The potential of fungi to control green leafhopper
Nephotettix virescens on rice
by
Jaime Augusto Jimenez Gomez
Thesis submitted to Imperial College, University of London in partial fulfilment of the requirement for the degree of
Doctor of Philosophy
1989
1 To
Gloria
for being Gloria
#
2 ABSTRACT
The green leafhopper (GLH), N ephotettix virescens is an important pest vector of several virus diseases in rice throughout Asia.
Cultures have been established on rice var. TNI and maintained throughout this study.
The entomogenous Deuteromycetes, Verticillium lecanii ,
M etarhizium anisopliae , M etarhizium flavoviride , Paecilom yces
farinosus, Tricho thee ium, Sporothrix insectorum and B e a u v e r i a b a s s i a n a , have been examined for pathogenicity to GLH under conditions of suboptimal humidity. B. bassiana isolates 110-82, 235-
85, 261-85, 266-85 and 268-85 were the most virulent strains with
LC50 values after 9 days at 25±1*C of 2-12X10e conidia per ml. M. a n i s o p l i a e 275-86 was highly virulent to GLH only at constant 100% relative humidity, (LC50 of 0.7X10e conidia per ml after 6 days).
Intraspecific differences were recorded in the growth response of
B. bassiana and M. anisopliae isolates to temperature, conidial survival, sporulation on insect cadavers and spread of mycosis. B. b a s s i a n a 268-86 had the optimum combination of characters and was selected for further detailed study.
Single spore isolates (SSI) of B. bassiana 268-86 had increased, similar or reduced virulence compared with the multi spore parent
(MSI). When 2 SSI's and the MSI were passaged through insects twice, virulence increased markedly. Subsequent passaging failed to maintain increased virulence and after 5 infection cycles virulence had reverted to the original level. Subculturing on SDA resulted in reduced virulence.
£?. b a s s i a n a 268-86 sporulated well on semi-solid media with yields after 21 days of 1.6 and 1.7X1010 on rice and wheat respectively. In submerged culture, B. bassiana 268-86 was able to produce hyphal bodies in complex media and submerged conidia in a defined medium.
These spores were 5 and 10 times as virulent as those produced on wheat respectively.
A rapid method for determining conidial viabilities of B. bassiana and M. anisopliae was developed using the optical brightener bi- striazinyl amino stilbene.
The prospects for using fungi for control of GLH are discussed.
_ 4 + ACKNOWLEDGEMENTS
I particulary wish to thank my supervisor, Dr. Adrian T. Gillespie,
for his continuous help, advice and support throughout the period of
research and during the preparation of this thesis.
I am indebted to the Colombian People who through the Instituto
Colombiano Agropecuario (ICA) and Federacion de Cafeteros de Colombia
(FEDECAFE) provided the financial support to make possible this study.
I would like to thank: Dr. Michael Way (Imperial College at Silwood
Park) for his guidance. Dr. Cristopher Payne for his interest in the
project. Dr. Richard Hall for his friendship and support. German
Valenzuela, Emilio Echeverry and Dr Gabriel Cadena (FEDECAFE) for
their interest and support to the project. John FenIon, Cathy Townley
and Mike Ledieu for excellent help with statistics and computing. Sue
Bowsey for neip with typing. Andy Smith for help with photography and
art work. Linda Holt and Rosemary Palmer for help with insect
rearing. Dave Chandler for his helpful criticism of the manuscript.
I am grateful to a number of people for technical and academic
advice, including: Drs. Denis Burges, Keith Sunderland, Martin Meadows
and N. Hywel-Jones as well as Paul Jarrett, Mike Challen and Neil
He Iyer.
I should like to thank my colleagues, Hussan, Ian, Beverly, Bergman,
Norman, Doreen, Luciano, Michelle, Jackie, Debbie, Paul, Edwin, Anna,
Dave, Mark and many others for their continued friendship during my
stay at IHR, making the work most enjoyable.
