the mean number of eggs laid per female C. storeyi at 28°C averaged 123 eggs on maize seedlings.
Rose (1972a) discovered that Cicadulina disperse downwind and that females fly farther than males. He also found that flight behavior was influenced by season, time of day, species, sex, presence or absence of mature ova in the females, age and conditions of food plants.
MSV is transmitted in a circulative and nonpropagative manner by eight Cicadulina species which belong to the subfamily Deltocephalineae and the tribe Macrostelini. The transmission of MSV by C. mbila was described by Storey in a series of classical experiments (Storey, 1925,
1928, 1932, 1933 and 1939) from which he concluded that the virus could be acquired in 15 sec and be inoculated in 5 min, the latent period of
MSV within leafhopper was short (10-24 hr) and the virus persisted in the leafhopper for its life span.
MSV can be controlled to some extent by agronomic practices such as planting dates, rotation and seed treatment with systemic insecticides. However, the best control strategy is the planting of MSV resistant varieties that perform well in most African countries. This approach particularly helps small-holding farmers who produce most of the maize in Africa and who cannot cope with the financial and technical burden of chemical pesticides. These resistant varieties have been developed by the International Institute of Tropical Agriculture (IITA) from MSV sources identified in the population Tropical Zea Yellow (IB32) and in the variety La Revolution from Reunion Island.
Studies were undertaken to gain futur understanding of the complexity of the interactions between the pathogen, the vector, the genotype and the environment. Specifically, studies were undertaken to
(i) determine seasonal occurrences of MSV and infective Cicadulina spp. populations and their associations with abiotic factors; (ii) estimate the dispersal patterns of Cicadulina populations; (iii) examine crop phenology, feeding, oviposition, preferences and development of
Cicadulina leafhoppers; (iv) study the biology and the transmission of
MSV by C. arachidis and C. ghaurii; (v) determine the transmission of
MSV from maize varieties and grass species to maize and (vi) spatially and temporally quantify MSV disease. CHAPTER I
STUDIES ON MAIZE STREAK VIRUS
AND CICADULINA LEAFHOPPERS
IN NIGERIA
INTRODUCTION
Maize streak virus (MSV) is an indigenous African geminivirus
transmitted by Cicadulina leafhoppers and causes sporadic disease outbreaks in sub-Saharan countries (Fajemisin and Shoyinka, 1976;
Guthrie, 1977; Kira et al., 1981). The virus probably first encountered raaize when this crop was introduced from its native Mesoamerica into
Africa during the 16th century (Guthrie, 1976; Buddenhagen, 1983).
Environmental conditions (e.g. rainfall, temperature and relative humidity) and host plants have marked effects on leafhopper populations and perhaps on species composition as shown by studies in Nigeria
(Dabrowski, 1987c; Okoth and Dabrowski, 1987) and Zimbabwe (Rose, 1973a;
1978). These studies established that leafhopper population densities were low at the onset of the rainy season and then rose gradually as host plants became abundant and succulent. Leafhopper populations sharply declined in the dry season due probably to the absence of hosts on which to feed and the prevailing adverse environmental conditions
(temperatures and wind). Rose (1972b and 1978), and Okoth and Dabrowski
7 8
(1987) indicated that there was a close relationship between Cicadulina population density and seasonal rainfall as well as host plants.
In the rainforest zone the unreliable short, August-break divides
the long bi-modal rainfall season into two growing seasons. The incidence and symptom severity of leafhopper transmitted virus like MSV
tend to fluctuate from year to year, season to season and area to area depending upon infective leafhopper population levels and their species composition and activity (Conti, 1976). MSV incidence and symptom severity also change according to maize genotypes and age of plant at the time of infection (Buddenhagen, 1983; IITA, 1984). In studies conducted in Nigeria, incidence of MSV ranged from 5% in experimental plots in Ibadan during the first growing season of 1973 to 100% in farmers' fields during the second season (Fajemisin et al., 1976). In contrast, in 1983 and 1984 the incidence of MSV in farmers' fields in
Ibadan varied between 20.0 and 25.0% during the first season to 26.1 and
28.4% during the second season, respectively (Okoth and Dabrowski,
1987). However, in the Guinea Savanna zone with a unimodal rainfall pattern, MSV incidences exceeding 75% were reported in maize fields planted late in 1983 (IITA, 1984).
Reports by Rose (1978), IITA (1985) and Dabrowski (1985) Indicated that MSV epidemics depend on Cicadulina populations which are favored by high temperature and low rainfall. Furthermore, Fajemisin et al. (1986) indicated that outbreaks of MSV are often associated with drought conditions or irregular early rains. Cicadulina spp. differ in their efficiency in transmitting MSV, and thus species composition presumably plays an important role In maize streak epidemics (Dabrowski, 1987c). Reports on field studies of Cicadulina spp. and MSV disease
severity and incidence across ecological zones in Nigeria are scarce.
This investigation was undertaken: (i) to measure MSV incidences and
symptom severity in maize plantings at four locations (Ikenne in the
tropical rain forest zone, Mokwa in the Southern Guinea Savanna zone,
Samaru and Funtua in the Northern Guinea Savanna zone); (ii) to estimate
seasonal occurrence of Cicadulina leafhopper populations in the various
ecological zones; (iii) to determine species composition and natural
infectivity of Cicadulina from grasses surrounding the maize plots; (iv)
to determine possible interactions of MSV incidence versus MSV infected
leafhopper populations, rainfall and temperature and also of leafhopper
population levels versus abiotic factors; and (v) to determine the
effect of infective leafhopper dose on the response of maize varieties with varying levels of MSV resistance.
MATERIALS AND METHODS
Ecological zones and sites. Sequential plantings and leafhopper
samplings were conducted at four sites in two ecological zones, one of which had northern and southern components (Fig. 1). The first was
Ikenne (lat. 06° 52', long. 03°42') located in the rain forest zone.
The average annual rainfall at Ikenne is approximately 1700 mm and the duration of the rainy season 8 months. The pattern of rainfall in this
ecological zone is described as bi-modal since there are two peaks of rainfall during the year with a short dry spell around August. The mean 10
SUMARU
Sahel zone Sudan savanna IKENNE Guinea savanna Rain forest
Fig. 1. Ecological zones and study sites in Nigeria 11 temperature for Ikenne is 26°C. The second site was Mokwa (lat. 09°19', long. 05°06') located near Bida in the Southern Guinea Savanna zone. It has an average annual rainfall of approximately 1100 mm and the duration of the rainy period is approximately 7 months. The rainfall pattern is unimodal. The mean temperature at Mokwa is 25°C. The third and fourth sites are Funtua (lat. 11°33', long. 07°19') and Samaru (lat. ll0llf, long. 07°38'), both located near Zaria in the northern Guinea Savanna zone. The average annual rainfall for both is approximately 850 mm and the duration of rainy period is approximately 5 months. The rainfall pattern is unimodal. The mean temperature for both locations is 24°C.
Sequential plantings. Three improved IITA developed maize varieties were used in this study: the open pollinated TZB-Gusao, and the hybrids 8329-15 and 8321-21. They were rated as susceptible, moderately and highly tolerant to MSV, respectively. Each monthly planting consisted of four 10 m rows of each variety (0.76 m x 0.30 m spacing). A randomized complete block design with four replications was used for each planting date. Two seeds/hill were planted and later thinned to one plant per hill. After planting, the herbicide Primextra was applied at 2.5 kg a.i./ha. Fertilizer was applied at a rate of 120 kg of N, 120 kg of P20s and 120 kg of K20 per hectare. A sidedressing of
78 kg of N (calcium ammonium nitrate) per hectare was applied at 6 wk after planting. Sequential plantings were done during the 1989 planting season (June to Sept.) at Mokwa, Samaru and Funtua. At Ikenne where irrigation facilities are available, plantings were done from December
1988 to September 1990. 12
MSV incidences and severity measurements. MSV incidence and symptom severity were evaluated in the central two rows of each maize plot at 21, 42 and 63 days after planting. At Ikenne, in addition to maize, incidences (number of infected plants/total number of plants x
100) of grasses with MSV-like symptoms were rated biweekly in areas surrounding maize fields within a 50 cm x 25 cm quadrat from May to
August 1990. At each assessment, 20 quadrats were randomly taken and nonparametric analysis was performed only on incidence data from quadrats with plants showing streak-like symptoms. A symptom scale of 0 to 9 was used where zero represented healthy plants, 1 equaled 1 to 5% of the leaf area covered by streak symptoms (very few broken lines mostly in the midrib of the upper leaves) and 2, 3, 4, 5, 6, 7 and 8 represented approximate streak coverage of 6-15, 16-30, 31-45, 46-60,
61-75, 76-90 and 91-100%, respectively. Nine represented 100% of the all leaves extensively streaked or necrotic indicating imminent death.
Seasonal MSV incidence curves were drawn. Arc-sine transformed
MSV incidence and MSV severity data (scale) were analyzed as split plot with variety as main plots and assessment dates as subplots. MSV incidence on TZB-Gusao at 21, 42 and 63 days after planting were regressed on rainfall, temperature, and leafhopper population density as well as on MSV infectivity of leafhoppers.
Grass hosts of MSV and Cicadulina leafhoppers. At each site, annual and perennial grasses showing maize streak-like symptoms were recorded. Grasses from which leafhoppers commonly were collected at the four sites were also recorded. 13
Cicadulina population samplings and species composition.
Leafhoppers were sampled from grasses surrounding maize fields with a net cage throughout the duration of the experiment, and in maize fields from May to September 1990 at Ikenne. The net cage used for sampling consisted of an aluminum frame of 1 m x 1 m x 1.5 m (IITA, 1984). The frame was placed over a grassy area and quickly covered with a cloth of dark cotton on three sides and the top. The fourth side was covered with a fine, transparent mesh. Leafhoppers and other insects inside were attracted by the natural light which entered through the transparent mesh. Cicadulina were collected selectively with an aspirator from the fine mesh. For each sampling date, 20 samples were taken from 20 (1 x 1 m) grass plots surrounding maize fields and later from maize at each location.
Field collected males at each site at each sampling date were identified to species. Females collected from the field were identified from F., males reared from the progeny of these females. Species identification was made by microscopic examination of male genitalia following the descriptions of Ruppel (1965), van Rensburg (1983) and
Dabrowski (1987d).
The mean leafhopper population density per 1 x 1 m plot was determined for each location and sampling date on species basis.
Seasonal abundance curves for monthly leafhopper collections per species were drawn and seasonal distribution of leafhopper population densities were regressed against rainfall and temperature.
Percentage of infective Cicadulina. Leafhoppers collected from grasses surrounding maize fields at each site and from maize fields 14 during May to September 1990 at Ikenne were tested for transmissibility of MSV for each sampling date. In such tests, leafhoppers were caged singly on healthy MSV-susceptible maize seedlings of variety Pool 16 within plastic tube cages. Leafhoppers were removed after 5 days of inoculation access period and caged plants were kept in the glasshouse and evaluated for symptom development at 3 wk after inoculation.
Healthy maize seedlings were included as checks to detect any possible accidental transmission during the process.
Weather data. Monthly rainfall and temperature readings were obtained from the Institute of Agricultural Research and Training at
Zaria and Ikenne and from the Weather Station Headquarters in Lagos.
Vector pressure. In order to test the effect of number of viruliferous leafhoppers on Incidence and severity of MSV in maize, a trial was conducted In a screenhouse at IITA, Ibadan. A split plot design was used where number of viruliferous leafhoppers was the main plot and maize varieties with different MSV-tolerance levels (TZB-Gusao,
8329-15 and 8321-21) were subplots. Maize was planted using a spacing of 0.41 m between rows (3 rows/subplot) and 0.13 m between plants in rows. Each treatment was replicated four times. After emergence, each subplot was covered with a cage measuring 1.35 ra x 1.35 m x 1.19 m made of metal frame covered with a fine insect-proof mesh. Cicadulina storeyi were given an acquisition access period (AAP) of 48 hr on maize infected with the MSV isolate maintained routinely at IITA for all testing and experimental work. Viruliferous leafhoppers (4, 8, 16 or
32) were released on individual maize plants 8 days after planting.
This was accomplished by walking into the cage and delivering the 15
leafhoppers into the maize whorl using a leafhopper dispenser vial. To prevent movement, leafhoppers were anesthetized with C02 prior to
release on maize plants (Leuschner and Buddenhagen, 1982). After a 24 hr inoculation access period (IAP), caged maize plants were sprayed with 1.5 ml of Rogor diluted in 1 liter of water to kill the leafhoppers before cages were removed.
A second experiment was carried out in the screenhouse where a
combination of different numbers of viruliferous leafhoppers (zero, two,
four or eight) and length of virus acquisition access period (6, 12 and
24 hr) were used as the main plot and maize varieties as the subplot.
Spacing, number of replications, length of IAP, and method of handling leafhoppers were as described above.
MSV incidence was evaluated for both experiments at 14 and 28 days after inoculation and MSV severity (scale 0 to 9) was rated at 28 and 56 days. Arcsine transformed MSV incidence and MSV disease severity data were analyzed using ANOVA procedure of SAS software (Statistical
Analysis System Institute Inc., Cary, NC, USA). Means were compared using polynomial orthogonal contrast.
RESULTS
Virus incidence. MSV incidence recorded at 63 days at Ikenne was low during the period surveyed (Fig. 2) except in the October 1989 planting when it reached 56% for TZB-Gusao. Likewise, highest incidence of MSV for TZB-Gusao was observed 63 days after planting in the July Fig. 2. Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, varieties maize for (MSV) incidence virus streak Maize 2. Fig. > 2 40 ■ 40 2 3 (A c c o o C o <3 20 Percent MSV incidence ‘ 60 0 2 - 40 60- 0-1— ■ - Dec.,1088 1 42 21 1 42 21 Feb.,1989 y atr planting after ays D y atr planting after ays D 8329-15 and 8321-21 planted at Ikenne at planted 8321-21 and 8329-15 E3 8321-21 □ ■ TZB -Gusao TZB -Gusao ■ 0 8329-15 8329-15 0 TZB-Gusao ■ 8321-21 □ 8329-15 8329-15 63 63 3
Percent MSV incidence t) u k. o c • c O 0 2 40- c 60 40 20 60 _ - __ * HKzZ2=i TZB-Gusao, 8329-15 and 8321-21 planted at Ikenne at planted 8321-21 and 8329-15 TZB-Gusao, Jul.,1989 1 2 63 42 21 21 Apr.,1989 as fe planting after Days as fe planting after Days __ r 42 □ E3 u 8321-21 8329-15 TZB-Gusao n e f J TZB-Gusao 8321-21 8329-15 3 6 2 CL s •> o o c C • c U 0 ■ 20 5 CO > "D « • c o • u c - 40 o 40- 0 2 60 60 • 60 - 0 - u.18 ® ® Aug.,1989 May.,1989 1 2 63 42 21 y atr planting after ays D as fe planting after Days 42 □ 8321-21 8329-15 -Gusao TZB TZB-Gusao 6321-21 8329-15 63 17 18
Fig. 2 (continued). Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Ikenne
60 60 ■ TZB-Gusao ■ TZB-Gusao Sept.,1089 Oct. .1989 0 8329-15 fZ 8329-15 o c □ 8321-21 □ 8321-21 H 40 I 40- o %
S 20 o c u
42 21 42 63 Days after planting Days after planting
60' • Dec.,1989 ■ TZB-Gusao t> • TZB -Gusao c □ 8329-15 o • c 8329-15 □ 8321-21 • o 40- 2 8321-21 o 40- c >c/> >CO _ 2 0 - * 2 0 - o c o IL CL 21 42 63 Days after planting 21 42 63 Days after planting 19
Fig. 2 (continued). Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Ikenne
60 ■ TZB-Gusao O Mar.,1990 c □ 8329-15 □ 8321-21 *52 c (A> 3 c 2 0 - o * a
21 42 63 Day* attar planting
60 * TZB -Gusao u Apr., 1990 c 8329-15 2• u 8321-21 c > cn 3 c L_O « a
42 63
Daya altar planting 20 planting at Funtua (43%) and in the August and September plantings at
Mokwa (50%) (Fig. 3-5). MSV incidence at Samaru (Fig. 4) remained relatively low for all plantings. At Ikenne from May to September 1990,
MSV incidence for maize was absent. However, biweekly evaluation of streak like symptoms in surrounding grasses indicated 8, 5, 3 and 2 quadrats out of 20 with plants showing streak-like symptoms for assessment dates from May 15 to September 1, 1990, respectively. The mean incidence of plants with streak-like symptoms in the quadrats (N -
9) in the May 15 evaluation (23% ±0.9) was significantly higher than those of the other five assessment dates conducted during the period May
30 to September 1, 1990. The latter mean incidences of grasses with
MSV-like symptoms varied from 8.3 ± 0.9 to 12.0% + 1.2 (with N varying from 2 to 8). When total number of healthy plants (3,465) and grasses with MSV-like symptoms (119) were considered in all 120 quadrats, the overall putative MSV incidence in grasses bodering maize fields was
3.4%.
Highly significant (p<0.01) interactions (variety x assessment date) for MSV incidence were observed for the October planting at Ikenne
(Table 1) and for the July and September plantings at Mokwa (Tables 2 and 4). MSV incidences for the susceptible variety TZB-Gusao and moderately tolerant variety 8329-15 were low when first recorded 21 days after planting and increased gradually reaching a maximum 63 days after planting. However, incidences for highly tolerant variety 8321-21 remained relatively low at all locations and assessment dates.
Significant differences (p<0.01) in MSV incidence were also found among maize varieties in the August planting at Mokwa (Table 3) and in the Fig. 3. Maize streak virus (MSV) incidence for maize varieties TZB-Gusao, varieties maize for (MSV) incidence virus streak Maize 3. Fig.
Percent MSV Incidence Percent MSV Incidence GO- - 0 4 0 2 - 0 4 GO - 0 2 - - 8321-21 □ Gusao TZB- ■ 8329-15 □ Jun.,1989 1 3 6 2 4 21 y atr planting after ays D y atr planting after ays D 391 ad82-1 lne t Mokwa at planted 8321-21 and 8329-15 2 4 Aug.,1989 8321-21 □ E3 ■ TZB-Gusao TZB-Gusao ■ 8329-15 8329-15 2 to > e u c 20 40- e 40 60 60 0 2
- - £ Z i n w 8321-21 □ E2 TZB-Gusao ■ Jul., Jul., 1989 1 2 4 21 1 3 6 2 4 21 8329-15 8329-15 y atr planting after ays D as fe planting after Days Sept.,1989 □ □ □ Gusao TZB- ■ 8321-21 8329-15 21 22
60'
Jun., 1969 u ■ TZB-Gusao c 40- □ 8329-15
□ 8321-21 > u a. BZQsa. T ~ 1 21 42 63 Days altar planting 60' Jui.,1989 ■ TZB-Gusao £3 8329-15 = 40- □ 8321-21 to 2 - 2 0 ' e u 21 42 63 Days attar planting 60' D TZB-Gusao Aug.,1989 □ 8329-15 1 40 i □ 8321-21 > to 2 £ 2 0 ' oIU E IU CL m * B n . 21 42 63 Days attar planting Fig. 4. Maize streak virus (MSV) Incidence for maize varieties TZB-Gusao, 8329-15 and 8321-21 planted at Samaru Fig. 5. Maize streak virus (MSV) incidence for maize varieties maize for (MSV) incidence virus streak Maize 5. Fig. TZB-Gusao, 8329-15 and 8321-21 planted at Funtua at planted 8321-21 and 8329-15 TZB-Gusao, Percent MSV Incidence Percent MSV Incidence Percent MSV Incidence 0 2 40 60 0 2 40- 60' 40- 0 2 60' - - * 8321-21 □ 8329-15 □ TZB-Gusao B 21 days 42 days 63 days days 63 days 42 days 21 Aug.,1989 1 2 63 42 21 Jun.,1989 as fe planting after Oays as fe planting after Days ee fe planting after Deye 42 8321-21 □ 8329-15 H ■ TZB-Gusao TZB-Gusao ■ E3 8321-21 □ ■ TZB-Gusao TZB-Gusao ■ Jul.,1989 8329-15 8329-15 Table 1. Maize streak virus (MSV) incidence and severity in October 1989 planting at Ikenne. Assessment date Variety 21 days 42 days 63 days Incidence8 Severity1* Incidence Severity Incidence Severity TZB-GUSAU 12.84 2.92 38.24 2.68 56.09 2.51 (21.04)c (38.21) (48.48) 8329-15 8.45 2.40 27.46 2.22 42.52 2.11 (16.88) (31.61) (40.59) 8321-21 2.39 1.42 10.28 1.47 12.73 1.67 (8.89) (18.69) (20.85) LSD(0.05) (4.08d> 0.82e 8 Percent infected plants. b 0-9 score (0-healthy, 9-100%leaves extensively streaked or necrotic). c arcsine transformed data. d and e LSD for MSV incidence and severity on the interaction (variety x assessment date) respectively. ro ■P* Table 2. Maize streak virus (MSV) incidence and severity in July 1989 planting at Mokwa. Assessment date Variety 21 days 42 days 63 days Incidence® Severity1* Incidence Severity Incidence Severity TZB-GUSAU 1.66 1.71 6.53 2.27 12.15 2.96 (7.35)c (14.83) (20.40) 8329-15 3.32 2.38 8.84 2.25 9.86 3.04 (10.54) (17.30) (18.34) 8321-21 0.49 0.00 0.70 0.25 1.40 0.50 (3.95) (4.83) (6-83) LSD(0.05) (3.06)d 1.44e 0 percent infected plants. b 0-9 score(0-healthy, 9-100% leaves extensively streaked or necrotic), c arcsine transformed data. d and e LSD for MSV incidence and severity on interaction (variety x assessment date) respectively. fO wn Table 3. Maize streak virus (MSV) incidence and severity in August 1989 planting at Mokwa. Assessment date Variety 21 days 42 days 63 days Mean Variety Incidence® Severity11 Incidence Severity Incidence Severity Incidence Severity TZB-GUSAU 4.54 3.23 42.07 2.41 49.65 2.66 28.87 2.77 (12.31)c (40.36) (49.65) (32.47) 8329-15 4.39 2.71 45.12 1.59 50.70 2.04 30.14 2.11 (12.05) (42.06) (45.43) (33.18) 8321-21 1.13 0.50 17.33 1.55 23.50 1.76 11.59 1.27 (3.95) (4.83) (6.83) (5.20) LSD(0.05) (5.82)d 0.98e Mean 3.14 2.15 34.05 1.85 40.80 2.15 (10.17) (35.68) (39.72) LSI)f(0.05) (4.40) (0.75) a percent infected plants b 0-9 score(0-healthy, 9-100% leaves extensively streaked or necrotic). c arcsine transformed data. d and e LSD for MSV incidence and severity on varieties, respectively. f LSD for MSV incidence and severity on assessment dates. to Table 4. Maize streak virus (MSV) incidence and severity in September 1989 planting at Mokwa. Assessment date Variety 21 days 42 days 63 days Incidence8 Severityb Incidence Severity Incidence Severity TZB-GUSAU 8.74 3.01 34.88 2.63 49.35 2.52 (17.19)c (36.15) (44.56) 8329-15 9.96 2.50 32.57 2.26 49.83 2.07 (18.40) (34.76) (44.82) 8321-21 2.08 0.95 14.77 1.19 19.08 1.56 (8.33) (22.58) (25.92) LSD(0.05) (3.73)d 0.83e 0 percent infected plants. b 0-9 score(0-healthy, 9-100% leaves extensively streaked or necrotic). c arcsine transformed data. d and e LSD for MSV incidence and severity on interaction (varieties x assessment date) respectively. 28 June, July and August plantings at Funtua (Tables 5, 6 and 7). There was no significant interaction for MSV incidence for these planting dates at Mokwa and Funtua. The maize variety 8321-21 differed significantly (p<0.01) in MSV incidence from varieties TZB-Gusao and 8329-15 (Tables 1-7). Symptoms severity. Significant differences (p<0.01) in MSV severity were noticed among varieties in the October planting at Ikenne (Table 1), August and September plantings at Mokwa (Tables 3 and 4), and June and August plantings at Funtua (Tables 5 and 7). MSV severity on varieties TZB-Gusao and 8329-15 was significantly greater than on variety 8321-21. Grass hosts of MSV and Cicadulina leafhoppers. At all four locations, MSV-like symptoms were observed on annual grasses, especially Brachiaria disticophylla, B. lata, B . deflexa, Digitaria horizontalis and Eleusine indica, bordering maize fields during the mid to late growing season (data not shown). Streak-like symptoms also were observed on perennial grasses Panicum maximum (all locations), Andropogon gayanus (Mokwa) and Paspalum scrobiculatum (Ikenne) (data not shown). In all the areas surveyed, leafhoppers were mostly collected from Brachiaria spp., Digitaria horizontalis and Eleusine indica. They were also found on Roettboellia cochinehinensis and Pennisetum spp., but only towards the end of the rainy season in the Guinea Savanna. Cicadulina populations and species composition. At Ikenne leafhopper population densities in grasses surrounding maize fields remained relatively low during the irrigated dry (January - March) and rainy (April - October) seasons of 1989 and 1990 reaching maxima at the Table 5. Maize streak virus (MSV) incidence and severity in June 1989 planting at Funtwa. Assessment date 21 days 42 days 63 days Mean variety Variety Incidence3 Severity1* Incidence Severity Incidence Severity Incidence Severity TZB-GUSAU 2.24 3.00 8.26 3.89 11.81 4.30 6.79 3.3 (8.62)c (16.68) (20.11) (15.14) 8329-15 3.14 2.88 9.04 3.20 17.20 3.58 8.94 3.22 (10.17) (17.47) (24.54) (17.39) 8321-21 0.54 0.00 2.61 1.75 5.61 2.13 2.50 1.29 (4.22) (9.34) (13.66) (9.08) (3.32)d 1.18® LSDC0.05) Mean 1.80 1.96 6.27 2.95 11.03 3.33 (7.67) (14.50) (19.44) LSI)f(0.05) (1.91) (0.77) a percent infected plants. b 0-9 score(0-healthy,9-100% leaves extensively streaked or necrotic) c arcsine transformed data. d and e LSD for MSV incidence and severity on varieties, respectively. f LSD for MSV incidence and severity on assessment dates. N5 VO Table 6. Maize streak virus (MSV) incidence and severity in July 1989 planting at Funtwa. Assessment date Variety 21 days 42 days 63 days Mean Variety Incidencea Severity13 Incidence Severity Incidence Severity Incidence Severity TZB-GUSAU 14.40 2.92 26.68 3.73 43.04 4.41 27.30 3.69 (22.3)c (31.1) (41.0) (31.5) 8329-15 13.20 2.85 22.33 3.41 39.09 3.94 24.10 3.40 (21.3) (28.3) (38.6) (29.4) 8321-21 2.39 0.94 6.70 2.00 11.70 2.05 6.27 1.66 (8.9) (15.0) (19.7) (14.5) LSD(0.05) (7.64)d 1.44e Mean 9.04 2.24 17.59 3.05 29.82 3.46 (17.5) (33.1) LS°f(0.05) (5.38) (0.62) a percent infected plants. b 0-9 score(0-healthy, 9-100% leaves extensively streaked or necrotic). c arcsin transformed data. d and e LSD for MSV incidence and severity on varieties, respectively. f LSD for MSV incidence and severity on assessment dates. Table 7. Maize streak virus (MSV) incidence and severity in August 1989 planting at Funtwa. Assessment date Variety 21 days 42 days 63 days Mean Variety Incidence0 Severity1* Incidence Severity Incidence Severity Incidence Severity TZB-GUSAU 3.20 2.39 13.91 3.44 20.33 3.20 11.36 3.01 (10.3) (21.9)c (26.8) (19.7) 8329-15 2.78 3.50 8.55 3.08 15.27 2.86 8.07 3.15 (9.6) (17.0) (23.0) (16.5) 8321-21 0.54 0.00 0.89 0.67 2.50 1.61 1.17 0.76 (4.2) (5.4) (9.1) (6.2) LSD(0.05) (5,47)d 1.45e Mean 1.94 1.96 6.53 2.40 11.25 3.46 (8.0) (14.8) (19.6) LSI)f(0.05) (5.96) (1.29) 0 percent infected plants. b 0-9 score(0-healthy, 9-100% leaves extensively streaked or necrotic), c arcsine transformed data. d and e LSD for MSV incidence and severity on varieties, respectively. 1 LSD for MSV incidence and severity on assessment dates. 32 end of the rains in December, 1988 and November, 1989 (Fig. 6). Leafhopper population densities were low in maize fields (0 to 0.12 leafhoppers/m2) and in grasses bordering them (0 to 0.4 leafhoppers/m2) during May to September, 1990. For the Guinea Savanna zone locations, the leafhopper population densities were low at the beginning of the rainy season, generally rising and reaching their peaks before the rains ended in July and September, 1989 at Mokwa (Fig. 7), in August at Samaru (Fig. 8) and in July at Funtua (Fig. 9). Monthly rainfall data during the sampling periods for these four sites are presented in Figs. 6-9. The species composition results showed that C. mbila was the predominant species in all ecological zones (Fig. 6-9). It appeared at all locations and for all sampling dates. C. storeyi was the least common species ranking after C. arachidis and C. similis in abundance in both the savanna and humid forest zones. C. storeyi, C. arachidis and C. similis occurred only during the dry season (Fig. 6) in the humid forest zone. The latter species was not found in Samaru (Fig. 8). C. ghaurii was restricted to Ikenne and found predominantly during the rainy season. Vector infectivity. For Cicadulina collected at Ikenne, the natural MSV infectivity rate was low in the first growing season (April- June), rising during the second (July-November) to reach peaks during January, 1989 and 1990 (Fig. 10). At Mokwa and Samaru the percentage of field-collected Cicadulina leafhopper transmitting MSV rose gradually during the growing season reaching maxima of 15% for October (Fig. 11) and 22% for September (Fig. 12), respectively. At Funtua, the 33 4 00“ £ 200 - 100 - De Ja Fe Ma Ap Ma Ju Ju Au Se Oc No De Ja Fe Ma Ap C. arachidis 8 “ C, mbila CM E ra c C. similis 3 T3 CO O ...... Q St0rQy j u L. at JO C. ghaurii E 3 C c ca at £ 2 - De Ja Fe Ma Ap Ma Ju Ju Au S e Oc No De Ja Fe Ma Ap 1988 1989 1990 Month Fig.6. Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Ikenne, 1988-1990 cc 100“ i.. iauia efopr est pr pce i gr s s sse ra g in species per density leafhopper Cicadulina Fig.7. Mean number Cicadullna/m2 0 0 2 300- - odrn mie n rifl dsrbto a Mokwa,1989 at distribution rainfall and maize bordering May June uy u Sept Aug July Aug. July Month Sept. -*« - « * - - Oct. C. arachidis C. C. mbilaC. c. C. storeyi C. similis Nov Nov. 34 35 150- E E 100 - ra c « EC 5 0 - Jun Jul Sep OctAug 8 C. arachidis CM C. mbila E c8 c 6 C. storeyi 3 •a a u O 4 CO a> 2 S 0 June July SeptAugust Month Fig.8. Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Samaru,1989 36 150" E E 1 00 - *-« c to DC Jun Jul Aug Sep Oct C. arachidis C. mbila CM E 6 M- C. similis ro C. storeyi TJ oco O 4 o n E 3 C CO 2 ...... - - ______0 Jun Aug S epJul Oct Month Fig.9. Cicadulina leafhopper density per species in grasses bordering maize and rainfall distribution at Funtua,1989 i.0 Ifcie iauia efhpes % ad Cicadulina and (%) leaf hoppers Cicadulina Infective Fig.10. Clcadullna lnfectlvlty(%) 10 0 2 25- 30 - 5 1 0 9 9 1 9 8 9 1 8 8 9 1 " - i —i > 3 h ——i —i —i “‘“CH-o 0 i— i— i— i— i— — i— — 0 C>“ th“ t3“ i— i— —i— De Ja Fe Ma Ap Ma Ju Ju AuSepOc No De Ja Fe Ma Ap Ma Fe Ja De No AuSepOc Ju Ju Ma Ap Ma Fe Ja De efopr est a lkenne,1988-1990. at density leafhopper % Cicadulina infectivlty Cicadulina % Cicadulina/m2 Month Cicadulina (m2 37 i.1 Ifcie iauia efhpes % ad Cicadulina and (%) leaf hoppers Cicadulina Infective Fig.11. Cicadulina infectlvlty {%) 0 1 25- 0 2 - - efopr est a Mka 1989. Mokwa, at density leafhopper May •• %Ccdln infectivity Cicadulina % □•••■ Jun O Cidulina Cidulina /m2 u u Sep Aug Jul Month Oct Nov -10 Cicadulina /m2 38 i.2 Ifcie iauia efopr () n Cicadulina and (%) leafhoppers Cicadulina Infective Fig.12. Cicadulina lnfectivlty(%) 20 30 efhpe dniy t aau 1989. Samaru, at density leaf hopper ue uy uut et Oct. Sept. August July June Cicadulina /m2 Cicadulina Cicadulina infectivity(%) Cicadulina Month Cicadulina /m2 39 40 percentage remained low throughout the growing season with a sharp increase to 16% at the end of the season (Fig. 13). High rates of MSV transmission did not correspond with high numbers of collected leafhoppers (Figs. 10-13). Regression analyses. Significant associations (p<0.001) were observed between MSV incidences on TZB-Gusao and leafhopper population densities (one month after each planting), percent transmission and rainfall at 42 days: MSV incidence »* -2.47 + 4.2 Cicadulina density - 0.27 transmission + 0.01 rainfall; R2 — 90.5%, (1) and at 63 days after planting at Ikenne: MSV incidence - -3.18 + 6.2 Cicadulina density -0.39 transmission + 0.01 rainfall; R2 - 91.6%. (2) Similarly a significant association (p<0.037) was observed between MSV incidence on TZB-Gusao at 21 days after planting and leafhopper population density sampled the same, day of planting at Funtua: MSV incidence — -2.06 + Cicadulina density; R2 - 99.7%. (3) A positive association (p<0.020) was also found between MSV incidence at 42 days after planting and mean temperature at Samaru: MSV incidence - 57.82 - 2.20 temperature; R2 — 99.9%. (4) Vector pressure. Significant differences (p<0.0001) in MSV incidences and severity were observed among maize varieties TZB-Gusao, 8329-15 and 8321-21 inoculated with different numbers of MSV-exposed leafhoppers at 14 days after inoculation (Tables 8 and 9). Incidence of MSV on variety 8321-21 was significantly lower than incidence of MSV on 8329-15 and TZB-Gusao. However, no significant differences in MSV t.3 Ifcie iauia efopr () n Cicadulina and (%) leafhoppers Cicadulina Infective Ftg.13. Cicadulina Infectlvity (%) 30 10 0 2 - - efopr est a Fnu drn 1989. during Funtua at density leafhopper Month Aug Cicadulina /m2 Cicadulina % Cicadulina /infectivity Cicadulina % eJ nJlOct SepJun Jul -10 Cicadulina Im2 41 Table 8. Maize streak virus (MSV) incidence on maize varieties at 14 and 28 days after inoculation with different numbers of MSV-exposed leafhoppers following a 24 hr inoculation access period (IAP). Maize variety Leaf hopper TZB- 4 85.0 95.0 82.5 92.5 67.5 85.0 78.33 90.80 (0.75)° (0.93) (0.73) (0.88) (0.62) (0.78) (0.70) (0.86) 8 97.5 97.5 97.5 97.5 80.0 97.5 91.7 97.5 (0.95) (0.95) (0.95) (0.95) (0.71) (0.95) (0.87) (0.95) 16 100.0 100.0 100.0 100.0 82.5 97.5 94.2 99.2 (1.00) (1.00) (1.00) (1.00) (0.73) (0.95) (0.92) (0.98) 32 100.0 100.0 100.0 100.0 97.5 100.0 99.2 100.0 (1.00) (1.00) (1.00) (1.00) (0.95) (1.00) (0.98) (1.00) LSD(0.05) (0.12)b (0.25)c Mean 95.6 98.1 95.0 97.5 81.9 95.0 (0.93) (0.97) (0.92) (0.95) (0.76) (0.92) LSDd(_0S) (0.05) (0.06) 0 arcsine transformed data. b and c LSD for MSV incidence on leafhopper number at 14 and 28 days respectively. d LSD for MSV incidence on varieties for 14 and 28 days after IAP. to Table 9. Maize streak virus (MSV) severity on maize varieties at 28 and 56 days after inoculation with diffferent numbers of MSV-exposed leafhoppers following a 24 hr inoculation access period(IAP). Maize varieties Leaf TZB-GUSAU 8329-15 8321-21 hopper ______number 28 days 56 days 28 days 56 days 28 days 56 days 4 4.98 6.1 2.65 3.85 1.72 2.48 8 5.18 6.50 3.08 3.65 1.60 2.50 16 5.48 7.08 3.00 3.83 1.65 2.60 32 5.40 7.23 3.00 3.83 2.15 2.98 LSD(0.05) °* 568 ° - 5lb and b LSD for MSV severity on interaction (leafhopper numbers x varieties) at 28 and 56 days after IAP respectively. LO■p - 44 incidence were found among these varieties at 28 days after inoculation. Significant differences (p<0.0001) in MSV incidence were also found among MSV-exposed leafhopper numbers at 14 days after infestation (Table 8). Incidences of MSV were significantly lower when low numbers of leafhoppers per plant (4) were used, compared to higher numbers (8, 16 and 32). There was a significant (p<0.0002) linear effect of log of numbers of MSV-exposed leafhopper on MSV incidence for all varieties at both assessment dates, although no significant differences in MSV incidence were observed among MSV-exposed leafhopper numbers at 28 days after infestation. Highly significant differences (p<0.0001) in MSV severity were noticed among varieties 28 days after inoculation, whereas no significant differences were detected among numbers of MSV-exposed leafhoppers. Significant (p<0.048) interactions (number of MSV-exposed leafhoppers x variety) for MSV severity were observed when MSV disease was fully expressed at 56 days after infestation. This implies that increasing the number of MSV-exposed leafhoppers did not have the same effect on all varieties. The effect of increasing leafhopper numbers on MSV severity was more pronounced on the susceptible variety TZB-Gusao than on varieties 8321-21 and 8329-15. At both assessment dates, MSV severity for 8321-21 was significantly lower than the MSV severity rating observed for 8329-15 or TZB-Gusao. The results of MSV incidence and severity on maize varieties inoculated with different numbers of MSV-exposed leafhoppers for various time periods are presented in Tables 10 and 11. Significant differences (p<0.0017) in MSV incidence were observed among varieties only at 14 days after infestation (Table 10). Variety 8321-21 had a significantly Table 10. Maize streak virus (MSV) incidence on maize varieties at 14 and 28 days after inoculation with different numbers of MSV-exposed leafhoppers at various time periods following a 24 hr inoculation access period (IAP). Leaf Maize varieties hopper numbers TZB-•Gusao 8329- 15 8321- 21 Mean X Time 14 days 28 days 14 days 28 days 14 days 28 days 14 days 28 days 2 x 6 16.04 20.00 25.83 29.65 8.82 17.50 16.90 22.38 (0.22)B (0.27) (0.33) (0.36) (0.16) (0.26) (0.24) (0.30) 2 x 12 16.00 16.52 19.72 24.72 16.90 16.90 17.54 19.38 (0.22) (0.23) (0.28) (0.32) (0.24) (0.24) (0.25) (0.26) 2 x 24 39.72 47.22 39.72 44.72 25.44 34.72 34.96 42.22 (0.43) (0.48) (0.43) (0.47) (0.32) (0.38) (0.39) (0.44) 4 x 6 39.72 42.22 33.72 35.72 20.00 34.72 31.14 37.53 (0.43) (0.45) (0.36) (0.40) (0.27) (0.38) (0.35) (0.41) 4 x 12 32.29 32.29 31.04 34.72 34.72 39.72 32.68 35.59 (0.34) (0.34) (0.33) (0.38) (0.38) (0.43) (0.35) (0.38) 4 x 24 49.72 57.22 59.72 72.22 44.72 62.22 51.39 63.89 (0.50) (0.55) (0.56) (0.65) (0.46) (0.58) (0.51) (0.59) 8 x 6 47.22 59.72 53.61 58.61 38.89 56.39 46.57 58.24 (0.48) (0.57) (0.52) (0.56) (0.41) (0.54) (0.47) (0.56) Ul Table 10. (continued): 8 x 12 52.22 57.22 54.72 57.22 40.00 52.77 48.98 55.74 (0.52) (0.56) (0.53) (0.55) (0.42) (0.52) (0.49) (0.54) 8 x 24 74.72 77.22 74.16 76.66 67.22 69.72 72.04 74.54 (0.68) (0.69) (0.67) (0.68) (0.62) (0.65) (0.66) (0.67) (0.18)b (0.16)c l s d (o.05> Mean 40.85 45.51 43.58 48.25 33.0 42.74 (0.42) (0.46) (0.45) (0.49) (0.37) (0.44) ( 0.04) (0.05) 8 arcsine transformed data. b and c LSD for MSV incidence on leafhopper numbers x time at 14 and 28 days after IAP respectively. d LSD for MSV incidence on varieties at 14 and 28 days after IAP. Table 11. Maize streak virus (MSV) severity on maize varieties at 28 and 56 days after inoculation with different numbers of MSV-exposed leafhoppers at various time periods following a 24 hr inoculation access period (IAP). Leaf Maize ■varieties hopper numbers TZB-•GUSAU 8329-15 8321-■21 Mean X Time 28 days 56 days 28 days 56 days 28 days 56 days 28 days 56 days 2 x 6 4.1 5.0 3.8 5.0 2.9 3.3 3.6 4.4 2 x 12 3.6 5.3 3.1 4.0 2.0 2.5 2.9 3.9 2 x 24 4.4 5.5 2.8 3.9 1.7 2.6 3.0 4.0 4 x 6 5.0 5.9 3.3 3.9 2.5 3.4 3.6 4.4 4 x 12 4.1 4.6 3.1 3.5 1.8 2.3 3.0 3.4 4 x 24 4.6 6.4 3.7 4.0 2.3 2.8 3.5 4.4 8 x 6 4.3 5.8 3.7 4.2 2.4 3.8 3.5 4.6 8 x 12 5.3 