Thanks especially to my parents, Pedro Nel and Emma Jimenez, my
sons, Carlos, Jaime and Pablo, and my wife Gloria for making it all
worthwhile. *
5 CONTENTS
PAGE
3 Abstract
5 Acknowledgements
6 Contents
15 List of tables
19 List of f igures
25 List of Plates
27 Introduction
SECTION I. LITERATURE REVIEW
29 1. Introduction to the rice crop and its main pests
31 2. The green leafhopper (N ephotettix spp): important
rice
36 3. The use of insecticides and the rice pest problem
38 4. Historical highlights of insect control by fungal
pathogens m 40 5. Taxonomic classification of entomogenous fungi 41 6. Class Deuteromycetes - Biology and life cycle
46 descriptions of important species
48 7. Fungal pathogens of rice pests
48 8. Development of mycoinsecticides
48 A. Selection of pathogens
50 B. Virulence and bioassay systems
54 C. Storage
55 D. Mass production
60 E. Formulation *
6 SECTION II. FUNGAL BIOLOGY
Materials and methods
62 1. General
62 A. Media
62 i. Sabouraud dextrose agar
62 ii. Yeast extract plus glucose liquid medium
63 iii. Sabouraud liquid medium
63 iv. TK1 broth defined medium
64 v. Grains used for semisolid culture
64 B. Media and equipment sterilization
64 C. Surfactant antifoam and buffer
64 D. Temperature humidity and pH measurement
65 E. Total spore counts
65 F. Assessment of spore viability
66 G. Production and harvesting of aerial conidia
66 H. Production and harvesting of hyphal bodies and
submerged conidia.
67 2. Source of strains used
67 3. Effect of temperature on germination and mycelial growth
67 A. The effect of temperature on spore germination
67 i. The SDA slide technique
69 ii. The lactophenol technique
69 B. Effect of temperature on mycelial growth
70 4. Effect of water activity on germination and mycelial
growth
70 A. Influence of water activity on germination
70 B. Influence of water activity on mycelial growth +
7 72 5. Shelf life experiments
72 A. Influence of temperature on survival of dried conidia
73 B. Influence of drying method and temperature on
germination and pathogenicity of stored conidia
74 C. Influence of temperature and antibiotic addition on
germination and pathogenicity of stored spore
suspensions
75 D. Influence of vacuum drying on viability of various B.
b a s s S a n a spore-types
75 6. Effect of SDA subculturing on production of conidia
76 7. Influence of ultra violet light on germination of B.
b a s s i a n a conidia, submerged conidia and hyphal bodies
77 8. Measurement of conidial germination by the optical
brightener technique
80 9. Viability of stored conidia as measured by the SDA slide
and the optical brightener techniques
81 10. Differentiation of B. bassiana 268-86 spore types
Results
82 11. Effect of temperature on germination and mycelial growth
82 A. Temperature influence on germination of fungi
84 B. Effect of temperature on mycelial growth
88 12. Effect of water activity on germination and mycelial
growth
88 A. Influence of water activity on fungal germination
92 B. Effect of water activity on mycelial growth
99 13. Storage experiments
99 A. Influence of temperature on survival of dried aerial
conidia *
8 99 B. Influence of drying method and temperature on
germination and pathogenicity of stored aerial
con id ia
111 C. Influence of vacuum drying on germination of spore
types
111 D. Influence of temperature and antibiotic addition
on germination and pathogenicity of liquid stored
spores
116 14. Effect of SDA subculturing on conidial production
116 15. Influence of ultra violet light on the survival of B.
b a s s i a n a 268-86 aerial conidia, submerged conidia and
hyphal bodies
119 16. Measurement of conidial viability by the optical
brightener technique
119 17. Viability of stored conidia as measured by the
fluorescent and the SDA slide methods
125 18. Differentiation of B. bassiana 268-86 spore types
131 Discussion
SECTION III. INSECT - FUNGUS INTERACTIONS
Materials and methods
141 1. General
141 A. Production of rice plants
144 B. Insect rearing and maintenance for bioassay
148 C. Bioassay of fungi to determine pathogenicity to N ephotettix virescens
148 i. Preparation and standardization of spore *
9 suspensions
148 ii. Assay procedures
149 a. Direct immersion in spore suspensions
149 b. Contagion from cadavers
149 D. Statistical analysis
149 i . Probit analysis
150 ii. LT50 or GT50 calculated by MLP
151 iii. Estimation of LT50 and GT50 by maximum
likelihood program
152 iv. Mean time of death
152 V. Analysis of variance in screening experiments
153 2. Initial selection of fungal strains
153 3. Quantification of pathogenicity
153 A. LC50
153 i. In suboptimal rh conditions
154 ii. In optimal rh conditions