5.9 3.8 4.6 2.3 2.9 2.8 3.8 8 x 24 5.1 5.4 3.8 4.1 2.7 3.0 3.9 4.2 LSD{0.05) 1.00a 1.21b Kean 4.5 5.5 3.4 4.1 2.3 2.9 LSDC(0.05) 0.37 0.43 a and b LSD for MSV severity on leafhopper numbers x time at 28 and 56 days after IAP respectively. c LSD for Msv severity on varieties at 28 and 56 days after IAP. 48 lower MSV incidence than varieties TZB-Gusao and 8329-15 only at 14 days after infestation. Irrespective of numbers of leafhoppers x MSV exposure time, significant differences (p<0.0001) in MSV severity ratings were observed among varieties at both assessment dates. The highly tolerant variety 8321-21 had a significantly lower severity rating than the intolerant variety TZB-Gusao and the moderately tolerant variety 8329-15. DISCUSSION The results of MSV incidences on the susceptible variety TZB-Gusao and moderately tolerant variety 8329-15 are in agreement with previous findings where low MSV incidences were noticed in early season plantings and high MSV incidences in late (Guinea Savanna) and in second season (rain forest) plantings (Fajemisin et al., 1967; Fajemisin et al., 1982; Okoth and Dabrowski, 1987). The lack of significant differences between TZB-Gusao and 8329-15 in terms of MSV incidence might be due to either the contamination of the susceptible, open pollinated variety TZB-Gusao with MSV-tolerance genes or the low frequency of MSV tolerance genes (especially modifiers) in the moderately tolerant hybrid 8329-15 (Soto et al., 1982; Kim et al., 1989). The low MSV severity rating recorded on the susceptible variety TZB-Gusao, was most likely due to late occurrence of the disease within the fields in these ecological zones (van Rensburg, 1981). MSV incidence and disease severity on variety 8321-21 remained relatively low across locations and sampling dates. This confirms the existence of two characteristics; tolerance and 49 tolremicity (low field virus incidence) that confer the overall MSV resistance (Buddenhagen, 1983). The screenhouse data showed a different trend in terms of MSV incidence. The variety 8321-21 significantly differed in MSV incidence from varieties TZB-Gusao and 8329-15 only in early growth stages (14 days after inoculation). Low virus incidence as a result of non preference for feeding by vectors would not occur when leafhoppers are confined to the maize plants as in these studies. The incubation period of MSV within 8321-21 appeared to be longer than within the other two varieties. Hence, the disease progress within the resistant variety had a marked lag phase as was also shown with a MSV-tolerant local variety in Mauritius (Autrey and Ricaud, 1983). The variety 8321-21 was significantly different in terms of MSV-symptom severity from the other two varieties although MSV severity increased with log infective leafhopper numbers. The results on leafhopper samplings carried out in Guinea Savanna locations were in accordance with previous reports indicating that leafhopper populations build up with rains because of the abundant suitable grass hosts for leafhopper development, survival and fecundity (Rose, 1978; Fajemisin et al., 1986; Okoth et al., 1987). In the humid forest zone, a different leafhopper population trend was observed with populations consistently low during the dry irrigated and first rainy season and a sharp increase at the end of the second rainy season (November - December). Guthrie (1976), Rose (1978) and IITA (1984) have reported collections of high numbers of leafhoppers in irrigated maize and/or wheat that harbor weeds which attract leafhoppers. However, this 50 was not the case with the surveys carried out at Ikenne where maize was grown under irrigation during the dry season. Low numbers of leafhoppers were collected on B. distichophylla, B. deflexa, D. horizontalis and E. Indica during the period surveyed at Ikenne except in December 1988 and November 1989. The reason for these low leafhopper population levels is not well understood, but it could be among other factors due to unsuitability of B. distichophylla the most abundant grass at this location, as an oviposition host of leafhoppers. Also heavy rainfall during most of the year could have resulted in high leafhopper mortality. In Zimbabwe, Rose (1972b; 1978) reported a positive correlation between Cicadulina numbers and rain during the preceding months. However, no such correlation was found in our studies, although it is well documented that rainfall distribution allows increased grass growth which in turn supports leafhopper population build u p . The sharp population peaks observed at the end of the rains at Ikenne might be in part due to leafhopper migrating from areas where grasses have started to dry out. All these aspects deserve further studying. C. mbila was the most common species in all ecological zones. The predominance of the species declined in the order C. mbila, C . arachidis, C. storeyi, C. similis and C, ghaurii. C. similis, previously observed only in the Guinea Savanna zone (Okoth and Dabrowski, 1987) was also found in the humid forest zone. In these surveys, there were seasonal differences in species composition as in the previous reports (IITA, 1985; Dabrowski, 1987c). C. ghaurii, which in our surveys was only found in the rain forest zone, had been reported 51 previously from other ecological zones in Nigeria and Cameroun (Dabrowski, 1987a). Evidence from this study support the view that leafhopper species are not confined to small geographical area but rather are distributed over large ones (Ruppel, 1965; Rose, 1978; Okoth and Dabrowski, 1987). The proportion of MSV inoculative leafhoppers was not consistent across locations and sampling times. The percentages of field-collected leafhoppers which were viruliferous were low at the beginning of the rainy season (0 to 4%) and reached high proportions (15 to 22%) toward the end of the rainy season. Okoth and Dabrowski (1987) reported similar results for Nigeria. High percentages of viruliferous leafhoppers toward the end of rainy season could suggest a broad reservoir of MSV in local grasses and maize or could just be due to the fact that as the season progresses, more leafhoppers have fed on an infected plant and therefore have become infective. Annual grasses such as D. horizontalis, E. indica, B. deflexa and B. distichophylla on which leafhoppers were often collected and MSV-like symptoms observed might have an epidemiological importance. B. distichophylla might not be a good oviposition hosts and was found to be a poor MSV sources (see Chapter I). The sources for MSV reoccurrence in maize following the dry season and the ways leafhoppers overseason in West Africa are not well understood. However, we believe it is possible that at the end of rainy season, infective leafhoppers migrate into moist grasses bordering rivers or into grassy weeds and cultivated maize in bottom lands with abundant residual moisture. These hosts then provide a mean for 52 survival of leafhoppers and perpetuation of MSV during dry season. In the humid forest zone, MSV may also bridge maize growing seasons on perennial grasses, including Coix lachryma-jobi, Axonopus compressus and Paspalum scrobiculatum. In irrigated areas where maize and grassy weeds (i.e. Brachiaria spp., Digitaria spp., and Eleusine spp.) grow side by side year around, both the leafhoppers and MSV might survive in both the crop and weeds. Indeed at Ikenne, leafhoppers were collected in all monthly (and later biweekly) samplings and MSV symptoms were always present on grasses and sometimes on maize. The regressions between MSV incidence on TZB-Gusao and leafhopper density and natural MSV infectivity as well as weather conditions were erratic. Generally, MSV incidences were closely associated with infective leafhopper population levels and weather factors (e.g. rainfall and temperature) in the rainforest zone and the Northern Guinea Savanna zone. Similar associations between MSV incidence and the above factors have been reported at IITA (Okoth et al., 1987). However, studies conducted in Reunion Island (Reynaud, 1988) have shown that there was not necessarily a direct association between MSV incidence on maize and infective leafhopper population density. There was also no close association between MSV incidence and viruliferous leafhopper populations and weather factors in Guinea Savanna zone. These regressions imply that the high MSV incidences recorded on TZB-Gusao and 8329-15 in the rain forest zone were most likely to have been due to high infective C. mbila leafhopper numbers. This is confirmed by the screenhouse studies where high MSV incidences on maize varieties were obtained with high numbers of viruliferous C. storeyi. This study 53 indicated that there was a linear relationship between MSV incidence and disease symptom severity on log of infective leafhopper numbers. These field and screenhouse results were in accordance with the report that MSV incidence and symptom severity increased with increasing viruliferous leafhopper numbers (Rose, 1974). Hao and Pitre (1970) reported similar results on corn stunt disease which is transmitted by the leafhopper Dalbulus maidis in the Americas. Low levels of infective Cicadulina spp. population could cause high MSV incidences on maize if the insects were more efficient in transmitting MSV and much more mobile (Cater, 1962; Fajemisin et al., 1976). This could be the case for Funtua where infective C, mbila were in low numbers, especially for July 1989 when we failed to trap any. At Samaru, MSV remained relatively low perhaps because of high proportion of C. arachidis which is a poor vector of MSV and low numbers of C. mbila and C. storeyi, In the rain forest zone, MSV disease on maize was virtually nonexistent in all plantings done from March to September, 1990, despite the presence of low MSV symptom incidence on grasses in proximity to fields. This could be due to low leafhopper population densities during the period and B. distichophylla with MSV-like symptom (presumably abundant virus source) was found to be a poor MSV source. CHAPTER II DISPERSAL OF CICADPLINA STOREYI IN MAIZE FIELD AND ITS BEHAVIOR IN RELATION TO MAIZE VARIETY, MAIZE GROWTH STAGE AND PRESENCE OF MAIZE STREAK VIRUS INTRODUCTION Maize streak geminivirus (MSV) is transmitted by Cicadulina species that are considered sedentary, although short and long distance fliers have been identified (Rose, 1973b and 1978). Rose indicated that proportions of short and long distance fliers in Cicadulina leafhopper populations varied according to species and seasons. Long distance fliers are always found farther away from the source and are predominantly females (Rose, 1978), whereas short distance fliers seem to be responsible for transmission of MSV within maize fields located in the vicinity of infected grasses (Gorter 1953). Movement and feeding behavior of Cicadulina leafhoppers, which are influenced by biotic and abiotic factors, play an important role in the spread of maize viruses including MSV in fields (Chiykowski, 1981). The known host range of Cicadulina leafhoppers includes only members of the Poaceae (Gramineae). However, their feeding, oviposition and nymphal development are affected by different host plants and by different varieties within the same host species (van Rensburg, 1982b; 54 55 Dabrowski, 1987b). It has been shown that the preferred host plant for feeding is not necessarily the best host plant for oviposition and nymphal development of Cicadulina storeyi (syn. C. triangula) (Dabrowski, 1987b). Feeding and oviposition behaviors of leafhoppers may be modified by MSV resistant cultivars or by the health of host plants. Mark, release and recapture techniques involving leafhoppers have been used to estimate population densities (Southwood, 1978; Sinsko and Graig, 1979) and to study dispersal in peach and cherry orchards and blueberry and paddy rice fields (Ito and Miyashita, 1961; Purcell and Suslow, 1982; Whitney and Meyer, 1987; Larson and Whalon, 1988). By using rubidium and flourescent dye dusts to mark leafhoppers, these authors have been able to evaluate leafhopper dispersal rate, distance and direction of movement within and outside fields or orchards. Despite widespread research on Cicadulina leafhoppers, little consideration has been paid to leafhopper movement and preference for feeding and oviposition on maize cultivars. This study was initiated, (i) to evaluate the temporal and spatial distribution and dispersal patterns of C. storeyi populations within maize fields and (ii) to determine the preference of C. storeyi for maize growth stages within the field and for feeding, oviposition and nymphal development on maize varieties (healthy or/and infected). 56 MATERIALS AND METHODS Leafhopper marking and capture. Cicadulina storeyi to be marked and released were obtained partly from the small scale colony established in the glasshouse from our field collections and from the IITA mass-reared colony. Leafhoppers were held in vials in groups of ca. 100 for marking. They were marked using one Day-GLOTM fluorescent powder dye (Corona pink). Leafhoppers were placed in dry vials with 0.250 g of dye. Vials were gently agitated for about 1 min and then placed together in a cage (0.40 m x 0.40 m x 0.75 m ) containing millet (Pennisetum americanum) seedlings. Controls (unmarked leafhoppers) used in survival, flight activity and transmission efficiency tests were handled in the same manner. Marked leafhoppers (ca. 8,000 for the first and ca. 20,000 for the second test) were released at dusk by dumping the leafhoppers onto white sheets placed on the ground at the release point. The next morning dead leafhoppers were counted (ca. 600 for the first and ca. 800 for the second trial). Effect of marking on survival. Three sets of 10 leafhoppers (five males:five females) replicated four times were treated with one of the two fluorescent powder dyes (Corona pink or yellow) or remained undusted (control). Test leafhoppers were caged in PVC tubes with maize seedlings and held in the transmission room at 26 + 1°C and 12 hr light and 12 hr dark photoperiod. Counts of surviving leafhoppers were made every 2 days for an 8 day period. A two way ANOVA was performed on the number of surviving leafhoppers. 57 Effect of marking on flight activity. Three sets of leafhoppers (ca. 100 per set) were marked with either Corona pink or yellow or were undusted (control). The leafhoppers were then placed on maize plants of variety Pool 16 in a cage (1.42 m x 0.80 m x 0.71 m) in the glasshouse. The trial involved four cages (replications) each with four yellow sticky traps hung at 0.50 m above the floor to monitor insect flight activity for 1 wk. Analysis of variance was performed on square root transformed leafhopper recapture numbers. Effect of marking on transmission efficiency. Three sets of 30 leafhoppers (15 males:15 females per set) replicated twice were dusted with one of the two fluorescent powder dyes (Corona pink or yellow) or were undusted (control). Marked leafhoppers were allowed a 48 hr acquisition access period (AAP) on MSV-infected maize plants (var. Pool 16). They were then confined on susceptible healthy maize seedlings (Pool 16) for a 24 hr inoculation access period (IAP). Folowing the IAP, maize plants were treated with granular Furadan to kill leafhoppers and then transferred to insect-proof cages for MSV-symptom development. Symptom ratings were taken 3 wk after inoculation. A two way ANOVA was performed on the number of infected plants. Field release and recapture. Leafhopper field releases were made in maize breeding fields (approximately 4 hectares) located at IITA, Ibadan. The maize in these fields had growth stages varying from 2.0 (30 days after emergence) to 4.0 (60 days after emergence) (Hanway, 1966). Yellow sticky traps (0.125 m x 0.075 m) were hung 0.50 m above the ground at distances of 5, 10, 20, 40, 80 and 160 m in four (first trial) and in eight directions (second trial) radiating from the release 58 point (Fig. 14). Yellow sticky traps were collected daily for 14 days. Sticky traps with leafhoppers were brought into the laboratory for examination. A long wave ultraviolet lamp (Black ray) was used to detect marked leafhoppers. Temperature, rainfall and wind speed were obtained from the IITA weather station and wind direction from the Moor Plantation Station at Ibadan. Regression equations (Ito and Mayashita, 1961; Freeman, 1977; Inoue, 1978; Stanner et al., 1983) were used to fit leafhopper recapture data over distance for each single day: logten(leafhopper count) - a + b logten(distance), (5) and for all the days: logten(leafhopper count) - a + b logten(distance) + c day + d day logten(distance), (6) and over time: leafhopper count - a + b day. (7) Leafhopper maize-growth-stage preference. This study was conducted in the West Bank, IITA, Ibadan. A randomized complete block design with four replications was used. Maize was planted eight times at weekly intervals from May 22 to July 10, 1990. Each planting consisted of 10 m rows (0.76 m x 0.30m spacing); two seeds/hill were planted and later thinned to one. Zero tillage was used, and herbicides, Primextra and Gramoxon (2.5 kg + 1.0 kg a.i./ha), were applied before planting. At planting time, fertilizer was applied at a rate of 120 kg of N, 120 kg of KjO and 120 kg of Pa0s per hectare. Six weeks after planting, the field was sidedressed with an additional 78 kg of N per hectare (calcium ammonium nitrate). Leafhoppers were released 59 o o 360 o N , i • 160 45 160 31i? • 160 • 80 .80 80 •4 0 ,40 '40 270° 20 020 * in *10 *20 160 80 40 90 • U A ‘, *20 *40 80 160 20 2 0 ® 20 40. 40 40 • 80, 80' 80 9 160_ 22E? 160 135 160 • 180 Fig. 14. Arrangement of yellow sticky traps* to captureCicadulina storeyi (second release) from release point41. 60 at dawn 1 wk after the last planting. Five releases were made for every two replications (a total of 10 point releases) to ensure equal exposure of all maize plots to leafhoppers. Approximately 300 leafhoppers were released at each release point. Leafhopper sampling was made for each maize plot at 1 day before release and at 3 and 6 days after release. Two samples were randomly taken with a net cage in each plot. Maize growth stages were determined as defined by Hanway (1963 and 1966). Data were transformed to log x +1 before analysis of variance and means were separated by polynomial comparisons. Leafhopper maize variety preference. Tests were carried out on preference of C. storeyi for three maize varieties with varying degrees of MSV tolerance (susceptible TZB-Gusao, moderately resistant 8329-15 and highly resistant 8321-21). Potted young healthy or infected maize plants of the three varieties were placed either separately (3 healthy or 3 diseased varieties) or together (6 healthy and diseased varieties) in a plastic cage (0.41 m x 0.41 m x 0.41 m) kept in a growth chamber at 26 + 1°C. Plants were arranged either in a triangular (healthy or infected) or in a circular (healthy and infected together) configuration. Settling preference of C. storeyi was also monitored on healthy versus infected plants of each variety. Then, 32, 48 or 96 insects (according to the test) were starved for 2 hr and released in the center of the cage. Numbers of leafhoppers present on each plant were counted after 6 hr. These tests were repeated three times over a period of 2 to 3 days. An ANOVA or a Chi Square Test was used to analyze the data. 61 Leafhopper maize varietal oviposltion preference. Ninety (45 males:45 females) C. storeyi were introduced Into a cage (0.50 m x 0.40 m x 0.70 m) with nine young maize plants of TZB-Gusao, 8329-15 and 8321- 21 (three plants per variety) arranged in a 3 x 3 Latin Square (free choice). The test was repeated twice. A separate test was conducted using 10 leafhoppers (5 males:5 females) caged on each variety (no choice). Young maize plants were arranged in a completely randomized design fashion with three replications repeated three times. These tests were conducted in the growth chamber at 26 + 1°C for 1 wk. After oviposition by leafhoppers plants were bleached in a mixture of 10% acetic acid and 75% ethyl alcohol, and then numbers of eggs laid per week were counted under a stereo microscope and recorded. An ANOVA was performed using Minitab program. Developmental period. Three sets of 100 (50 males:50 females per set) of C. storeyi were confined each in a cage (0.43 m x 0.30 m x 0.70 m) containing young maize plants of either TZB-Gusao, 8329-15 or 8321- 21. These cages were placed in a growth chamber at 26 + 1°C to allow leafhoppers to deposit eggs for 2 days. Leafhoppers were then removed and caged with plants were transferred to the glasshouse where they were frequently checked for the emergence of the adults that developed from eggs laid earlier by females. Once these first adults were noticed, daily counts of adults were made until all adults eclosed. 62 RESULTS Effects of marking on survival, flight activity and transmission efficiency. The survival of C. storeyi treated with two fluorescent powder dyes did not significantly differ (p>0.05) from that of undusted leafhoppers (Table 12) indicating that dusting had little if any adverse effect on adult survival. The number of leafhoppers captured on yellow sticky traps as a measure of their flight activity also was not significantly different between marked and undusted insects (Table 13). Marking did not hamper the ability of C. storeyi to transmit MSV (Table 13). Male and female leafhoppers both transmitted MSV as efficiently as did their unmarked counterparts. Temporal patterns. The recapture rates for released leafhoppers were 1.88 and 6.23% for the first and second release, respectively. There were differences in leafhopper recaptures over time. The numbers of leafhoppers recaptured over 14 days declined exponentially following their release (Fig. 15). This is confirmed by the linearized form of the exponential model (ln(Nt)] evaluated for goodness of fit of leafhopper recaptures and which yielded a fairly straight line (Fig. 16). It is known that rainfall and temperature directly affect leafhopper survival and movement, respectively. Heavy rainfall causes leafhopper mortality by drowning, while cold temperatures force leafhoppers to hide in maize whorls. During the first release, mean monthly temperature was 27.5°C and fields were irrigated, since there had been no rains. During the second release, mean temperature varied from 24.0 to 27.0°C and there were only two rains of approximately Table 12. Effect of flourescent dye on survival of Cicadulina storeyi. Days after marking Dye Treatment 2 4 6 8 Control 9.25a 8.25 4.25 2.50 (unmarked) Corona pink 8.75 7.75 4.75 2.25 Yellow 9.00 7.25 5.25 2.25 (SE-0.51)b Mean+SEC 9.00+0.29 7.75+0.29 4.75+0.29 2.33+0.29 0 Numbers of surviving leafhoppers. b n- 4 c n- 12 cr» LO Table 13. Effect of fluorescent dye on Cicadulina storeyi flight activity and virus transmission efficiency. Number of infected plants Dye Mean leafhopper treatment Male Female Mean+SEa recaptured+SEd control (unmarked) 5.5 5.5 5.5+0.53 10.75(3.25+ 0.30)e corona pink 5.5 6.5 6.0+0.53 9.25(2.95+0.30) yellow 4.5 5.5 5.0+0.53 8.50(2.88+0.30) (SE-0.75)b Mean+SEC 5.2+0.43 5.+0.43 a n (dye) equal Four. b n (sex x dye) equal Two. c n (sex) equal Six. d n (flight activity) equal Four. e are square root transformed data. d\ i.5 Nme o Ccdln soei efopr recaptured leafhoppers storeyi Cicadulina of Number Fig.15. Clcadullna storey] recapture numbers 0 0 2 100 300“ 400“ 500 “ “ 0 vr ie fe hi release. their after time over 2 4 as olwn lahpe release leafhopper following Days = *10A{-0.13649x) RA2 0.963 6 9 . 0 = 2 A R ) x 9 4 6 3 1 . 0 - { A 0 1 * 7 3 . 3 2 4 = y 6 1 1 1 1 1 20 18 16 14 12 10 8 65 Log number of Cicadulina storeyi recaptured 0.0 0.5- 2 1 2.5- 3.0 1.5“ . . 0 i.6 Lnaie fr o epnnil oe o number on model exponential of form Linearized Fig.16. 0 - - 0 fe hi release. their after f iauia try lahpe rcpue oe time over recaptured leafhopped storeyi Cicadulina of 2 4 as olwn lahpe release leafhopper following Days 6 8 = X 9 5 6 3 1 . 0 - 7 5 2 6 . 2 = y 0 2 4 6 8 20 18 16 14 12 10 RA2 0.963 6 9 . 0 = 66 67 8.0 nun. These weather conditions, which were fairly uniform within each release period, are considered to have not altered leafhopper catches over time (14 days). Spatial patterns. There were significant differences (p<0.001) in leafhopper recaptures among distances over the 14 day period. Trap catches of marked leafhoppers were highest at sticky traps nearest the release point and decreased linearly with distance for days 1 to 9 (Fig. 17). This Figure presents actual leafhopper counts. This linearity is also observed in the plot of "logten" leafhopper counts against logten distance for each day (Fig. 18). After day 9, leafhoppers recaptured over distance became evenly distributed (Figs. 17 and 18). No significant linear regression was found between logten leafhopper and logten distance from day 10 through day 14. The partial regression coefficient (d) of the interaction of regression equation of leafhopper catches over distance for all the days was highly significant (p<0.0001) implying that the decline in the number of leafhoppers recaptured at the farthest distance from the release point diminished over time (Fig. 17). The number of leafhoppers recaptured (N) best fits the equation: N - exp[8.17 - 1.681ndistance - 0.57daylnl0 + 0.13 day ln(distance)], (8) where exp - exponential and In - natural logarithm. Rate of dispersal of leafhoppers as determined by dividing i distance recaptured from the release point (m) by the day following release (Larsen and Whalon, 1988) was equal to 2.55 m/day and 2.76 m/day for the first and the second release, respectively. The rate of movement of dispersing leafhoppers also varied with distance. The mean l.7 Nmes f iauia try lahpes N recaptured (N) leafhoppers storeyi Cicadulina of Numbers Flg.17. Numbers of Cicadulina storeyi recaptured 200 100 - 1 t ifrn dsacs vr 4 as olwn release. following days 14 over distances different at N=exp(8.17-1.68ln(distance)-0.57day+0.125day!n(distance) o dsac () rm efopr ees point release leafhopper from (m) distance Log 2 3 m day7 day9 day3 day! 54 6 68 Logten leafhopper count Logtanleafhopper count Fig. Fig. 0 2 3 1 . 05 . 15 2.0 1.5 1.0 0.5 0.0 0.0 otn itne m fo rlae point release from (m) distance Logten otn itne m fo rtae point reteaae from (m) distance Logten 18 day 3 day . Numbers of of Numbers . lge) tvros itne rm ees point release from distances various at (logten) y - 2 . 4 1 3 8 -1.0005X -1.0005X 8 3 1 4 . 2 - y 0.5 T o ifrn as fe efopr release leafhopper after days different for 1.0 3.5173 - 2.0076X 3.5173 0.937 iauia storeyi Cicadulina A - RA2 1.5 0.871 2.0 -I £ a. a. o. O D 9 c o O 3 c o □. o o 2 3 1 0 2 3 1 0.0 . 08 . 12 . 16 1.8 1.6 1.4 1.2 1.0 0.8 0.6 otn itnem fo rlae point release from distance(m) Logten otn ltnam fo rlae point release from dlstanca(m) Logten lahpes recaptured leafhoppers day 2 day day 4 day - -2.4684X X 4 8 6 4 - 8 . 8 2 9 2 . 4 - y y - 2.6243 - 1 - 2.6243 y - 0.5 1.0 .2594k .2594k A - RA2 * 0.952 - R*2 1.5 996 .9 0 2.0 69 Logten leafhopper count Lo9tan Iwfhopper count of Numbers (continued). 18 Fig. 0 2 3 1 0 3 2 1 0.0 0.0 recaptured (logten) at various distances from release point release from distances various at (logten) recaptured otn itne m fo rlae point release from (m) distance Logten otn itne m fo rlae point release from 3 3 y - 1.6165 - 0.92291X R*2- 0.928 y - 0.92536 - 0.45116* R*2 . 0.544 2 2 a. dayS Q. day 10 1 1 cn 0 0 0.0 1.0 1.50.5 2.0 0.0 0.5 1.0 1.5 2.0 Logtan dlatanca(m) from ralaaaa point Logtan dlatanca (m) from ralaaaa polnl 3 3 S. 2 2 day 11 a day 12 1 1 0 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Logtan dlatanca (m) from ralaaaa point Logtan dlatanca (m) from ralaaaa point 72 Fig. 18 (continued). Numbers ofCicadulina storeyi leafhoppers recaptured (logten) at various distances from release point for different days after leafhopper release 3 3c oO km 2 day 13 a OQ. £ a c 1 o> _lo o 0.0 0.5 1.0 1.5 2.0 Logten distance (m) from release point 3 *4 3c uO - 2 aCL o day 14 c 1 *.03 O -I 0 0.0 0.5 1.0 1.5 2.0 Logten distance (m) from release point 73 rate of movement for each capture distance increased with distance from 1.44 m/day at 5 m to 13.62 m/day at 160 m (Fig. 19). During the period of the second release, the wind was blowing from the West and South-West toward the East and North-East except on June 21 and 28, 1990 when it blew from the North and South. The wind speed varied from 4.1 to 5.3 km/hr. Trap captures of marked leafhoppers were higher from North-East and East (318 leafhopper recaptured in 12 days) compared to the West and South-West (158 leafhoppers). Leafhopper maize-growth-stage preference. Leafhopper population density/m2 1 day before, 3 and 6 days after releases are presented in Table 14. Before leafhopper releases, very few insects were found in the maize plots. Highly significant differences (p<0.001) in leafhopper counts were observed among maize growth stages at 3 days after release. Polynomial orthogonal comparisons revealed a significant (p<0.001) quadratic effect of log leafhopper population density on maize growth stages (Fig. 20) indicating that leafhoppers preferred maize at growth stages 1.0 to 3.0 (2 to 6 wk after planting). Six days after releases, the numbers of leafhoppers markedly declined, most likely due to heavy rains which fell 2 days before sampling. Leafhopper maize variety preference. The test for homogeneity of ratio (1:1:1) on the number of leafhoppers found on three healthy maize plants of three varieties indicated that data from these sets were heterogeneous. Another test for homogeneity of ratio (1:1:1) on the number of leafhoppers settling on diseased maize plants of the same varieties also showed that data from these sets were heterogeneous. Data sets did not share a common ratio for each host settling preference i.9 Rt o dsesl mdy o Ccdln storeyi Cicadulina of (m/day) dispersal of Rate Fig.19. Mean leafhopper dispersal rate (m/day) sac () rm ees pit hr lahpes ee captured were leafhoppers where point release from (m) istance D efopr a vros itne fo rlae point. release from distances various at leafhoppers 1 2 4 8 160 80 40 20 10 5 74 Table 14. Cicadulina storeyi population density in maize field with different growth stages. Cicadulina population density/m2 Maize growth stage One day before Three days after Six daysafter release release release +0.5(1 week) 0.00 0.13(0.10+0.51)a 0.00 +1.0(2 weeks) 0.50 3.38(1.46+0.51) 0.88 +1.5(3 weeks) 0.38 9.13(1.85+0.51) 1.75 +2.0(4 weeks) 0.75 3.63(1.37+0.51) 1.38 +2.5(5 weeks) 0.75 3.88(1.55+0.51) 1.50 +3.0(6 weeks) 0.50 2.88(1.28+0.51) 0.25 +3.5(7 weeks) 0.50 1.00(0.55+0.51) 0.00 +4.0(8 weeks) 0.50 1.25(0.72+0.51) 0.00 a logx+1 transformed data plus SE and n-4. Log leafhopper count 0.00 0.25- - 0.50 0.75- 2.00 1.25- 1.50- 1.75* i.0 Qartc fet f o Ccdln storeyi Cicadulina log of effect Quadratic Fig.20. 0 efopr on o mie rwh stage growth maize on count leafhopper 1 2 eky plantings Weekly 7 3 5 4 6 8 76 77 experiment. Therefore, Chi squares from these sets for each experiment cannot be pooled together. Examination of these individual X2 showed that there was no consistent preference of C. storeyi for any variety either healthy or diseased. In some data sets, there was a preference for either TZB-Gusao, 8329-15 or 8321-21. The reasons for these discrepancies are not clear. In addition, ANOVA was performed on square root transformed leafhopper counts for the two experiments and the results showed again that there was no preference of C. storeyi for TZB- Gusao, 8329-15 or 8321-21, whether infected with MSV or healthy (Tables 15 and 16). However, on diseased plants, the mumber of leafhoppers setling on variety 8321-21 was approximately half of those on the other two varieties (Table 16). The test of homogeneity of ratio (1:1:1:1:1:1) of the number of leafhoppers found on maize plants (healthy and diseased together) in seven sets was not significant (the seven sets of data shared a common ratio) . Thus X2 from all the sets of data were pooled together (X„2 — 98.32) and compared to the tabular X2 (57.34). The number of leafhoppers on infected TZB-Gusao was significantly higher than any other treatment combination (healthy x diseased plants). The ratio is about 2:1:1:1:1:1 in favor of diseased TZB-Gusao. Similarly, tests for homogeneity of ratio (1:1) on leafhopper counts on healthy versus diseased plants of TZB-Gusao, 8329-15 and 8321-21 in seven sets each were not significant. Pooled X2 from each seven sets of data for each test (54.64, 27.44 and 78.86) were larger than the corresponding tabular X2 at 1% level (18.47) confirming that there was preference of C. storeyi for diseased maize plants (TZB-Gusao, 8329-15 and 8321-21). The 78 Table 15: Number of Cicadulina. storeyi settling on healthy maize varieties. Days Maize variety Mean+SEd 1 2 3 TZB-Gusao 28.8(5.2)° 26.0(5.0) 29.0(5.3) 27.9(5.1+0.4) 8321-21 27.0(5.0) 17.3(4.2) 28.3(5.3) 24.4(4.8±0.4) 8329-15 21.7(4.5) 27.3(5.1) 16.0(3.8) 21.7(4.4+0.4) (SE-0.82)b Mean+SEC 25.8(4.9±0. 5) 23.6(4.7+0 .5) 24.4(4.8+0.5) a Square root transformed data. b n—3 c and d n-9 Table 16. Number of Cicadulina storeyi settling on maize varieties infected with maize streak virus (MSV). Maize variety Mean leafhopper counts+SEb TZB-Gusao 12.50(3.51±0.36)° 8321-21 7.33(2.63+0.36) 8329-15 13.17(3.52+0.36) a Square root transformed data+SE. b n-6 79 ratio of preference seems to be about 3:1 in favor of diseased vs healthy TZB-Gusao and diseased vs healthy 8321-21 and 2:1 in favor of diseased vs healthy 8329-15. Host oviposition preference. There were no significant differences in number of eggs laid by C. storeyi on TZB-Gusao, 8329-15 and 8321-21 (Tables 17 and 18). However, when the insects were given a free-choice they seemed to prefer TZB-Gusao (Table 17) more than the other two. Developmental period (egg to adult) of C. storeyi. Mean and median developmental periods (days) of C. storeyi (Table 19) on TZB- Gusao, 8329-15 and 8321-21 were not found to be significantly different. DISCUSSION Marking leafhoppers with fluorescent dye had no significant adverse effects on leafhopper survival, flight activity and transmission efficiency of MSV. These results were in agreement with previous reports for other leafhoppers (Purcell and Suslow, 1982; Larsen and Whalon, 1978). There was a rapid decline in adult survival which might have been due to other factors including low relative humidity. The recapture rate of marked leafhoppers was higher for the second than first release. This probably was the result of overcrowding (due to great numbers of released leafhoppers the second time) which might have enhanced leafhopper escaping behavior, and of placing yellow sticky traps in four more directions. The recapture rates of C. storeyi (1. 88 and 6.23%) for the first and the second release experiments were similar Table 17. Number of eggs laid per female of Cicadulina storeyi on three maize varieties over 7 day oviposition period(free-choice test). Experiments Maize Mean ±SEa variety 1 2 TZB-Gusao 174.0 107.0 140.5+20.2 8321-21 94.0 112.3 103.2+20.2 8329-15 114.3 82.7 98.5+20.2 (SE-34.9)b Mean+SEC 127+16.5 100.7+16.5 a n- 6 b n (interaction)- 3 c n - 9 0 0 o Table 18. Number of eggs laid per female of Cicadulina storeyi on three maize varieties over 7 day oviposition period (No-choice test). Experiments Maize variety Mean+SEa 1 2 3 TZB-Gusau 145.7 136.7 142.7 141.7+13.0 8321-21 146.3 128.0 128.0 134.1+13.0 8329=15 152.0 168.7 121.0 147.2±13.0 (SE=22.4)b Mean+SEC 148.0+0.13 144.4+0.13 130.6+0.13 a and c n=9 b n-3 00 Table 19. Developmental period(egg to adult) of Cicadulina storeyi on maize varieties. Maize variety Mean emergence + SE8 Median emergence+SE TZB-Gusao 24.18+0.31 24.3+0.30 8321-21 24.56+0.31 24.3+0.30 8329-15 25.03+0.31 25.0+0.30 a n-3 (experiments) ; 50 couples of C . storeyi per experiment. 00 ro 83 to those of Paraphlepsius irroratus (Say) (Larsen and Whalon, 1988), and Colladonus montanus (Purcell and Suslow, 1982), but significantly lower than those of Nephotettix cincCiceps (Ito and Miyashita, 1961) and Scapbytopius spp. (Whitney and Mayer, 1987). Temperature and rainfall, known to affect directly leafhopper survival and movement were fairly uniform within each release period and apparently did not alter leafhopper daily catches over time. The number of leafhoppers recaptured over time decreased exponentially following their release. This trend could be expected since senescing leafhoppers are dying and prey to predators over time. Similar exponential curves had been observed with P. irroratus (Larsen and Whalon, 1988). Flourescent dye which adhered on the cervix and on the sternum between the bases of legs of C. storeyi did not wash off allowing easy identification of marked leafhoppers during the release period. Leafhoppers catches decreased linearly with distance from the release point. These results agree with the report by Rose (1978) that short and long fliers exist in Cicadulina spp. The steep decline of leafhopper captures with distance suggests the predominance of short distance fliers in C. storeyi population. This is not surprising since most of C, storeyi used were from IITA mass reared colony where the rearing conditions have probably resulted in selection for short distance fliers over years. These results were in accordance with previous findings that C. mbila were caught in highest number nearest bordering grasses and steeply declined with distance from them (Gorter, 1953; Rose, 1973b and 1978). Similar linear decline of leafhopper catches with distance from release point has been observed for P. 84 irroratus (Larsen and Whalon, 1988). Our data Indicated that 10 days after release, leafhoppers were evenly distributed throughout the maize field. Leafhopper movement could have been affected either by different maize growth stages, although most of them were in the range of attractiveness for C. storeyi, or by physiological status of female C, storeyi (presence of mature ova). The rates of leafhopper dispersal were similar for the first and the second release. However, they varied with distance from 1.44m/day at 5m to 13.6m/day at 160m. Wind direction as indicated by numbers of leafhoppers recaptured (318) in East and North-East directions toward which the wind was blowing had a remarkable effect on leafhopper movement. However, no such indication could be detected with the first release most likely because of the low numbers of leafhoppers recaptured (1.88%). The influence of wind direction on leafhoppers movement was also shown by the high numbers of C. storeyi caught down wind from breeding grasses in Zimbabwe (Rose, 1974). The results of this experiment indicated that maize is more attractive or susceptible when it is at the growth stages varying from 1.0 to 3.0. When maize is before growth stage 1.0 visual and olfactory cues might not be strong enough to attract the leafhoppers. After growth stage 3.0 maize becomes unsuitable for feeding and oviposition (Rose, 1978) and as a result leafhoppers migrate to palatable host plants. These results were consistent with the observations made by Sedlacek and Freytag (1986) that Graminella ntgrifrons was found in greater numbers in maize at growth stage 1.0. C. storeyi preferred 85 neither very young nor old maize plants, thus, caution should be taken not to grow maize at preferred growth stages at the vicinity of plots used for MSV resistance screening. C. storeyi did not show any varietal preference for any of the three healthy or MSV-infected maize varieties. The results failed to show preference among the maize varieties because there was perhaps not enough data. However, we believe that Cicadulina leafhoppers do prefer MSV susceptible varieties over resistant ones. Feeding preference of C. mbila