154 B. Relationship between spore concentration and number
of spores adhering to insects (Determination of LD50)
155 4. Effect of temperature on infection 4 156 5. Influence of relative humidity on infection
156 A. Infection at optimal and suboptimal relative humidity
156 B. Influence of high humidity period on pathogenicity
157 6. Improvement of pathogenicity
157 A. Effect of adding nutrients to spore suspensions
157 B. Effect of pregerminating conidia
158 C. Experiments with single spore isolates
158 i. Selection of single spore isolates
159 ii. Standardization of suspensions by weight 4
- 1 0 160 lii. Pathogenicity of single spore isolates to adult N. virescens
160 D. Correlation between pathogenicity and rate of conidial
germination
161 7. The use of the optical brightener bi-striazinyl amino
stilbene to study spore germination i n v i v o
161 A. The infection process as revealed using the optical
brightener
162 B. Correlation between germination rate on agar and
insect wings of B. bassiana spores
163 8. Assessment of disease spread
163 A. Spore production on insect cadavers
164 B. Sporulation on insect cadavers and attachment to
plants
lb4 C. Correlation between number of dead sporulating
insects and disease spread to nymphs
165 9. Effect of subculturing and passaging on pathogenicity of
strains
166 10. Comparison of the pathogenicity of Hyphal bodies, aerial
and submerged conidia
Results
167 11. Production of plants and insects
167 12. Selection of fungal isolates pathogenic to N ephotettix v i r e s c e n s
169 13. Quantification of pathogenicity
169 A. LC50
169 i. Under sub-optimal relative humidity
173 ii. Under optimal relative humidity «
- 1 1 176 B. Relationship between spore concentration and number
of spores adhering to insects. Determination of LD50
181 14. Effect of temperature on mortality
181 15. Influence of relative humidity on infection
181 A. Infection at optimal and subcpt.imal relative humidity
185 B. Influence of high humidity period on pathogenicity of
fungi to N ephotettix virescens
185 16. Improvement of pathogenicity
185 A. Effect of adding nutrients to spore suspensions
188 B. Effect of conidial pregermination
188 C. Selection of single spore isolates (SSI)
188 i. Standardization of single spore isolates by
weight
188 ii. Pathogenicity of Beauveria bassiana 268-86
single spore isolates to adult N ephotettix v i r e s c e n s
191 D. Correlation between pathogenicity and rate of conidial
germination on SDA * 196 17. The use of the optical brightener bi-striazinyl amino- stilbene to study spore germination I n v i v o
196 A. Conidial germination i n v i v o under optimal and
suboptimal relative humidity
200 B. Correlation between germination i n v i t r o and i n v i v o
18. Assessment of disease spread
200 A. Spore production on insect cadavers
208 B. Selection of strains by number of dead insects
producing conidia and infection of nymphs
213 C. Correlation between number of sporulating and stuck
- 12 » GLH cadavers and disease spread to nymphs
220 19. Effect of subculturing and passaging on pathogenicity
of Beauveria basslana and M etarhlzium
a n i s o p l i a e to adult N ephotettix virescens
222 20. Pathogenicity of Beauveria bassiana 268-86 hyphal bodies,
aerial and submerged conidia to adult N ephotettix v i r e s c e n s
225 Discussion
SECTION IV. PRODUCTION OF FUNGI ♦
Materials and methods
238 1. General
238 2. Preparation of inoculum
238 3. Production on semi-solid medium
0*50 A. General
241 B. Effect of increasing inocula on spore yields
241 C. Production of B. bassiana on different cereal grains
241 D. Effect of shaking on production of Beauveria bassiana
con idia
242 E. Effect of oil on yields of Beauveria bassiana
242 F. Effect of adding perlite on yields of B e a u v e r i a b a s s i a n a
243 4. Production of Beauveria bassiana spore types in
submerged culture
243 A. Production using Erlenmeyer flasks
B. Medium scale production using 1.6 litre fermenters
246 5. Production of conidia on semisolid medium
1 3 - » 246 A. Effect of increasing inoculum on conidial yields
246 B. Production of B. bassiana on cereal grains
249 C. Effect of shaking on B . b a s s i a n a yields
249 D. Effect of adding oil on B. bassiana yields
249 E. Effect of adding perlite on yields of B. bassiana
254 6. Production of spores in liquid culture
254 A. Erlenmeyer flasks
256 B. Using 1.6 litre fermenters
260 Discussion
^ 265 SECTION V. GENERAL DISCUSSION AND RECOMMENDATIONS
272 BIBLIOGRAPHY
*
1 4 - LIST OF TABLES
PAGE No
33 1 Duration of N ephotettix virescens life cycle on rice
68 2 Origin of fungal isolates
71 3 Water activity data for aqueous glycerol solutions
83 4 Estimated time needed for 50 or 95% germination of
fungal strains on Sabouraud dextrose agar
85 5 Estimated time needed for 50 or 95% germination of
strains with high pathogenicity to N ephotettix v i r e s c e n s
110 6 Pathogenicity of fungal strains (aerial conidia)
stored in dry conditions to adult N ephotettix v i r e s c e n s
112 7 Effect of vacuum drying on survival of B e a u v e r i a
b a s s i a n a 268-86 spores as indicated by germination
on Sabouraud dextrose agar
115 8 Pathogenicity of Beauveria bassiana 268-86 spores
stored in liquid conditions to adult N ephotettix v i r e s c e n s
122 9 Conidial viabilities of Beauveria bassiana and
M etarhizium anlsopliae using Sabouraud dextrose agar
and fluorescent techniques
123 10 Viabilities of fungal conidia using fluorescent and
Sabouraud dextrose agar methods after lyophilization
and storage at 5*C
124 11 Viabilities of fungal conidia using fluorescent and
- 1 5 - # Sabouraud dextrose agar methods after vacuum
drying and storage at 5*C
126 12 Viabilities of fungal conidia using fluorescent and
Sabouraud dextrose agar methods after lyophilization
and storage at 20*C
127 13 Viabilities of fungal conidia using fluorescent and
Sabouraud dextrose agar methods after vacuum drying
and storage at 20*C
142 14 Compost used for rice cultivation
143 15 Composition of rice liquid feed
# 146 16 Relative humidities provided by closed, ventilated
and open covers
168 17 Pathogenicity of 29 strains of fungi to adult N ephotettix virescens
17\y l u Approximate times needed to achieve 50% mortality
of adult N ephotettix virescens after treatment with
fungi
171 19 Pathogenicity of fungi to adult N ephotettix
v i r e s c e n s in suboptimal relative humidity conditions
174 20 Pathogenicity of fungal strains to adult N ephotettix
■ v i r e s c e n s in subop timal relative humidity conditions;
combined data
175 21 Pathogenicity of fungal strains to adult N ephotettix
v i r e s c e n s in optimal rh conditions
177 22 Relationship between spore concentrations and number
of conidia adhering to adult N ephotettix virescens
after immersion in spore suspensions
179 23 Virulence of fungi to adult N ephotettix virescens at #
- 1 6 # optimal and suboptimal relative humidity. LD50
182 24 Effect of temperature on pathogenicity of fungi to
N ephotettix virescens adults as measured by
estimated LT50
183 25 Analysis of variance of several factors which
influence the mortality of N ephotettix virescens
adults after conidial applications with four fungal
strains and incubation at optimal and suboptimal rh
conditions
187 26 Effect of adding nutrients to spore suspensions on
# the virulence of fungi to adult N ephotettix v i r e s c e n s
189 27 Effect of pregerminating conidia on the virulence of
fungi to adult N ephotettix virescens
192 28 The effect of single and multispore isolates of
Beauveria bassiana 268-86 on N ephotettix virescens
194 29 Virulence of Beauveria bassiana multi and selected
single spore isolates to adult N ephotettix virescens
195 30 Virulence of Beauveria bassiana and M etarhizium * a n i s o p l i a e single and multi spore isolates to adult N ephotettix virescens
197 31 Comparison of GT50 and 95 values with estimated
LT50s obtained from multi and single spore isolates
of Beauveria bassiana 268-85
199 32 Germination and mycelial growth of B e a u v e r i a
b a s s i a n a 268-86 and M etarhizium anisopliae 275-86
on adult N ephotettix virescens at optimal and
suboptimal rh. #
17 " 206 33 Relationship between germination of B e a u v e r i a
b a s s i a n a 268-86 spores on Sabouraud dextrose agar
and insect wings
209 34 Disease transmission from infected adult N ephotettix
v i r e s c e n s to nymphs in M etarhizium anisopliae and Beauveria bassiana
224 35 Pathogenicity of Beauveria bassiana (268-86) spores
produced in semisolid or submerged culture to adult N ephotettix virescens
248 36 Production of Beauveria bassiana on different cereal
grains
259 37 Biomass of Beauveria bassiana (268-86) spores produced
in liquid culture
- 18 - * LIST OF FIGURES
PAGE No
86 1 Effect of temperature on mycelial growth of
V erticillium lecanii 11-73 and 19-79, and
M etarhizium anisopliae 83-82 and 275-86 on
Sabouraud dextrose agar
87 2 Effect of temperature on mycelial growth of
M etarhizium anisopliae 300-86, M . flavoviride 203-
84, Paecilom yces farinosus 104-82 and Trichothecium # 213-85 on Sabouraud dextrose agar
89 3 Effect of temperature on mycelial growth of
Beauveria bassiana 110-82, 138-83, 235-85 and 261-85
on Sabouraud dextrose agar
90 4 Effect of temperature on mycelial growth of
Beauveria bassiana 266-85, 268-86, 269-86 and 270-
86
91 5 Effect of temperature on mycelial growth of