and C. storeyi for susceptible maize varieties has been demonstrated using electronic feeding monitor (Mesfin and Bosque Perez, personal communication). There was no oviposition preference of a susceptible variety over resistant ones, since C, storeyi laid similar numbers of eggs on all of the varieties, although they laid more eggs on the susceptible variety when they were given a choice. Also the nymphs developed faster on all maize varieties. These results were in accordance with the findings of Collins and Pitre (1968) that susceptible hybrids were not preferred over corn stunt virus (probably maize chlorotic dwarf machlovirus) resistant hybrids by G. nigrifrons or Dab lulus maidis. Nevertheless, Sogawa (1982) reported that brown planthopper, Nilapavarta lugens preferred susceptible over resistant rice varieties probably because the former had phagostimulants (sucrose) that elicited and sustained sucking by N. lugens. Dabrowski (1987b) has also indicated different oviposition preferences by C. storeyi to millet (Pennisetum americana) cultivars used in leafhopper mass rearing. This could be due to strong visual and olfactory stimuli in some of these varieties which attracted leafhoppers. The influence of visual 86 and olfactory stimill during close-range host finding of D. maidis has been reported in the laboratory bioassays (J.L. Todd, personal communication). C. storeyi leafhoppers showed preference for MSV-infected maize plants especially TZB-Gusao when they were given a choice between healthy and infected maize varieties either separately or together. These results were consistent with the report by P.G. Markham (personal communication') that C. mbila showed a preference for infected maize plants over healthy maize initially based on color and then on feeding. Similar results were reported with three species of aphids, Myzus persicae, Aphis fabae and Aulacorthum solani which preferred leaves showing pronounced yellows from virus infection over healthy ones (Carter, 1962). Viral infection may induce chemical changes in infected plants resulting in accumulation of free amino acids and sugars such as sucrose (Harpaz and Klein, 1966; Sogawa, 1982). Therefore, maize varieties infected with MSV might produce significant increases in these phagostimulants which would arrest the departure of C. storeyi leafhoppers. CHAPTER III CICADULINA BIOLOGY AND ITS TRANSMISSION OF MAIZE STREAK VIRUS INTRODUCTION Temperature is a major environmental factor that affects directly leafhopper fecundity, longevity and developmental period, as well as the virus, the host, the transmission process, and/or the disease process (Damsteegt, 1980 and 1983). Several species of Cicadulina, including £7. mbila, C. storeyi, £7. similis and C . ghaurii, have been reported as vectors of maize streak geminivirus (MSV), a virus prevalent in sub-Saharan African countries and neighbouring islands (Rose, 1978; Okoth et al., 1985; Dabrowski, 1978c). Much work has been done on the biology of C. mbila, £7. storeyi, C. parazeae, C. china and £7. bipunctella zeae for which similar results have been obtained (van der Merwe, 1926; Ruppel, 1965; Rose 1973a; Ammar 1975 and 1977; van Rensburg, 1982a, 1982b and Dabrowski, 1985). Delong (1971) pointed out that host plants on which leafhoppers develop may influence their fecundity, survival and developmental period. Cicadulina species that are more fecund, survive longer and develop faster may play an important role on the epidemiology of MSV (Rose, 1973b and 1978). Cicadulina leafhoppers, which are composed of active and inactive populations (Storey, 1932), differ in their MSV transmission efficiency. 87 88 MSV, which they transmitted in a circulative relationship can be acquired by feeding on infected plants for as short as 15 and 30 sec for C, mbila and C. storeyi, respectively (Storey, 1925 and 1939; Seth et al., 1972; Guthrie, 1976; Rose, 1978; Zagre, 1983). Okoth (1985) found that there were no significant differences in virus transmission efficiency amongst various Cicadulina populations from different ecological zones in Nigeria. Previous authors also indicated that Cicadulina leafhoppers can remain infective for life, once they aquire the virus. The pattern of MSV transmission could be either intermittent or continous depending upon the length of the inoculation access period (Okoth, 1985). Ling (1969) has reported a continuous transmission of rice tungro virus by green leafhoppers (Niphotettix species) when given two day serial transfers. There has been, however, none or little work done on the biology and transmission efficiency of MSV by C . arachidis and C. ghaurii. Hence, this study was performed: (i) to determine and compare adult survival, fecundity and developmental time (egg to adult) of C. storeyi, C. arachidis and C. ghaurii; (ii) to describe MSV transmission by C. arachidis and C. ghaurii; (H i ) and to compare the transmission efficiency of different species as well as of males and females of Cicadulina leafhoppers. 89 MATERIALS AND METHODS Culture of Cicadulina spp. Small colonies of C. arachidis and C, mbila were maintained on millet (P. typhoides) plants inside 0.50 m x 0.40m x 0.70 m cages in a growth chamber at 26 + 1°C. That of C. ghaurii, however, was reared on maize plants in 0.50 m x 0.40 m x 0.70 m cages kept in a growth chamber at 30 + 1°C. Colonies of these species were established from field collected adults. C. storeyi was obtained from the IITA mass rearing facilities. Developmental period (egg to adult). Three sets of 100 (50 males: 50 females per set) leafhoppers for each of the two species (C. arachidis and C. storeyi) and three sets of 20 (10 males: 10 females per set) C. ghaurii were confined in PVC tube cages containing maize seedlings in a growth chamber at 26 + 1°C to allow leafhoppers to lay eggs for 2 days. After 2 days, the insects were removed and the maize plants for each species and set of leafhoppers were placed in 0.43 m x 0.30 m x 0.70 m wooden mesh cages and transferred to growth chambers at 20, 25 or 30°C. Each cage was checked periodically for the emergence of adults from eggs deposited earlier by females. Once nymphs started to eclose, adults were removed and counted until all eclosed. Adult survival and fecundity. One hundred newly eclosed adults (50 males:50 females) each of C. arachidis and C. storeyi, and 20 emerging adults (10 males:10 females) of C. ghaurii were released in separate PVC tube cages containing maize seedlings (var. Pool 16). The PVC tube cages were placed in growth chambers set at 20, 25 or 30°C. Maize plants were removed weekly, and surviving male and female leafhoppers were counted and 90 placed in cages with new maize plants. The removed plants were placed in bottles containing a mixture of 10% acetic acid and 75% ethyl alcohol to bleach leaves. Weekly counts of eggs deposited in maize leaves was made under a stereoraicroscope. The experiment was terminated when all females died. A series of transmission tests was conducted in the growth chamber at 26 + 1°C with a 12 hr light and 12 hr dark photoperiod as described by Zagre (1983) and Okoth et al. (1987), At the end of inoculation access periods (IAP), leafhoppers were removed from the test plants which were then treated with granular Furadan and transferred to insect-proof cages for MSV symptom expression and evaluation after 3 wk. A maize variety (Pool 16) infected with MSV was used as the virus source. The net reproductive rate (Ro), the capacity for increase (rc) and the mean adult age of egg-laying females (tc) were calculated from fecundity table data (Madden, 1985). Acquisition access period (AAP). Nonviruliferous leafhoppers (ca. 120 for C. arachidis and ca. 60 for C. ghaurii) were starved for 2 hr and then given AAP on MSV-infected maize for various periods ranging from 15 sec to 96 hr for a total of 12 different AAP. For AAPs of 15 sec to 1 min, individual leafhoppers confined in the vials were observed on infected maize leaf segments and the leafhoppers were removed after feeding for the desired period. At the end of these different AAPs, leafhoppers were caged singly in healthy maize seedlings of MSV- susceptible hybrid FR114 x FR303 for an inoculation period (IAP) of 24 hr. Inoculation access period. Leafhoppers (ca. 120 C. arachidis and ca. 60 C. ghaurii) were given a 48 hr AAP on MSV-infected maize. After 91 the AAP, leafhoppers were starved for 2 hr and then were individually caged on healthy maize seedlings of hybrid FR114 x FR303 for an IAP varying from 1 min to 96 hr for a total of 10 IAP. For the 1 min IAP, Individual leafhoppers were observed on healthy maize leaf segments as reported above. Latent Period (LP). Virus-free C. arachidis and C, ghaurii were starved for 2 hr and then given an AAP on MSV-infected maize for 1, 2 or 8 hr (C . arachidis) or for 8 hr (C . ghaurii) . Leafhoppers were then caged singly on healthy maize seedlings of hybrid FR114 x FR303. A serial transfer was made every 2 hr for the first 14 hr (three to six times according to AAP) followed by a 10, 24 or 48 hr IAP. Test plants were treated with granular Furadan and kept in the growth chamber at 26 + 1°C and 12 hr light and 12 hr dark photoperiod for MSV symptom development and evaluation after approximately 3 wk. Retention of MSV by Cicadulina spp. Newly emerged leafhoppers (25 C. arachidis and 10 C, ghaurii) were allowed to acquire MSV from Infected maize plants for 24 hr. They then were caged singly on healthy maize seedlings of hybrid FR114 x FR303 for 5 days. Leafhoppers were transferred to new test plants every 5 days after which most insects had died. Transmission efficiency of Cicadulina species. Nonviruliferous leafhoppers of C. mbila, C . storeyi, C. arachidis and C. ghaurii were starved for 2 hr and then given an AAP on MSV-infected maize plants for times ranging from 1 to 48 hr for a total of five. At the end of these AAPs, insects were Individually caged on healthy maize seedlings of hybrid FR114 x FR303 for an IAP of 24 hr. The number of test plants varied 92 between eight and 60 plants according to the availability of each Cicadulina species. Transmission efficiency of male and female Cicadulina spp. Virus- free leafhoppers were given a 48 hr AAP on MSV-infected maize and then caged singly on healthy maize seedlings (hybrid FR114 x FR303) for a 24 hr IAP. The number of test plants per sex varied between 13 (£7. ghaurii) and 30 (£7. mbila, £7. storeyi and £7. arachidis). RESULTS Developmental period (egg to adult). C. storeyi developed much faster than C. arachidis and £7. ghaurii at the three temperature regimes (Table 20). While the latter two species had similar developmental times at 30°C, they differed from each other in their growth rate at 25 and 20oC. Mean developmental period decreased remarkably (p<0.001) from low (20°C) to high temperatures for all the three species. These leafhopper species, especially C. arachidis, had low developmental rates at 20°C. Nevertheless, the three temperatures were sufficient for development. The best temperature for the three species was approximately 30°C. Adult survival and fecundity. £7. storeyi generally survived better at all temperatures than did £7. arachidis and £7, ghaurii (Table 21). The former species overlapped in survival times (t25 at 30°C, t50 at 25°C, and t75 at 20°C) with £7. arachidis. However, £7. ghaurii, did not live as long as £7. arachidis, except at 30°C (t75) . Female leafhoppers of these species lived longer than males for all these temperatures. At 20°C £7. ghaurii, C. storeyi and £7. arachidis females Table 20. Mean and median developmental period (number of days) of Cicadulina species reared on maize (variety Pool 16) at different temperatures. Temperature Cicadulina 30°C 25°C 20°C species Mean±SE Median Mean+SE Median Mean+SE Median C. storeyiB 21.6+0.1 21 28.8+0.2 29 51.8+0.3 52 C. arachidisb 23.7+0.1 23 29.7+0.3 29 59.9+0.3 60 C. ghauriic 23.1+0.2 23 31.1+0.3 31 54.9+0.6 55 8 n equal 239,123 and 130 for C. storeyi at 30, 25 and 20°C, respectively. b n equal 150,60 and 139 for C, arachidis at 30, 25 and 20°C, respectively. c n equal 51,43 and 31 for £7. ghaurii at 30, 25 and 20°C, respectively. Table 21. Survival time (t25,t50 and t75 in weeks) of Cicadulina spp reared on maize at different temperatures . Temperatures Cicadulina species 30°C 25°C 20°C t25a t50b t75c t25 t50 t75 t25t 50t 75C C. storeyi 3 5 7 4 5 6 3 5 7 C. arachidis 3 4 5 3 5 5 2 5 7 C . ghaurii 1 3 5 2 3 4 1 3 5 a, b and c equal the time(wk) when 25,50 and 75% of 100 C. storeyi and C.arachidis and 20 C, ghaurii are dead,respectivily. 95 lived for 7, 11 and 12 wk, respectively. C. ghaurii, which thrives best at high temperatures in its natural habitat (Port Harcourt in South Eastern Nigeria) (Dabrowski, 1978a) did not live longer at 30°C The fecundity of Cicadulina species as measured by the average number of eggs laid per female per generation is given in Table 22. C. storeyi and C. ghaurii females laid more eggs during their life times at high than at low temperatures. However, C. storeyi laid more eggs than did C. ghaurii at all temperatures. C. arachidis with a different trend of egg-laying compared to the other two species, had the same average number of eggs (Ro) at 30°C and 25°C (64.8). C. storeyi and C. ghaurii had their maximum capacity of increase per week (rc) at 25°C, whereas <7. arachidis had its at 30°C. A low rate of increase for all species was obtained at 20°C. The mean adult age (Tc) of an egg laying female (Table 22) was higher at 20°C than at 25°C and 30°C for C. storeyi and C. arachidis. However, survival was low at all temperatures for C. ghaurii. Acquisition access period. Ten percent of C. arachidis acquired MSV in 1 hr following a 24 hr IAP (Table 23). In spite of the low transmission efficiency by this species over time (varying between 3.3 to 13.3%), there was a significant (p<0.001) linear association between this factor (y) and log time (x): y - 5.4 + 1.2x; R2 - 67.2%. (9) In contrast, C. ghaurii acquired virus within 15 min (25% transmission efficiency). Its transmission efficiency remained fairly constant after AAPs ranging from 15 min to 48 hr after which it increased to a maximum of 37.5% when given a 96 hr AAP (Table 23). The transmission efficiency Table 22. Fecundity parameters(Ro,tc and rc) of Cicadulina. spp at different temperatures. Temperatures 30°C 25°C 20°C CicadulinaS species Rob rcc tcd Ro rc tc Ro rc tc C. storey 93.7 1.39 3.26 59.9 1.47 2.79 57.0 1.14 3.55 C. arachidis 64.8 1.46 2.85 64.8 1.33 3.14 45.4 0.87 4.40 C. ghaurii 84.9 1.65 2.69 46.2 1.70 2.26 37.8 1.36 2.68 a 50 couples of C. storeyi and C . arachidis and 20 couples of C, ghaurii. b Mean number of eggs laid by a Cicadulina spp during its life time c capacity for increase per week d the mean adult age in wk of egg-laying female. VO o\ 98 (y) of this species was also found to be closely associated with log time (x) : y - 18.9 + 3.2x; R2 - 75.8%. (10) Inoculation access period. C. arachidis transmitted MSV after a minimum IAP of 1 hr following a 2 hr starvation and a 48 hr AAP (Table 24). The transmission efficiency of this species (y) increased over time (x) reaching a maximum of 30% when given a 96 hr IAP: y - 8.4 + 3.6x; R2 •- 85.1%. (11) Similarly, C. ghaurii transmitted the virus after a 1 hr IAP. Its transmission efficiency increased considerably over time. A significant association (p<0.001) was observed between transmission efficiency (y) by this species and log time (x): y - 16.9 + 71x; R2 - 85.0%. (12) Latent period.The results of MSV transmission by C. arachidis following 2 hr and 8 hr AAPs are presented in Table 25. Only one out of 15 Insects transmitted MSV to the first test plants after a 1 hr latent period. The latent period of MSV in this leafhopper was completed between 24 and 48 hr. For the three sets of tests a total of 10 leafhoppers transmitted the virus to the first test plants. The time when 50% of these leafhoppers (in this case 10 leafhoppers) transmitted MSV to the first test plant is known as median latent period (LP50) . Hence, the results of these tests showed that LPS0 of MSV for C. arachidis is between 14 and 24 hr. In the case of C. ghaurii only three out of 10 insects transmitted MSV to the first test plants after an 8 hr AAP due presumably to low numbers of leafhopper tested (Table 26). Two leafhoppers completed their latent period between 10 and 12 hr and Table 23. Transmissibility of maize streak virus (MSV) by Cicadulina arachidis and C. ghaurii given variable acquisition access periods (AAP). Percent transmission AAP C. arachidisa C. ghaurii 15 sec 0.00 0.00 30 0.00 0.00 1 min 0.00 0.00 15 0.00 25.00 30 0.00 25.00 1 hr 10.00 25.00 2 6.70 12.50 4 3.30 25.00 12 6.70 25.00 24 13.30 25.00 48 10.00 25.00 96 10.00 37.50 0 30 C. arachidis and 8 C. ghaurii were tested at each AAP, respectively. Table 24. Transmissibility of maize streak virus by Cicadulina arachidis and C. ghaurii given variable inoculation access periods (AAP). Percent transmission IAP C. arachidis8 C. ghaurii 1 min 0.00 0.00 15 0.00 0.00 30 0.00 0.00 1 hr 10.00 20.00 2 10.00 20.00 4 10.00 20.00 12 20.00 40.00 24 20.00 40.00 48 20.00 60.00 96 30.00 60.00 8 10 and 5 C. arachidis and C. ghaurii were tested at each IAP, respectively. VO yo Table 25. Maize streak virus (MSV) latent period for Cicadulina arachidis after a 2 hr and a 8 hr-AAP. Acquisition Numbers Serial transfers (hr) access of C. __ period arachidis 4 10 12 14 24 48 96 2 hr 1 2 3 4 +° + + + 5 6 + - + + + 7 8 9 10 11 12 13 14 15 8 hr 1 2 3 4 5 6 7 8 9 10 11 12 + 13 + 14 + 15 a No symptoms. b First test plant showing symptoms. Table 26. Maize streak virus (MSV) latent period for Cicadulina ghaurii after a 8 hr acquisition access period. Numbers of C. ghaurii Serial transferts (hr) 1 2 3 4 5 6 7 8 9 10 10 B 12 -- +b - + -- - 14 --- + + --- 24 -- - + + - -- 48 - -- + + - - - 96 --- + + --- 0 Test plant not showing symptoms. b First Test plant showing symptoms. 102 another between 12 and 14 hr. These results suggest that the latent period of MSV for £7. ghaurii is between 10 and 12 hr. Persistence of MSV in Cicadulina species. Two insects out of 25 C. arachidis transmitted MSV to FR114 x FR303 seedlings following a 24 hr AAP (Table 27). One insect died after 5 days, another one continuously transmitted MSV during its life span until it died after 25 days. Four insects out of 10 £7. ghaurii transmitted the virus and retained it until their death following a 24 hr-AAP (Table 27). The 5 day serial transfers used accounted for the frequent rather than intermittent transmissions registered for both species. MSV transmission efficiency by Cicadulina species. The results (Table 28) revealed that the transmission efficiency of these species increased over time. C. storeyi had similar MSV-transmission efficiency to £7. mbila. £7. ghaurii was as efficient in MSV transmission as £7. mbila and £7. storeyi. However, £7. arachidis transmitted MSV inefficiently compared to the other species. MSV transmission efficiency of male and female Cicadulina species. Percent transmission by four Cicadulina species (Table 29) indicated that both females and males had similar abilities to transmit MSV. DISCUSSION £7. storeyi appeared to develop faster than £7. arachidis and £7. ghaurii at all temperatures. The latter two species overlapped in their development times in some temperatures. The three temperature regimes were suitable for the development of the three leafhoppers species. The Table 27. Persistence of maize streak virus (MSV) in Cicadulina spp given a 24 hr acquisition access period. Numbers of leafhoppers Species Test of inoculativitv after8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 C. arachidis 1-5 -b - -- - +c -- - ...... + - - - 5-10 -- -- + - - - ...... o - - - - 10-15 0d - - 0 - + -- - 0--00...... 00 15-20 0 0 0 + - 0 - - - . . . 0 - 0 - 0 20-25 + - - - C. ghaurii 1-5 + - --- + + - + - 5-10 + - --- + + - + - 10-15 + - --- + + - 0 - 15-20 0 - - -- + + - 0 20-25 - -- - + + - 8 given in days b (-) Plants not showing symptoms c (+) Plants showing MSV symptoms d (0) leafhoppers dead Table 28. Transmission of maize streak virus (MSV) by four Cicadulina spp given variable aquisition access periods and a 24 inoculation access period. Acquisition access period Species 1 hr 12 hr 24 hr 48 hr C. mbila 20.00° 25.00 40.00 40.00 C. storeyi 10.00 20.00 30.00 45.00 C. arachidis 10.00 6.70 15.00 13.00 C. ghaurii 25.00 25.00 25.00 40.00 ° Percent plants showing MSV symptoms. n varied between 8 to 60 according to the availability of each Cicadulina species. Table 29. Comparison of maize streak virus (MSV) transmission by males and females of four Cicadulina. species. Species Observations(n) Transmission8 male female male female C. mbila 30 30 33.30 46.7 C. storeyi 30 30 46.70 43.30 C. arachidis 30 30 13.30 13.30 C. ghaurii 13 13 38.5 30.8 8 Percent of inoculated maize plants showing MSV symptoms. 106 optimum temperature appeared to be approximately 30°C. The mean development periods from egg to adult of the three species were similar to those reported for C. mbila on maize (Rose, 1973b; van Rensburg, 1982a; Mesfin, 1987), £7. bipunctella on barley (Ammar, 1977), £7. triangula (syn. £7. storeyi) on maize (Dabrowski, 1985), and £7. ghaurii on maize (Dabrowski, 1987a). All Cicadulina species survived and reproduced readily at all temperatures tested. C, storeyi survived better followed by C. arachidis. The former species occurs in all ecological zones in Nigeria although it has been suggested that it performs better in areas where the daily temperature is higher than 28°C (Dabrowski, 1987c). However, C. ghaurii which thrives better at high temperature and high relative humidity in its natural habitat (Port Harcourt) (Dabrowski, 1987a) did not live longer in these studies. The mean adult age (Tc) of egg laying females of this species was low for all temperatures. This could be due to the relative humidity in the growth chamber which has not been high enough (70-85%) for optimum survival and fecundity. Generally, £7. storeyi was more fecund than the other two species. C. ghaurii, which has high temperature requirements, deposited almost twice as many eggs at high temperature than at low ones. In contrast, £7. arachidis laid similar number of eggs at all temperatures. This species, which has been found predominantly in the savanna zone where mean temperatures during the rainy season are lower than in the forest zone, probably gave its maximum egg production between 25 and 30°C. Nault and Madden (1985) and Damsteegt (1984) reported that constant low temperatures increased leafhopper development time and longevity and 107 reduced leafhopper fecundity. Similar results were found in this study. Cicadulina species which develop more quickly, have a short life span, have a relatively high fecundity and colonize unstable agricultural habitats can be considered as r-strategists. This is in accordance with the prediction by Southwood (1977) that animals which occupy stable habitats will be K-strategists, whereas those in short lived, disruptive habitats will be r-strategists. This is also consistent with the finding by Nault (1985) that the leafhoppers D. maidis and D. eliminatus, both maize specialists, are r-strategists, whereas Dalbulus which are Tripsacum specialises are K-strategists. Cicadulina leafhoppers are known to have short and long distance fliers, although the proportions of these forms vary according to the species (Rose 1973b). Therefore, only the long distance fliers would be axpeted to migrate into green grasses or into grassy weeds and cultivated maize in the bottom lands with residual moisture or in irrigrated areas and then move back into young maize the following season. C. ghaurii and C. arachidis acquired MSV in 15 min and 1 hr, respectively after 24 hr IAP. However, Storey (1925) and Zagre (1983) indicated that C. mbila and C. triangula ( syn. C. storeyi) were capable of acquiring the virus from infected plants within 15 and 30 sec, respectively. Similarly, Okoth (1985) reported a minimum of either 30 sec or 15 min by various populations of C. mbila and C. triangula. Like C. triangula (Zagre, 1983), C. ghaurii and C. arachidis were able to transmit MSV to healthy plants after a minimum IAP of 1 hr following a 48 hr AAP. However, this IAP was longer than the 5 min 108 reported for C. mbila (Storey 1938) and shorter than the 2 hr for various populations of C. mbila and £7. triangula (Okoth et al., 1987). The transmission efficiency of £7. arachidis and £7. ghaurii increased with increasing duration of AAP and IAP as previous reports for other leafhopper species have indicated (Bennett, 1971; Goodman, 1981; Bock, 1982; Harrison, 1985; IITA, 1985). Bock et al (1974), Goodman (1981), Tonkyn and Whitcomb (1987) have reported that geminiviruses are always concentrated in the phloem although some of them are found in the xylera and the mesophyll. However, Markham, P.G. (personal communication) has recently reported that leafhopper-transmitted MSV is found only in the mesophyll cells of infected maize leaves. This explains why leafhoppers pick up MSV in a short time, since they may feed during the initial contact with infected plants in the mesophyll cells. Nevertheless, inoculation of MSV to healthy maize can only take place when the leafhoppers salivate in the phloem tissue hence much more time Is needed. The discrepancies between leafhopper species in terms of the minimum AAP and IAP could be due to several factors including leafhopper feeding behavior on a particular plant, the concentration of virus in the plant and the charge of virus within the leafhoppers. Both Cicadulina species retained MSV throughout their life. This was reported by Storey (1928) for £7. mbila, Zagre (1983) for £7. triangula, and Ling (1969) for other leafhopper-borne viruses. The pattern of MSV transmission by £7. arachidis and £7. ghaurii was continuous. Similar patterns were observed with MSV and maize mosaic virus when C. mbila and Peregrinus maidis, respectively, were given 2 109 day serial transfers (Goodman, 1981). The minimum latent period of MSV in C. arachidis and C. ghaurii was between 8 and 10 hr and 10 and 12 hr, respectively. Median latent period for C. arachidis was between 14-24 hr. Storey (1932) reported a minimum latent period of 6 hr for C. mbila while Zagre (1983) found a minimum latent period between 10 and 12 hr for C. triangula. Conti (1985) pointed out that the length of latent period, which is the time it takes the virus to circulate through the insect body and reach the salivary glands (Storey, 1933), is peculiar for each virus-vector combination. Like C. mbila (Storey, 1932) and in C. storeyi (Zagre, 1983), active and inactive transmitters occurred in both C. ghaurii and C. arachidis. However, there were fewer active C. arachidis transmitters in our colony compared to C. ghaurii, C. mbila, C. storeyi and C. ghaurii were more efficient in transmitting MSV than C. arachidis. The latter species was a poor vector. This agrees with the report by Okoth and Dabrowski (1987a) that a lower percentage of C. arachidis actively transmitted MSV compared to C. mbila, C. triangula (syn. C. storeyi) and C. ghaurii. Significant differences in transmission efficiencies of MSV for different MSV isolates were observed with C. mbila being the best vector followed by C. storeyi and C. bipunctella (Markham et al., 1984; Mesfin, 1987). However, the efficiencies in transmission of MSV among C. mbila, C. storeyi and C, ghaurii appeared similar in this study. Unlike the report from Dabrowski (1987c) that females Cicadulina spp are more efficient in transmitting MSV than males, the present data 110 showed that males are as efficient as females. However, Boulton and Markham (1986) reported higher concentration of the virus in males than in females given an AAP of less than 4 days. CHAPTER IV TRANSISSION OF MAIZE STREAK VIRUS ISOLATES BY CICADULINA STOREYI INTRODUCTION Maize streak geminivirus (MSV) is indigenous in African grasses and several host specific strains have been reported (Bock, 1974; Rose, 1978; Damsteegt, 1983). These include strains from Zea mays L. (form A and B) , Saccharum officinarum, Pennisetum typhoides and Digitaria longiflora. All are transmissible to maize. Strains of the virus from Panicum maximum (Storey and McClean, 1930; IITA, 1985) and other wild grasses, such as Rottboellia cochinchinensls and Brachiaria nutica (IITA, 1985), have been reported not to infect maize. Isolates from Eleusine indica and Brachiaria distichophylla while able to infect maize, cause only mild and transient symptoms (IITA, 1984). More recently, the report by Pinner et al. (1988) failed to confirm that grass specific virus isolates occurred since all isolates could be transmitted to maize. In studies conducted in Nigeria 18 grass isolates have been transmitted to maize (T. Mesfin and B. Bosque Perez, personal communication^. The maize strain exists as variants or substrains (Bock, 1982) and may have different transmission characteristics depending on Cicadulina species. Ill 112 Enzyme linked immunosorbent assay (ELISA) which is a simple, rapid, sensitive and quantitative assay has been used for virus detection in crop plants, including maize (Clark and Adams, 1977; Clark and Barbara, 1987; Pinner and Markham, 1990). Close and distant relationships between various MSV isolates from maize and grasses using ELISA have been determined (Ngwira, 1988; Pinner et al., 1988; Dekker et al., 1988; Pinner and Markham, 1990; Mesfin, T. et al., personal communication) . ELISA has also been used to detect the virus in the insect vectors of plant viruses (Mumford, 1982; Gordon et al., 1985; Falk and Tsai, 1985; Nault and Gordon, 1988). These following studies were conducted to determine the transmissibility of some MSV isolates from three maize varieties varying in virus susceptibility and from four grass species and to determine MSV titer in the maize varieties with varying virus susceptibility and to detect MSV in Cicadulina leafhoppers. MATERIALS AND METHODS Transmission. Experiments to test the ability of Cicadulina species to transmit MSV isolates from maize varieties (TZB-Gusao, 8329- 15 and 8321-21) and from grass species (Digitaria horizontalis, Brachiaria distichophylla, Eleusine indica and Axonopus compressus) were conducted in a transmission room maintained at 26 + 1°C and 12 hr light:12 hr dark photoperiod. AAP, IAP, virus source and Cicadulina species are stated In each experiment described below. After Inoculation, test plants were treated with granular Furadan and 113 transferred to insect proof-cages for evaluation of MSV symptoms at 3 wk following inoculation. Acquisition by C. storevi of MSV from maize varieties with different levels of MSV tolerance. Seven hundred sixty C. storeyi (ca. 22 leafhoppers/infected plant) were given acquisition access period (AAP) of 6, 12 and 24 hr on seedlings of maize varieties TZB-Gusao, 8329-15 and 8321-21 upon full expression of MSV symptoms. Twelve MSV- infected plants per variety were used. MSV-exposed leafhoppers originating from each combination of variety x time (9 combinations) were then caged individually on healthy maize seedlings of TZB-Gusao, 8329-15, 8321-21 or FR114 x FR303 (MSV susceptible hybrid) for a 24 hr inoculation access period (IAP). Twenty test plants were used for each treatment giving a total of 720 plants for the 36 treatments. Inoculation of MSV to maize varieties with different levels of MSV tolerance by C. storevi. Three hundred sixty C. storeyi (30 leafhoppers/infected plant) were given a 96 hr AAP on maize seedlings of TZB-Gusao, 8329-15 or 8321-21 showing MSV symptoms. Four MSV-infected plants were used per variety and set of 120 leafhoppers. MSV-exposed leafhoppers were caged singly on healthy maize seedlings of the above varieties plus FR114 x FR303 for 6, 12 or 24 hr IAP. Ten test plants were used for each treatment giving a total of 360 plants for the 36 treatments. Transmission characteristics of MSV grass isolates. Grass infected with MSV-like symptoms were collected from farmers' fields in Southern Nigeria and kept in the screenhouse. Isolates of MSV from these grass species were identified based on MSV symptoms on maize 114 (either severe or mild) and transmission (of MSV-like symptoms from grass species) to maize using Cicadulina species. The procedures of transmissibility of these isolates by C. storeyi and C. mbila were identical to those described in Chapter III. Acquisition access period. In these tests 480 C. storeyi and 180 C. mbila were starved for 2 hr and then given an AAP varying from 15 sec to 96 hr on MSV-infected D. horizontalis, E. indica, B. distichophylla or A. compressus. MSV-exposed leafhoppers were then caged singly on FR114 x FR303 seedlings for 24 hr. Five and 10 test plants per treatment were used with C. mbila and C. storeyi, respectively. Inoculation access period. In this test 480 C. storeyi were given a 48 hr AAP on one of each of the MSV-infected grass species listed above. After this AAP, MSV-exposed leafhoppers were caged singly on FR114 x FR303 seedlings for varying periods from 1 min to 96 hr. Similarily, 120 C. mbila were given a 48 hr AAP on MSV-infected D. horizontalis or E. indica and the IAP tested as described for C. storeyi. Ten plants per treatment for C. storeyi and five plants for C. mbila were used. Latent period of MSV grass isolates in C. storevi. After starvation for 2 hr, 15 leafhoppers (per host) were given an 8 hr AAP on MSV-infected D. horizontalis, E. indica, A. compressus or B. distichophylla. Subsequently, MSV-exposed leafhoppers were confined singly on healthy FR114 x FR303 maize seedlings for 2 hr and serially transferred every 2 hr for the first 16 hr, followed by serial transfers of 16, 24 and 48 h r . These tests were conducted in a growth chamber at 26 + 1°C and 12 hr light:12 hr dark photoperiod. 115 Retention of MSV grass Isolates by C. storevi. Fifteen newly emerged £7. storeyi were given a 48 hr-AAP on one of each of the above MSV-infected grass species. After the AAP, vlruliferous leafhoppers were confined Individually on healthy FR114 x FR303 maize seedlings for 5 days. Serial transfers were made at 5 day intervals until most of the leafhoppers had died. Transmission of MSV grass isolates by Cicadulina spp. £7. storeyi, £7. mbila, C. arachidis and £7. ghaurii were given a 72 hr-AAP on each of the four grass species (D . horizontalis, E. indica, B. distichophylla and A. compressus) infected with MSV. At the end of the AAP, five exposed leafhoppers from each species were caged separately on healthy FR114 x FR303 seedlings for a 72 hr-IAP. Five test plants were used in tests of £7. ghaurii, and 10 test plants for each of the other leafhopper species. Serology. ELISA was used to determine the titer of MSV for the three maize varieties (TZB-Gusao, 8329-15 and 8321-21), and to detect MSV in Cicadulina leafhoppers extracts, MSV titer in maize varieties. TZB-Gusao , 8329-15 and 8321-21 seedlings at the one leaf stage were Inoculated with MSV. Three wk later when symptoms were fully developed, 0.5 g of Infected and healthy young leaf tissues were taken from each variety and ground in 4.5 ml of 0.1M sodium citrate, pH 7.0, using a mortar and pestle. Three samples of infected leaf tissues per variety were provided. Seven 5-fold (dilutions 1:50; 1:250; 1:1,250; 1:6,250; 1:31,250; 1:156,250 and 1:781,250) of the infected-leaf crude extracts per sample were prepared with the 0.1M sodium citrate buffer. A double sandwich ELISA (Clark 116 and Adams, 1977) as described in the following, was performed. Polystyrene microtiter plates were coated with MSV antibody (1 ug/ml) raised against Nigerian isolate of MSV (provided by G. Thottappilly) diluted in 0.05M sodium bicarbonate (NaHCOa) buffer, pH — 9.6. The plates were incubated overnight at 4°C and washed carefully and thoroughly with distilled water containing 0.05% Tween 20. Unbound sites were blocked with 0.05M NaHC03 plus 1% bovine serum albumine (BSA) and the antigen dilution series (1:50; 1:250; 1:1,250; 1:6,250; 1:31,250; 1:156,250) were added to weels (one dilution per well). The plates were incubated overnight at 4°C and washed thoroughly. Enzyme specific (alkaline phosphatase) conjugated antibody diluted at 1:1040 in phosphate-buffered saline, 0,05% Tween (PBS-T) was added and incubated for about 4 hr at room temperature. Unreacted conjugate was removed by washing wells three times and p-nitrophenyl phosphate at 1 mg/ml in 1M diethanolamine buffer, pH 9.8, was added. Absorbances at A405 nm for well contents were measured using the Titertek Multiskan photometer. For each dilution series an one way analysis of variance of the absorbance values was carried out using Minitab program, a general purpose statistical computing system (Minitab, Inc., State College, PA, USA). The least significant difference (LSD) was calculated for each dilution series where significant differences in absorbances were observed among varieties. MSV titer in Cicadulina species. Two sets of leafhoppers were prepared for the serological assays. In the first, C. storeyi and C. arachidis were given 6, 12 or 24 hr AAP on MSV-infected maize (variety Pool 16) after which they were caged individually on healthy maize 117 seedlings of FR114 x FR303 for a 24 hr-IAP. Then MSV-exposed leafhoppers were removed, put in vials and kept in the refrigerator at 4°C until tested by ELISA. The second set of leafhoppers consisted of C. storeyi given an AAP of 6, 12, 24 or 48 hr on MSV-infected TZB-Gusao, 8329-15 or 8321-21. Viruliferous leafhoppers were killed and kept separately in groups according to the AAP and maize variety. Prior to ELISA, leafhoppers were individually homogenized In 0.3 ml of 0.025M Tris, pH 8.0, 0.15M NaCl, 0.05% Tween 20 (TBS-T) using a 7 -ml tissue homogenizer. F(ab')2 ELISA (Ammar et al., 1989; Barbara and Clark, 1982) was used to test homogenates for MSV and consisted of the following steps: Polystyrene microtiter plates coated with F(ab')2 units against a Malawian MSV isolate (provided by D.T. Gordon) at 1 ug/ml in 0.05M sodium bicarbonate (NaHC03), pH 9.6, were incubated for 2 hr at room temperature and later washed thoroughly with TBS-T. Wells were blocked with 150 ul of 1% BSA in 0.05M NaHC03, pH 9.6, for 1 hr and rinsed with TBS-T. Leafhopper homogenates were added to the plates at 50 ul/well and plates were then incubated overnight at 4°C. After washing plates three times with TBS-T, detecting antibodies to the IITA MSV isolate (provided by D.T. Gordon) were added at 1:400 in TBS-T plus 1% BSA and plates incubated for 2 hr. Following plate wash with TBS-T, the bound detecting antibodies were labeled with biotinylated protein A at 1:1500 in TBS-T for 2 hr. Bound biotinylated protein A was labeled with a horse radish peroxidase (HRP)-streptavidin conjugate in TBS-T at 1:1000 for 30 min. Plates were washed and the substrate mixture which consisted of 20 ml of distilled water, 20 ul 0.1% H202, 2 ml 0. 