Sporothrix insectorum 299-86
93 6 The effect of water activity on conidial germination
of M etarhizium anisopliae 275-86
94 7 The effect of water activity on conidial germination
of Beauveria bassiana 268-86
95 8 The effect of water activity on mycelial growth of
M etarhizium anisopliae 275-86
97 9 The effect of water activity on mycelial growth of
Beauveria bassiana 268-86
1 0 0 10 Survival of vacuum dried conidia of V erticilliu m
- 1 9 • ■ l e c a n i i 11-73 and 19-79, Paecilom yces farinosus
104-82, Trichothectum 213-85, M etarhizium
a n i s o p l i a e 83-82 and 260-85, M etarhizium
flavoviride 259-85 and Beauveria bassiana 261-85
stored at 30*C
101 11 Survival of vacuum dried conidia of V erticilliu m
l e c a n i i 11-73 and 19-79, Paecilom yces farinosus
104-82, Trichothecium 213-85, M etarhizium
a n i s o p l i a e 83-82 and 260-85, M etarhizium
flavoviride 259-85 and Beauveria bassiana 261-85 + stored at 20 X
102 12 Survival of vacuum dried conidia of V erticilliu m
l e c a n i i 11-73 and 19-79, Paecilom yces farinosus
104-82, Trichothecium 213-85, M etarhizium
a n i s o p l i a e 83-82 and 260-85, M etarhizium
flavoviride 259-85 and Beauveria bassiana 261-85
stored at 5 X
103 13 Survival of lyophilized conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and « M etarhizium anisopliae 275-86 stored at 30*C
105 14 Survival of vacuum dried conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and
M etarhizium anisopliae 275-86 stored at 30X
106 15 Survival of lyophilized conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and
M etarhizium anisopliae 275-86 stored at 20X
107 16 Survival of vacuum dried conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and «
- 2 0 » M etarhizium anisopliae 275-86 stored at 20’C
108 17 Survival of lyophilized conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and
M etarhizium anisopliae 275-86 stored at 5*C
109 18 Survival of vacuum dried conidia of B e a u v e r i a
b a s s i a n a 110-82, 235-85, 266-85, 268-86 and
M etarhizium anisopliae 275-86 stored at 5*C
113 19 Survival of Beauveria bassiana 268-86 spores
stored in water at 5*C
114 20 Survival of Beauveria bassiana 268-86 spores
stored in water at 20*C
117 21 Effect of one and ten SDA subcultures on conidial
production of multi and single spore isolates of
Beauveria bassiana 268-86 and M etarhizium aniso
p l i a e 275-86
118 22 The effect of ultraviolet light on survival of
Beauveria bassiana 268-86 spores: submerged
conidia, aerial conidia and hyphal bodies # 120 23 Dispersion diagram of expected and observed viabilities of Beauveria bassiana 268-86 using agar
and fluorescent techniques
121 24 Dispersion diagram of expected and observed
viabilities of M etarhizium anisopliae
275-86 using agar and fluorescent techniques
180 25 Strain comparison of haemocytometer and Petri plate
counts to estimate number of conidia from B e a u v e r i a
b a s s i a n a and M etarhizium anisopliae adhering to
N ephotettix virescens adults *
2.1 - # 184 26 Mortality of adult N ephotettix virescens under
optimal and suboptimal rh, after conidial application
of Beauveria bassiana 110-82, 268-86 and
269-86, and M etarhizium anisopliae 275-86
186 27 The effect of varying periods of high humidity on
the virulence of Beauveria bassiana 268-86 and
M etarhizium anisopliae 275-86 to adult N ephotettix v i r e s c e n s
190 28 Number of conidia per mg found in nine single
spore isolates (SSI) and multispore isolate (MSI) of • Beauveria bassiana 268-86
198 29 Relationship between germination rate and time of
death in ten isolates of Beauveria bassiana 268-86
205 30 Relationship between germination rates i n v i t r o and
i n v i v o for Beauveria bassiana 268-86
submerged conidia, aerial conidia and hyphal bodies
207 31 Spore production on adult N ephotettix virescens
infected with Beauveria bassiana 110-82, 138-83,
235-35, 261-35, 266-35, 263-36, 269-86 and 270-86 • or M etarhizium anisopliae 82-82, 83-82 and 275-86
212 32 Adhesion of adult N ephotettix virescens to rice
plants after treatment with Beauveria bassiana
110-82, 268-86 and 269-86 or M etarhizium
a n i s o p l i a e 275-86 and maintenance at optimal or
suboptimal relative humidity.
214 33 Sporulation of Beauveria bassiana 110-82, 268-86
and 269-86 or M etarhizium anisopliae 275-86 on
adult N ephotettix virescens after maintenance at •
- 2 2 optimal or suboptimal relative humidity
215 34 Spread of mycosis on nymphs after treatment of
adult N ephotettix virescens with Beauveria bassiana
110-82, 268-86 and 269-86 or M etarhizium
a n i s o p l i a e 275-85 and maintenance at optimal or
suboptimal relative humidity.
216 35 Relationship between number of adult N ephotettix
v i r e s c e n s cadavers with sporulating mycelium of
Beauveria bassiana and attached to the rice plant
with spread of mycosis to nymphs under optimal
relative humidity
217 36 Relationship between number of adult N ephotettix
v i r e s c e n s cadavers with sporulating mycelium of
Beauveria bassiana and attached to the rice
plant, with spread of mycosis to nymphs under
suboptimal rh
218 37 Relationship between number of adult N ephotettix
v i r e s c e n s cadavers with sporulating mycelium of
Beauveria bassiana and spread of mycosis to nymphs
under optimal rh
219 38 Relationship between number of adult N ephotettix
v i r e s c e n s cadavers with sporulating mycelium of
Beauveria bassiana and spread of mycosis to nymphs
under suboptimal rh
221 39 Effect of subculturing on Sabouraud dextrose agar
and passaging through insects on the pathogenicity
of two single spore isolates (SSI) and multispore
isolate (MSI) of Beauveria bassiana 268-86 to adult
- 23 * isolate (MSI) of Beauveria bassiana 268-86 to adult
N ephotettix virescens, compared with MSI maintained
in liquid nitrogen
223 40 Effect of subculturing on Sabouraud dextrose agar
and passaging through insects on virulence of SSI's
1, 3 and multispore isolate of M etarhizium
a n i s o p l i a e 275-86, compared with isolates maintained in
liquid nitrogen
247 41 Effect of 0.1, 1.0 and 5.0 ml inocula on semisolid
media yields of Beauveria bassiana 268-86 and
M etarhizium anisopliae 275-86
250 42 Yields of Beauveria bassiana 268-86 from static
and shaken cultures of wheat with different
amounts of oil
251 43 Effect of adding sunflower oil on yields of
Beauveria bassiana 268-86 on wheat or rice, after
14 or 21 days
252 44 Effect of adding sunflower oil on yields of B e a u v e r i a
b a s s i a n a 268-86 on wheat or rice, after 28
or 35 days
253 45 Effect of adding perlite on yields of B e a u v e r i a
b a s s i a n a 268-86 obtained on semisolid media at 14,
21 and 28 days fermentation
255 46 Production of Beauveria bassiana 268-86 spores in
TK1, YG and SL using 250 ml Erlenmeyer flasks
257 47 Production of Beauveria bassiana 268-86 spores in TK1,
YG and SL media using 1.6 litre fermenters.
*
- 24 LIST OF PLATES
PAGE No
34 1 Green leafhopper (.N ephotettix virescens) adults, left
male, right female (X19)
78 2 Aerial conidia of Beauveria bassiana 268-86 stained with
bi-striazinyl amino stilbene and observed under UV light
(X250)
79 3 Aerial conidia of M etarhizium anisopliae 275-86 stained
with bi-striazinyl amino stilbene and observed under # combined UV and normal light CX250)
96 4 Mycelial growth of M etarhizium anisopliae 275-86 on SDA
after 30 days at 23±1*C and water activities between
0.91 and 1.00
98 5 Mycelial growth of Beauveria bassiana 268-86 on SDA
after 30 days at 23±1’C and water activities between
0.91 and 1.00
128 6 Beauveria bassiana 268-86 aerial conidia (X250)
129 7 Beauveria bassiana 268-86 spores produced in submerged
culture showing hyphal bodies and "conidia like" spores
(X250)
130 8 Beauveria bassiana 268-86 spores produced in submerged
culture showing hyphal bodies and "conidia like" spores
(X250)
147 9 Types of plastic propagators used to provide three
levels of rh
201 10 Germination of aerial conidia on GLH wings after 12
hours at 25±1*C and optimal rh (X150)
- 25 - • 202 11 Germination tubes growing on GLH wings after 14 hours
at 25+1‘C and optimal rh (X250)
203 12 External colonization of GLH wings by mycelia after 40
hours at 25±1’C and optimal rh (X250)
204 13 Mycelial colonization within GLH wings after 60 hours at
25114C and optimal rh (X150)
210 14 GLH cadavers attached to a rice plant with sporulating
mycelia of Beauveria bassiana 268-86. The arrows
indicate GLH nymphs that have also became infected (X6)
211 15 GLH cadaver attached to a rice plant with sporulating
• mycelia of M etarbizium anisopliae 275-86 (X7)
240 16 Production of Beauveria bassiana 268-86 and M etarbizium
a n i s o p l i a e 275-86 on semisolid media using 250 ml
Erlenmeyer flasks
24-0 17 Submerged fermentation of Beauveria bassiana 2b;'--8b
using a 1.6 litre fermenter.