1M sodium acetate, pH 5.8, and 200 ul tetramethly benzodine (TMB) was added at 118 100 ul/well. The reaction was stopped by adding 30 ul/well of H2S04 when the absorbance at 405 ran (A405) of the most colored control well equalled 1.00. Absorbances were recorded with a Model E1309 Bio-Tek automated microplate reader (Bio-Tek Instruments Inc., Winoski, Vermont, USA) coupled to a Hewlett Packard HP3000 computer terminal. Data were analyzed by statistic program EIAS (D.T. Gordon, personal communication') . RESULTS Acquisition by C. storevi of MSV from maize varieties. Differences in virus transmission efficiency by C . storeyi were observed for MSV acquisition from maize varieties TZB-Gusao, 8329-15 and 8321-21 for the 6-hr AAP (Table 30). However, differences were less marked when a 24-hr AAP was used. Inoculation of maize varieties with MSV by C. storeyi. When C. storeyi acquired virus from either MSV-infected TZB-Gusao or 8329-15 and then transmitted it at various lAPs to TZB-Gusao, 8329-15, 8321-21 or FR114 x FR303, the transmission efficiency was between 20-60% (Table 31). However, the transmission efficiency varied from 0 to 40% for leafhoppers given the AAP on 8321-21 and a 6-hr IAP on the same variety. Acquisition access periods of MSV grass isolates. Transmission efficiencies of C. storeyi after various AAPs on MSV-infected grass species indicated that C. storeyi acquired MSV from infected D. horizontalis and A. compressus in as short a period as 2 hr (Table 32). In contrast, MSV was acquired by C, storeyi from E. indica and B. Table 30. Transmission by Cicadulina storeyi of maize streak virus (MSV) given acquisition access periods (AAP) of 6, 12 or 24 hr on three maize varieties infected with MSV and a 24 hr inoculation access period. Inoculated maize varieties with MSV symptoms <%) MSV-infected maize and AAP TZB-Gusau 8329-15 8321-21 FRll4xFR303(S) TZB-Gusau(S)a 6 hr 20b 15 10 15 12 30 20 30 45 24 30 30 25 40 8329-15(IR) 6 hr 5 20 10 10 12 12.5 25 10 25 24 30 45 20 30 8321-21(HR) 6 hr 15 5 5 10 12 10 20 10 25 24 30 35 25 20 0 S, IR and HR - susceptible, intermediate resistant and highly resistant, respectively. b N- 20 for each percent. Table 31. Transmission by Cicadulina storeyi of maize streak virus (MSV) given a 96 hr acquisition access periodon three maize varieties and a 6, 12 and 24 hr inoculation access period (IAP). Inoculated maize varieties given different IAPs and showing MSV symptoms (%) TZB-Gusau(S)8 8329-15(IR) 8321-21(HR) FRll4xFR303(S) MSV source varieties 6hr 12hr 24hr 6hr 12hr 24hr 6hr 12hr 24hr 6hr 12hr 24hr TZB-Gusau 50b 40 60 50 60 60 20 40 50 50 30 60 8329-15 30 50 60 30 50 50 20 30 60 20 60 60 8321-21 20 10 30 20 30 40 0 30 40 10 40 40 0 S, IR and HR - susceptible, intermediateresistant and highly resistant, respectively. b N-10 for each percent. Table 32. Transmission of four maize streak virus (MSV) isolates acquired from four grass species given various acquisition access periods (AAP) following a 24 hr inoculation access period (IAP) on maize test plants by Cicadulina spp. Cicadulina species/MSV infected grass species and percent of inoculated maize plants showing MSV symptoms C. storeyi C. mbila AAP D, horizona E. indicab B. disticc A. compresd D. horizon E. indica A. compres 15 sec 0.0e 0.0 0.0 0.0 0.0f 0.0 0.0 30 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1 min 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 30 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1 hr 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2 20.0 0.0 0.0 10.0 0.0 0.0 0.0 4 10.0 0.0 0.0 10.0 0.0 0.0 20.0 12 30.0 20.0 0.0 20.0 20.0 20.0 20.0 24 50.0 20.0 10.0 40.0 20.0 20.0 20.0 48 50.0 20.0 10.0 30.0 20.0 20.0 40.0 96 60.0 30.0 20.0 20.0 60.0 20.0 60.0 a Digitaria horizontalis b Eleusine indica c Brachiaria disticophylla d Axenopus compressus e n- 10 for percent infected by C. storeyi. f n- 5 for percent infected by C. mbila. 122 distichophylla after 12 and 24 hr AAP, respectively. C. mbila acquired MSV from A. compressus in a 4-hr AAP and from D. horizontalis and E. indica in a 12-hr AAP. Significant (p<0.003) associations between transmission efficiencies of MSV isolates from Z). horizontalis: y - 18.5 + 5.8X; R2 - 71%, (13) E. indica: y - 7.6 + 2.6X; R2 - 60%, (14) and A . compressus: y - 10.9 + 3.2X; R2 - 63%, (15) by C. storeyi and log acquisition time were observed. Parallely, significant (p<0.003) linear regressions between rate of transmission of MSV isolates from E. indica: y - 6.7 + 2.2X; R2 - 59%, (16) and A. compressus: y - 13.5 + 4.5X; R2 - 60.6%, (17) by C. mbila and log acquisition time were also noticed. Inoculation access periods of MSV grass isolates. C. storeyi transmitted MSV from infected D. horizontalis and A. compressus to healthy maize seedlings when given a minimum IAP of 30 mln and 1 hr, respectively (Table 33). MSV isolates acquired from E. indica and B. distichophylla were transmitted by C. storeyi following a 48-hr AAP in a minimum IAP of 12 and 24 hr, respectively. Also following an 48-hr AAP, C. mbila transmitted both MSV isolates from D. horizontalis and E. indica when given a minimum IAP of 12 hr. Significant (p<0.006) associations between rate of transmission of MSV isolates from D. horizontalis: Table 33. Transmission by Cicadulina spp of maize streak virus (MSV) isolates acquired from four grass species given a 48 hr acquisition access period (AAP) followed by various inoculation access periods (IAP) on maize test plants. Cicadulina species/MSV infected grass species and percent of inoculated maize plants showing MSV symptoms C. storeyi C. mbila IAP D. horizon- E. indica- B. distic- A . compres- D. horizon E. indica 1 min 0.0e 0.0 0.0 0.0 0.0f 0.0 15 0.0 0.0 0.0 0.0 0.0 0.0 30 20.0 0.0 0.0 0.0 0.0 0.0 1 hr 10.0 0.0 0.0 10.0 0.0 0.0 2 30.0 0.0 0.0 10.0 0.0 0.0 4 20.0 0.0 0.0 10.0 0.0 0.0 12 40.0 10.0 0.0 20.0 20.0 20.0 24 40.0 30.0 10.0 30.0 40.0 20.0 48 60.0 40.0 20.0 20.0 60.0 20.0 96 60.0 50.0 40.0 60.0 40.0 40.0 Brac hiaria disti cophyll aa Digitaria horizontalis D Eleusine indica Brachiaria disticophyllaa d Axenopus compressus e n- 10 for each percent resulting from inoculation by C. storeyi. f n- 5 for each percent resulting from inoculation by C. mbila. 124 y - 20.4 + 7.6X; R2 - 85%, (18) E. indica: y - 7.2 + 5.8X; R2 - 64%, (19) and A. compressus: y - 10.4 + 5.6X; R2 - 66%, (20) by C. storeyi and log inoculation time were found. Also significant relationships were observed between transmission efficiencies of MSV isolates from D. horizontalis: y - 9.3 + 6.7X; R2 - 63%, (21) and E. indica: y - 5.7 + 4.3X; R2 - 65%, (22) by C. mbila and log inoculation time. Latent period of MSV grass isolates in C. storevi. Following 8 hr AAP, eight, five and two out of 15 C. storeyi transmitted MSV acquired from D. horizontalis, A. compressus and E. indica, respectively, to the first maize test plants (Table 34). The latent period for which 50% of the transmitting leafhoppers (i.e. eight, five and two) transmitted the virus to the first test plants was between 16-32 hr for both MSV isolates acquired from D. horizontalis and A. compressus, while the minimum latent period for MSV isolate acquired from E. indica was between 56-104 hr. However, none of the plants inoculated with MSV acquired from B. distichophylla showed MSV symptoms following an 8-hr AAP. Because of the absence of the transmission, the latent period for this isolate was not determined. Persistence of MSV grass isolates within leafhoppers. Following a 48 hr AAP, two, four, nine and 12 out of 15 leafhoppers retained MSV Table 34. Latent period of maize streak virus (MSV) isolates acquired from three grass species by Cicadulina storeyi. Grass species C. storeyi number from which MSV was Serial transfer acquired period(hr)0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Digitaria 10 b horizontalis 12 14 + 16 ------+ -- 32 - + - + ----- 56 - +c + + + + + - + -- 104 + + + + - - - + + + - + - - Eleusine indica 10 12 14 16 - 32 56 104 + Axonopus 10 compressus 12 14 16 + + - 32 - + ------56 + + + ------104 "h + - - + + ~ ~ - ~ + ■ a C. storeyi was given a 8 hr acquisition access period (AAP) followed by inoculation access period of the intervals indicated. b No symptoms. c Inoculated test plant showing symptoms. 126 acquired from infected B. distichophylla, E. indica, A. compressus and D. horizontalis until death (Table 35). For all isolates, the transmission pattern of MSV was persistent. Transmission of MSV-grass isolates by Cicadulina spp. C. storeyi and £7. mbila transmitted MSV from all four grass species (Table 36). However, C. ghaurii transmitted MSV from only three grass species (D. horizontalis, E. indica and A. compressus) and C. arachidis from two species (D. horizontalis and E. indica). MSV acquired from B. distichophylla was poorly transmitted by C. storeyi and C. mbila and not at all by either C. ghaurii or C. arachidis. MSV titer in maize varieties. In the two experiments, significant (p<0.002) differences in absorbances at A405 were observed among varieties for the four dilutions (1:10, 1:50, 1:250 and 1:1,250) tested (Table 37). TZB-Gusao had significantly greater absorbance values than 8329-15 or 8321-21. The relative concentration values (dilutions) at A405 equal to 0.46 and 0.90 were 1:500 and 1:278 for TZB-Gusao, 1:244 and 1:46 for 8329-15 and 1:100 and 1:9 for 8321-21, respectively for the first and second experiment. MSV titer In Cicadulina leafhoppers. In the first set of leafhoppers, MSV was detected in just one C. arachidis by ELISA (data not shown). This same leafhopper also tested positive for MSV infectivity by bioassay. None of the C, storeyi homogenates tested positive by ELISA, not even those from leafhoppers which transmitted MSV in the infectivity test. In the second set of leafhoppers, MSV was detected by ELISA in one C. storeyi (data not shown) given a 24 hr AAP on 8329-15. None of the leafhoppers given the various AAPs on Table 35. Persistence of maize streak virus (MSV) isolates acquired from four grass species by Cicadulina storeyi. Grass species C. storeyi number from which MSV Serial transfer was acquired period (days)a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Digitaria 1-5 +b + + + + - + + + + + -- + + horizontalis 5-10 + + + + + - + + + + + - - + + 10-15 + + + + + - + + + 0 + - - + + 15-20 0C + 0 + + - + + + + - 0 + + 20-25 + + + - 0 0 + + - 0 0 25-30 + + + - + + - _d Eleusine indica 1-5 --- + - + ------+ + 5-10 - -- - + - + 0 + + 10-15 --- 0 + - + ----- + + 15-20 -- 0 0 - + -- 0 - 0 + + 20-25 0 -- + -- 0 0 0 25-30 -- + -- Brachiaria 1-5 ------+ ------distichophylla 5-10 - - 0 - 0 -- 0 + + 10-15 -- 0 - - + + -- 0 -- 15-20 - - - 0 + 0 - - 0 0 20-25 - 0 - + 0 - 25-30 - - + - Axonopus 1-5 + + - + - + - - - + + - + + + compressus 5-10 0 0 - + - + -- - + + - + + + 10-15 - + - + 0 -- + + - + + + 15-20 - + - + - - + + 0 + + + 20-25 - + - + 0 0 + + + + + 25-30 - + - + + + + + + a persistence of inoculativity in days. b (+) Inoculated test plant showing MSV symptoms. c (0) Dead leafhopper. d (-) Inoculated test plant showing no symptoms. Table 36. Transmission by Cicadulina species (group of five leafhoppers per species) of maize streak virus (MSV) given a 72 hr acquisition access period (AAP) on four grass species followed by a 72 hr inoculation access period (IAP). MSV grass isolates Cicadulina Digitaria Eleusine Brachiaria Axonopus species horizontalis indica distichophylla compressus C. storeyi8 +b + + + C. mbila + + + + _c C. arachidis + + - C. ghaurii + + - + 8 leafhopper species transmitting MSV from grass species to maize. b Inoculated maize showing MSV symptoms. c Inoculated maize showing no MSV symptoms. Table 37. Absorbances at 405nm (A405) of dilutions of extracts from maize streak virus (MSV) infected maize varieties as determined by enzyme linked immunosorbent assay using MSV antiserum. A405 for dilutions0 Experiment Maize No variety 1:10 1:50 1:250 1:1250 1 TZB-Gusau 0.936 0.883 0.549 0.309 8329-15 0.734 0.735 0.457 0.243 8321-21 0.685 0.677 0.266 0.168 LSD<0.05> 0.093 0.104 0.091 0.063 2 TZB-Gusau 1.776 1.264 0.966 0.513 8329-15 1.232 0.877 0.450 0.289 8321-21 0.969 0.534 0.252 0.193 l s d (005) 0.122 0.157 0.162 0.106 a N-12 per dilution and n-3 per treatment. 130 MSV-infected 8321-21 or TZB-Gusao tested positive for MSV by ELISA (data not shown). DISCUSSION Short acquisitions feedings on MSV-infected resistant variety 8321-21 by £7. storeyi resulted in lower transmission efficiency compared to acquisition from the other two varieties. This was also true when £7. storeyi acquired the virus from the resistant variety 8321-21 and transmitted it into the same variety after a short IAP. This could probably be due either to low concentration of the virus in the mesophyll tissue of the resistant variety or to the feeding behavior of £7. storeyi on the resistant variety. Mesfin T. and N. Bosque Perez (personal communication1) have observed using electronic feeding monitor a non-preference for feeding of C. storeyi and C. mbila on the resistant variety 8321-21. Mesfin (1987) has also reported that £7. mbila probed less, fed longer from the phloem tissue of its preferred hosts and switched to mesophyll from phloem tissues on less preferred host. It is, therefore, possible that £7. storeyi did not reach the phloem tissue of 8321-21, a less preferred host, in most of the time during Inoculation feedings resulting in less Infection. These events may result in an overall lower transmission of MSV by Cicadulina species in the field planted with MSV resistant maize varieties as shown by our results on MSV incidence and symptom severity on 8321-21 described in Chapter I. 131 C. storeyi acquired MSV more quickly from infected D. horizontalis and A. compressus than did C. mbila. However, both species of Cicadulina required longer acquisition access periods to transmit MSV from infected E. indica and B. distichophylla than from the other two grass species. C. storeyi was capable of transmitting MSV from infected D. horizontalis and A. compressus to healthy maize after a shorter IAP compared with C. mbila. However, C. storeyi compared with C. mbila transmitted MSV from infected E. indica and B. distichophylla after a longer IAP. These minima AAP and IAP were longer than those reported by Storey (1926) for C. mbila and Zagre (1983) for C. triangula (syn, C. storeyi). Conti (1985) reported that minima AAP and IAP of leafhopper borne-viruses including MSV depended upon vector-virus combination. Not suprisingly, the rates of transmission of these MSV grass isolates by C. storeyi and C. mbila increased with increasing AAP and IAP and were in agreement with the previous reports (Storey, 1926; Zagre, 1983; Okoth et al., 1983). These AAP and IAP results generally showed that D. horizontalis was the best virus source and most susceptible to infection followed by A . compressus, E. indica and B. distichophylla. C. storeyi appeared to be more efficient than C. mbila in transmitting some of MSV grass isolates (e.g. D. horizontalis and E. indica) to maize. This was not in agreement with the finding by Markham (1984) and Mesfin (1987) that C. mbila was more efficient than C. storeyi in transmitting various MSV isolates from maize and grasses to maize. Each MSV grass isolate produced typical symptoms in infected maize FR303 x FR114 as reported by Mesfin (1987). MSV D. horizontalis or E. indica isolates produced mild symptoms in maize compared with the severe symptoms produced by MSV B. 132 distichophylla and A. compressus Isolates. Symptoms produced by the latter two grass isolates were similar to those produced by Nigerian MSV isolates from maize. The minimum latent periods of MSV isolates acquired by C. storeyi from D. horizontalis, A. compressus and E. indica were between 12-14 hr, 14-16 hr and 56-104 hr, respectively. The short latent period of MSV isolates from the former two grasses was similar to those reported by Storey (1928) for C. mbila and Zagre (1983) for C. storeyi involving MSV isolates from maize plant hosts. C. storeyi persistently transmitted MSV isolate from the four grasses until its death. C. storeyi, C. mbila, C, arachidis and C. ghaurii were all capable of transmitting MSV isolates from D. horizontalis and E . indica, whereas only C. storeyi, C. mbilaand C. ghaurii were able to transmit the MSV isolate from A. compressus. C. arachidis Is a poorer vector than the preceding three species. However, C, storeyi and C. mbila were poor vector of the MSV B. distichophylla isolate. Thus, the efficiency of transmission of different MSV grass isolates by Cicadulina leafhoppers varies according to species. Mesfin (1987) previously reported that C. mbila efficiently transmitted MSV Panicum maximum isolate to maize cultivar (Golden Bantam), whereas C. storeyi (syn. C. triangula) failed to do so. ELISA data clearly indicated that the concentration of MSV was highest in the susceptible variety TZB-Gusao (1:500 and 1:278 dilutions), intermediate in the moderately tolerant variety 8329-15 (1:244 and 1:46 dilutions) and lowest in the highly resistant variety 8321-21 (1:100 and 1:9 dilutions). These results are in agreement with 133 those of Baker and Harrison (1985) who reported that potato leafroll virus (PLRV) concentration was lower in tolerant than susceptible genotypes. They also observed an increase in PLRV concentration with increasing severity of leaf symptoms. Fargette et al. (1986) reported that very little African cassava mosaic virus was detected by ELISA in dark green areas compared with yellow or yellow-green areas. Likewise, Storey (1938) reported that MSV could be found mainly in the chlorotic portions of maize leaves. Therefore, the low concentration of MSV in the resistant variety may be due to the fact that most of the leaf area of this variety remains dark green with only few chlorotic broken lines which presumably contained little or no MSV. F(ab')a ELISA allowed the detection of MSV in single C. storeyi and C, arachidis leafhoppers. The ELISA technique has been used before to detect Beet Curly Top geminivirus (BCTV) in their leafhopper vectors, Circulifer tenellus (Mumford, 1982). Both geminiviruses, MSV and BCTV are circulative non propagative within their leafhopper vectors. Not suprisingly, circulative propagative and transoavarial viruses as well as mollicutes have been detected in their leafhopper and planthopper vectors (Archer et al. 1982, Gordon et al. 1985; Nault and Gordon, 1988). Boulton and Markham (1986) reported the detection of MSV in C. mbila, C, storeyi (syn. C. triangula) and C. china by using DNA dot-blot hybridization method. They also reported that the concentration of MSV acquired by C. mbila increased with increasing AAP. Although MSV was detected in two leafhoppers by ELISA, we believe that most lost their virus because approximately 3 months elapsed between the time they acquired the virus and the time ELISAs were 134 performed. In addition, these leafhoppers were retained for inspection by the US Department of Agriculture Animal and Plant Health Inspection Service (APHIS) and we do not know the conditions under which they were kept during retention. It is expected that assaying newly collected leafhoppers by F(ab')a ELISA will reveal the relationship between MSV concentration and different AAP lengths for Cicadulina spp. This investigation showed that MSV Isolates from maize and grasses have varying transmission efficiencies and consequently might have different importances in MSV epidemiology. CHAPTER V MAIZE STREAK VIRUS DISEASE PROGRESSION CURVE AND PATTERN OF SPREAD UNDER FIELD CONDITIONS IN A TROPICAL RAIN FOREST LOCATION INTRODUCTION Virus diseases are seldom uniformily distributed in the field and tend to aggregate in areas where conditions are favorable for their spread (Thresh, 1978). Vectors and virus sources are among those factors that influence the pattern of maize stresk virus spread. Indeed, clusters of MSV infected plants have been reported in maize fields located in the immediate vicinity of the source. In contrast, diseased plants were randomly distributed when immigrant leafhoppers from distance sources inoculated maize field (Rose, 1978, Autrey and Ricaud, 1983). Gorter (1953) used the doublet method to determine the spread of MSV in maize fields in South Africa. However, Madden et al. (1982) found that doublets and corrected doublets gave highly arbitrary results. Therefore, they proposed instead ordinary runs and point pattern analysis to detect clustered patterns of infected plants. Disease progress curves of many plant diseases have been analyzed and described using different growth models, Vanderplank (1963) 135 136 proposed two models, notably, the monomolecular and logistic models to describe plant diseases that increase in simple interest or compound interest fashion, respectively. In addition, plant diseases have been accurately described with models (including Gompertz and Richard) adopted from other scientific disciplines such as ecology and statistics (Madden, 1980). No attempt has been made in the past to quantify spartially and temporally maize streak virus in West Africa, therefore, this investigation was conducted to analyze and describe MSV disease progress in time and pattern of spread. MATERIALS AND METHODS Field trial. The experiment was conducted at Owo (near Akure) located in the rain forest zone. The field was planted with MSV- susceptible and downy mildew (Peronosclerospora sorghi) resistant variety of maize (Suwan 2) at the spacing of 0.75 m between rows and 0.25 m between plants. Two seeds/hill were planted and later thinned to one. The field was fertilized with 120 kg of N, 120 kg of P20E and 120 kg of KjO at planting and side dressed with 78 kg of N at 45 days after planting. After planting the herbicide Primextra (25 kg a.i./ha) was applied. The maize field was surrounded by 10m strip of grasses composed mostly of Brachiaria spp, Digitaria horizontal is, Eleusine indica and Panicum maximum. During planting time 2, the number of leafhoppers/m2 was determined and the incidence of MSV symptoms was determined on E. indica and Brachiaria spp. in the grass strip. Prior to the experiment, no maize plants with MSV symptoms were observed at 137 the location. Approximately 1,000 virus-free C. storeyi were released in the grass strip and MSV-infected maize seedlings (variety 8329-15) in pots were placed in the grass area to increase the chances of MSV spread. Analysis of MSV Incidence data to determine the pattern of MSV spread. A field of 0.25 hectare was divided into 85 9 x 3 m quadrats. Within each quadrat, four adjacent rows of 25 plants each were visually assessed for MSV symptoms at 3, 5, 7, 9 and 11 wk after planting. The spatial pattern of MSV spread was assessed using ordinary run analysis (Madden et al., 1981). To explain the method of analysis, in a quadrat MSV-infected plants were designated as 1, whereas healthy plants were designated as 0. Thus, in an ordered sequence of 100 diseased and healthy maize plants from four rows the total number of runs (u) was obtained on the basis that a run is a succession of one or more identical symbols (1 or 0), which are followed and preceded by a different symbol or no symbol at all. The expected run (E(u)) is calculated as: E(u) - 1 + 2m(N - m)/N (23) where ra is the number of infected plants and N is the total number of plants in an ordered sequence (100). The standard deviation of the number of runs is given by: su - (2m(N - m)[2m(N - m) - N]/[N (N - 1)]) (24) The standarized run is then calculated as follows: Zu - [u + 0.5 - E(u)]/su (25) When z<-l,64, the spread of infected plants within the quadrat was non- random (clustered). Conversely, when z>-1.64, the distribution of 138 infected plants was random. The pattern of MSV spread was also assessed by point pattern analysis (Madden et al., 1987) which consisted of determining the variance (s2) and the mean (X) of diseased plants in all quadrats. The variance to mean ratio (VTM), Lloyd's index of mean crowding: (X + s2/X - 1) (26) and Lloyd's index of patchiness: (X + s2/X - 1)/X (27) were calculated on the basis of these statistics (variance and mean). Analysis of field data to determine MSV disease progress curve (temporal pattern). Maize plants infected with MSV in the entire field were marked and their number counted every week. MSV disease assessment began a week after planting and continued up to flowering. MSV incidence (y) and absolute rate of MSV disease increase (dy/dt) were plotted against time (t). A model describing MSV incidence was developed by plotting transformed y(y*) versus t. The model yielding a straight line was conducted to provide the best fit of MSV incidence data. Regression analyses of linearized forms (Campbell and Madden, 1990) of logistic model: ln[y/(k - y)] - ln[yo/(k - yo] + rLt (28) Gompertz model: -ln[-ln(y/k)] - -ln(yo/k)] + rGt) (29) and Richard model, if m In[ 1/[ 1 - (y/k)1 -"]] - ln[l/[l - (yo/ky ■•]] + rRt (30) and if m>l: 139 ln[l/[(y/k)1 - 1]] - ln[l/[(yo/k)1 '“ - 1]] + rBt (31) where m — shape parameter, y - MSV disease incidence and k — maximum MSV disease infection. Coefficient of determination Ra, back transformation of R2(R*2) , mean square of error (MSE), t-test ratio, standardized residual plot and autocorrelation of residual were determined. RESULTS Pattern of MSV spread. The results of the ordinary runs (u) test for 85 quadrats of MSV infected plants, assessed five times, indicated randomness at the beginning of disease spread when there were low numbers of MSV-infected plants (X •* 3.5) (Table 38). As MSV-disease incidence increased with time, the percent of quadrats presenting clustering rose gradually from 11.52 to 38.82%. Aggregation of MSV- diseased plants was detected as early as 3 wk after planting and reached the maximum when maize was at the pollen-shed stage. At that time, the number of MSV-infected plants within quadrats varied from 12 to 61 (X - 33.0), while the maximum incidence for the entire field was 31.0%. For the last two observations (4th and 5th) , most of the clusters occurred in the quadrats where 26 to 50 plants were infected with MSV. In the point pattern analysis, VTM showed a highly aggregated pattern (all X2 calculated > X2 0.01(84) - 117) (Table 39). In all these assessment times, patchiness was greater than one (p<0.05) even though it declined to a value near the expected value for a random point pattern at the Table 38. Ordinary run analysis on a sequence of 100 MSV-infected and healthy maize plants for 85 quadrats. Percent quadrats Observation (weeks after planting) Random Non random 3 88.46 11.52 5 80.52 19.48 7 64.70 35.30 9 68.24 31.76 11 61.17 38.82 140 Table 39. Point pattern analysis on maize streak virus infected maize plants in all quadrats. Aggregation indices Observation (weeks after planting) Variance to mean Index of mean Lloyd's index ratio crowding of patchiness 3 2.50 5.04 1.42 5 4.37 11.25 1.43 7 3.65 25.99 1.11 9 3.44 32.88 1.10 11 3.48 35.56 1.08 141 142 last evaluation time. Patchiness and variance to mean ratio increased to a maximum at the second assessment time and then declined for the remainder of the observations. MSV disease progress curve. The plot of MSV incidence (proportion) against time (days) gave an s-shaped disease progress curve which showed a leveling off of MSV incidence at high values of time (Fig. 21). The calculated rate of MSV-disease increase (Fig. 22) revealed that the maximum estimated rate fell more or less in the middle part of the epidemic. The disease progress curve also showed a maximum infection well below 100% (1.0), hence an asymptote (k) was estimated by evaluating a set of k values varying from 0.311 to 0.380, assuming the Gompertz model. By analyzing the linear regression statistics of the different equations, the k value from the equation which yielded the highest R2 and R*2, the smallest MSE, the greatest t-test ratio for the slope and the best residual plot proved to be 0.320. The linearized forms of Logistic, Gompertz and Richard models were evaluated (with k - 0.320) for goodness of fit of the MSV disease progress data using Minitab. The Logistic's and Richard's (m - 0.5 and m - 3) plots displayed (Figs 23 and 24) gave curved lines, whereas that of Gompertz (Fig. 25) yielded a fairly straight line indicating that Gompertz model provided the best fit for the MSV disease data. Moreover, the linear regression statistics of these models are summarized in Table 40. These results clearly showed that Gompertz mode had the highest R*2' the lowest MSE, the greatest t-test ratio on the slope, the best residual plot (Fig. 26) and a non significant autocorrelation of residual (<0.70). Consequently, it was judged the most appropriate model for MSV Incidence (proportion) i.1 Mie tek iu (S) ies pors curve progress disease (MSV) virus streak Maize Fig.21. 0 0.3- 0.4 . 2 - o h ri frs oe (Owo). zone forest rain the for ie dy atr planting) after (days Time 20 060 40 80 143 Rate of MSV disease Increase (dy/dt) 0.00 0.01 0.02 i.2 Rt o mie tek iu (S) disease (MSV) virus streak maize of Rate Fig.22. - 0 nrae d/t frte oe zn (Owo). zone fores the for (dy/dt) increase 20 ie dy atr planting) after (days Time 060 40 80 144 tn (MSV Incidence / (k-MSV Incidence)) i.3 Te oitc i o mie tek iu disease virus streak maize of fit Logistic The Fig.23. -4- 12 k (asymptote) =0.32 (asymptote) k rges aa ih , prmtr o maximum for parameter a k, with data progress amount of disease. of amount 24 ie dy atr planting) after (days Time 36 48 60 72 145 1-m 1-m In (1/((MSV Incldence/asymtote) -1)) In (1/(1*{MSV Incidence/asymptote) ) -5- i.4 Te ihr ft f az sra vrs disease virus streak maize of fit Richard The Fig.24. 12 rges aa ih (hp prmtr =0.5 parameter) (shape m with data progress asymptote(k)=0.32 upr lt ad = (oe plot). (lower m=3 and plot) (upper 24 20 asymptote (k)=0.32 asymptote ie dy atr planting) after (days Time ie dy atr planting) after (days Time 660 36 40 48 60 72 80 146 i.5 Te oprz i o mie tek iu dsa progress diseas virus streak maize of fit Gompertz The Fig.25. •In (-In (MSV Incidence/asymptote)) 2 0 2 1 3 4 1 12 aa ih , prmtr o mxmm mut f disease. of amount maximum for parameter a k, with data smtt (k)=0.32 asymptote 24 ie dy atr planting) after (days Time 36 48 60 72 147 Table 40. Linear regression statistics for different models describing the maize streak virus disease progress curves. Model Statistic ______Logistic Gompertz Richard0 m-3 m-0.5 Rz(%) 96.1 99.4 91.5 90.2 R*2(%) 97.0 99.8 56.0 3.5 MSE 0.426 0.022 0.208 2.58 t test ratio 14.02 36.69 10.40 8.60 Residual plot usc s us us Autocorrelation of residual 0.57 0.10 0.59 0.55 a Richard model has a flexible shape parameter(m less or greater than one) c us-unsatisfactory when standardized residuals form a pattern and s- satisfactory when standardized residuals are scattered. i.6 Rsda po (admy itiue) o Gmet' fit Gompertz's for distributed) (randomly plot Residual Fig.26. Standardized residual f az sra vrs ies pors data. progress disease virus streak maize of ie dy atr planting) after (days Time 149 150 describing MSV disease spread. DISCUSSION MSV disease progression over time was best described by the Gompertz model, an appropriate model for compound interest diseases such as MSV in which there is spread from plant to plant within the field. The Gompertz model has also provided better fit to some maize dwarf mosaic disease epidemics (Madden et al., 1986) and to African cassava mosaic virus epidemic (Fargette et al., personal communication) than other models. Similarly, the Gompertz model provided a better fit for progress curves of several other plant diseases (Berger, 1980). The Gompertz model has an absolute rate of disease increase (dy/dt) greater than the logistic model during the early phase of an epidemic. In addition, its dy/dt reaches a maximum earlier (skewed to the right) than the dy/dt of the logistic model (Madden et al., 1986). In contrast, Vanderplank (1963) indicated that logistic model was the most appropriate in describing disease epidemics of compound interest. Conversely, Autrey and Ricaud (1983) reported that the MSV disease progress curve in Mauritius was either linear or exponential depending on whether invading leafhoppers are from a nearby or distant source. The plotting of the rate of MSV disease increase versus time resulted in a bell-shaped curve. The maximum estimated rate (or inflection point) occured more or less in the middle part of the curve suggesting the possibility of the logistic model. However, the decision in selecting the appropriate model for MSV incidence data could not be 151 based on these data alone because a large rate, due to a limited number of observations, could have occurred earlier or later than the time encompassed by the curve of absolute rate. This proved to be true because the curve of the absolute rate of MSV disease increase was asymmetrical. Although virus-free C. storeyi and MSV source were introduced to the grass strips around the maize field, the initial (yo) MSV disease was zero and the maximum disease intensity was still low (ca. 31%) with apparent rate of infection (r) equals 0.086. This is in contrast to the finding of Autrey and Ricaud (1983) who reported a fairly high initial MSV infection resulting in linear build up when viruliferous leafhoppers invaded maize field from perennial grasses or previous crop located in its immediate vicinity. It could be possible that physical factors and biotic factors (predators and preferred grass hosts) prevailing in the grass strips affected flight and feeding behavior as well as survival of Cicadulina species, especially C, storeyi. The asymmetrical sigmoid shaped curve showed that early infection rate was very low and started to increase significantly, probably when nymphs from eggs deposited on grass hosts emerged and adults moving away from grasses picked up the virus from the source within maize and spread it. Ordinary runs analysis indicated random patterns in most of the quadrats during the early phase of MSV infection. However, as disease intensity increased over time, randomness declined indicating a tendency towards clustering of infected maize plants later in the epidemic. The point patterns analysis, however, revealed clustered pattern of infected maize plants even at low disease intensity (mean diseased plants - 3.4) 152 indicating that MSV was predominantly spreading from plant to plant within the field. MSV, which is a polycyclic disease, was initiated in maize field (initial infection) from outside MSV source located in the grass strip. Leafhoppers which are transient on maize plants fed on MSV-infected plants randomly distributed within the field and spread MSV to healthy plants causing further disease increase. Therefore, farmers should avoid planting maize at the proximity of MSV and leafhopper sources located either in the grasses or previous crops. In the case there is no any other alternative, they must leave a strip of bare ground of approximately 10 m from the source as reported by Gorter (1953) and the fields should be kept clean from grasses. Temporal and spatial quantification of MSV disease progression allowed a better understanding of the interactions between the vector, virus, host plant and environmental factors. EPILOGUE The findings presented in this dissertation increased our understanding of maize streak virus (MSV) epidemiology in West Africa. In Chapter I, data on monthly plantings of three maize varieties with varying MSV susceptibility showed high MSV incidences on TZB-Gusao in second season plantings at Ikenne and late plantings at Mokwa and Funtua. The resistant variety 8321-21 had significantly lower MSV incidence and symptom severity than TZB-Gusao and 8329-15. The screenhouse results also showed significantly lower MSV severity rating on 8321-21. The results in this chapter also demonstrated that leafhopper population densities remained low during the rainy season and peaked at early dry season in the rainforest zone, whereas in the savanna zone, their numbers were low at the beginning of the rainy season and rose gradually reaching their maximum before the rains ended. Significant qualitative and quantitative differences in species composition within Cicadulina populations were observed with C. mbila, the commonest species. The MSV transmission by wild leafhoppers rose gradually as the rainy season progressed. Significant associations were found between MSV and leafhopper components as well as with physical factors shown for some of the test locations and for screenhouse trials. Future MSV epidemiological studies should focus on these relationships between MSV, infective Cicadulina populations and weather factors, and 153 154 on oversuramering of Cicadulina species and MSV especially in the Guinea Savanna zone. Findings of some investigators have supported the idea that leafhoppers carried aloft by strong harmattan winds, which blow southward, migrate to the South at the end of the growing season and then move back to the North at the beginning of the next rainy season. Evidences for this hypothesis could be sought by conducting aerial samplings of leafhoppers using long-distant traps (ca. 15 ft) at different distances from the North to the South once every 2 wk during the period from flowering time to the beginning of next season. The results in Chapter II indicated that C. storeyi counts decreased linearly with distance from the release point and exponentially with days following their release. Their dispersal rate increased with distance. This technique, which was found to be efficient with such small insects (ca. 3 mm), is strongly recommended for future dispersal studies of Cicadulina species for better understanding their movement activity, a contributing factor to MSV epidemiology. The time of the day that Cicadulina fly is an important feature for sampling or releasing leafhoppers and may be determined by using Johnson Taylor suction trap. By using this technique, C. Rodriguez, R. Taylor and L.R. Nault foersonnal communication) have discovered that Gramnella nigrifrons and Dalbulus species are crepuscular fliers. Furthermore, the results presented in these studies indicate that C. storeyi equally preferred TZB-Gusao, 8329-15 and 8321- 21 for settling, ovlposition and nymphal development. However, they preferred younger (growth stage 1.0 to 3.0) over older maize and MSV- infected over healthy maize plants. It is assumed that physiological 155 and chemical changes taking place in MSV- infected plants result in accumulation of phagostimulants such as sugars and free amino acids. Therefore, efforts should be made to determine the chemical composition of MSV-infected maize plants compared to healthy ones. The results on the biology of C. arachidis and C. ghaurii given in Chapter III showed that these two species had a similar developmental periods as compared to C. storeyi and other Cicadulina species, but that they appeared to differ from each other and from C. storeyi in their fecundity patterns. Data on MSV transmissibility by C. arachidis and C, ghaurii indicate that £7. arachidis is an inefficient vector, while £7. ghaurii is as efficient as C. storeyi and C. mbila. In the light of these findings, I recommend that preliminary studies be undertaken to determine optimum artificial rearing conditions (host plant, temperature, and relative humidity) for C. similis and to some extent C. ghaurii before the biology and the transmissibility of MSV by these species is more thoroughly studied. Leafhopper transmission of MSV isolates from maize varieties with different levels of resistance and from grass species are described in Chapter IV. Leafhopper transmission efficiency of MSV isolate from resistant variety was lower compared with that from moderately resistant and susceptible varieties. Data of MSV titer in these varieties showed that the concentration of MSV in the resistant variety was lower than in the other two varieties. This probably would explain the lower efficiency of this variety as source of MSV. The results on leafhopper transmission efficiencies of MSV isolates from the four grass species showed that D. horizontalis was the best source of MSV, followed by A. 156 compressus, E. indica and B. distichophylla. The latter was found to be a poor source of MSV. Therefore, effort should be made to study feeding, oviposition and nymphal development host suitability for Cicadulina species. X also suggest that the concentration of MSV at different leaf position (young to old leaves) and at different growth stages be determined for improved, MSV-resistant and susceptible maize varieties. 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Ingenieur Agronome These, Universite Nationale du Benin.