m
- 26 # INTRODUCTION
Widespread concern over the safety of chemical pesticides to the
farm worker, the consumer and the environment; phy to toxicity problems
and the continuing increase of pest resistance have prompted
considerable research over the last two decades into "Integrated pest
management" insect control is based primarily on close monitoring of insect populations and control measures should only be used where essential. Control methods used should permit natural control agents to have the maximum effect in regulating pest population. IPM programmes do not exclude the use of chemical insecticides but emphasize the establishment of economic threshold levels for insect species as parameters f^r making decisions and ^he use of biological control methods as the basis for the program. A complete revision of the biological control strategies was made by DeBach (1964) who considered that biological control should be the basis for all pest management systems. Ishii (1984), suggested that further studies are needed on varietal resistance, biological control methods and the use of chemicals before effective IPM strategies can be adopted on rice. A similar view was presented by Metcalf (1984). The need for alternative and ecologically bound pest control in rice has been highlighted recently in Indonesia, where the use of many chemical insecticides has been banned and an urgent IPM strategy to control the brown planthopper (BPH) has been adopted (Southern, 1987). Insect pathogens have been used for pest control and fit _ 27 perfectly into the IPM philosophy, with fungi offering particular advantages in the rice environment. Fungi infect insects by penetration through the cuticle, rather than by oral ingestion, so they can infect sucking insects, such as hoppers, while bacteria and viruses need to be ingested. Natural epizootics of entomogenous fungi are often observed in plant and leafhopper populations on rice, suggesting the rice environment is favourable for the growth of entomogenous fungi, (Rombach, 1987). Entomogenous fungi are generally safe for important groups of natural enemies such as predators and parasites as well as to mammals and they can often be isolated and grown in artificial media. The green leafhopper (GLH), N ephotettix virescens, is an important vector of various virus diseases in rice and in many countries has shown resistance to conventional insecticides (Metcalf, 1984). Many species of fungi have been reported infecting this pest in the field (Rombach, 1987) and it has been suggested that entomogenous fungi could form the basis of an IPM program for control of this pest (Aguda, 1984). The present study examines the potential of entomogenous fungi for control of GLH. As fungal strains vary greatly with respect to virulence, response to temperature, humidity, spore survival and other parameters, special effort has been made to select suitable strains for control of this pest. 28 - SECTION I LITERATURE REVIEW 1_. Introduction to the rice crop and its main pests Rice belongs to the genus O r y z a and comprises 25 species distributed throughout tropical and subtropical Asia, Africa, Central and South America and Australia. There are two cultivated species, Oryza glaberrim a Steud. and Oryza sativa Linn. 0. glaberrim a is grown in West Africa as an upland crop (5% of the world area), but it is gradually being replaced by 0 . s a t i v a (Grist, 1986). Rice is the world's most important food crop, and more than half the earth's population depend on it as their main carbohydrate source. It is grown in a wide range of climates, from the tropics to 53 degrees north (North eastern areas of China), and 35 degrees south of the Equator (New South Wales, Australia) (Scott, 1985). Hill (1983) gave a list of 48 insect species considered major rice pests and 47 regarded as minor pests. Considering this large number there are surprisingly few key pests, defined as those occurring on a regular basis, causing intolerable losses and not provoked directly through man's activities (Huffaker and Smith, 1980). Of course every pest on cultivated crops can simply be regarded as resulting from man's activities; he planted the crop as a monoculture in the first place. However, virtually all rice pests occur due to crucial changes in agricultural practices. Kiritani (1986) reviewed changes in the status of rice pests in. various cultivation systems in Japan. He + found that from 1945-70, when Increased production and stable yields were associated with the use of insecticides, vinyl covers and fertilizers, Scirpophaga incertulas, Scotinophara lurida and O x y a sp. decreased in numbers. In contrast Chilo suppressalis and N ephotettix cinctinceps increased markedly. Since 1970, rice growing has been restricted because of over supply and the aim has been to produce high-quality rice with the minimum of labour. This again caused changes in the status of rice pests with C. suppressalis becoming of reduced importance while N. cinctinceps and some polyphagous, multivoltine, Heteroptera have remained common or increased. Practices which affect pest status include the injudicious use of chemical pesticides, cultural measures such as double cropping, widespread planting of single, or genetically similar rice varieties, planting of long duration rice varieties, ratooning (growth of a second and third crop from stubbles of the first crop), and excessive use of fertilizers. At present, by far the most widely distributed and most important key pests are stemborers (Pyralidae and Noctuidae), leaf-folders and infrequently, armyworms, semi-loopers and hairy caterpillars (Noctuidae). The green leafhopper(GLH), N ephotettix virescens and the brown planthopper (BPH), N ilaparvata lugens, are both vectors of important viral diseases in rice and have been considered secondary or "induced" pests (Rombach, 1987). Some other key pests are of local importance, e.g. the gall midge, (.O rseolia oryzae ) in India, the whorl maggot (.H ydrellia philippina) in the Philippines, the rice hispa, (.Dicladispa arm igera ) in parts of Bangladesh and India, and white grubs (H olotrichia spp.) in watershed projects in Indonesia and upland areas of the Philippines. In the latter country, the malayan rice bug 30 - (MRB) CScotlnophara coarctata) can be regarded as a key pest on the island of Palawan; the insect was accidentally introduced about a decade ago, and natural enemies have not yet given sufficient control. However, it should be noted that in its native distribution area, which includes Malaysia, this insect only causes occasional damage. 2_. The green leafhopper (N ephotettix spp): important pests of rice Although BPH is considered the most important "hopper" pest in many countries of Asia, other "hoppers" are of increasing importance. Of these, N ephotettix spp are considered second in importance only to BPH. Hill (1983) mentioned the species: N. virescens, N. nigropictus (= A*', a p i c a l i s ), N. cinctinceps and N. im picticep -- as important vectors of viruses that cause rice dwarf, transitory yellowing, tungro and yellow dwarf diseases. N ephotettix spp. belong to the family cicadellidae and have rice as their primary host, but can also be found on several other plants which act as alternative hosts. Distribution of the insect includes: India, Pakistan, Burma, S.E. Asia, Philippines, S. China, Indonesia, Papua, New Guinea and West Iran. Shrivastava and Mathur, 1986), studied the biology and host specificity of N. virescens in field conditions of Madhya Pradesh (India). They found that although it can feed on 10 species of weeds which are very common in rice fields, it only completed its life cycle on Echinochloa colonum, Oryza spontanea, Leersia hexandra and rice. Significant variation in oviposition, incubation period, nymphal duration and adult longevity were noted on the different host plants. Egg hatch was significantly prolonged when insects were reared on hosts other than rice, while the oviposition duration was shortened. Although the number of nymphal instars was the same, their duration was significantly influenced by the host and marked variation was observed in the cases of 4th and 5th instars. Details of the life cycle of N ephotettix virescens on rice are provided in Table No 1. Nymphs and adults cause direct damage by sucking the sap from young leaves, but this mechanical damage is considered negligible compared with the potential damage of virus transmission. Eggs are laid in the leaf sheaths. Nymphs have a varied colour pattern in the notum and pass through five instars before they become adults. Adults are 3.2 -5.3 mm long, green with black spots on the wings and black wing tips. Female insects are larger than males (Plate 1). Longevity was reduced when it was reared in laboratory conditions. V'cile and Kuno (lfJ84) made a comparative study of the biology of N. nigroptctus , N. virescens and N. cinctinceps on rice maintained at 25±1*C and a 16 hour photoperiod and found the mean durations of the egg stage were 7.81, 9.42 and 8.31 days, respectively. The nymphal period averaged 19.60, 18.43 and 16.94 days for the males and 21.50, 19.11 and 17.83 days for the females respectively, while equivalent preoviposit ion periods were 4.91, 5.93 and 5.25 days. Females laid 198, 165.55 and 140.19 eggs respectively, adult males lived for 13.85, 22.68 and 15.55 days, and adult females for 19.35, 21.72 and 16.82 days. With the exception of the preoviposit ion period between the three species and fecundity between N. virescens and N. cinctinceps, differences between the three species were significant. Rice plant quality affected the behaviour and biology of N. v i r e s c e n s . Khan and Saxena (1985) used a choice test consisting of 32 TABLE 1. Duration of Nephotettix virescens life cycle on rice* Duration in days ** Event Range Mean Oviposition period 34-55 47.6 Incubation period 5-8 5.7 Nymphal period (instars) I 2-3 2.4 II 2-4 3.1 III 3-4 3.5 IV 3-4 3.5 V 4-6 4.7 Total 14-21 17.3 Female longevity 52-68 63.5 Male longevity 43-57 50.2 * Taken from Shrivastava and Mathur (1986) ** Average of ten replications 3 3 - PLATE 1 Green leafhopper (Nephotettix virescens) adults Left male, right female (X 19). 34 healthy and tungro virus-infected plants and found that initially diseased plants attracted significantly more GLH individuals than did healthy plants. However, after 24 h the situation was reversed and more individuals settled on healthy plants. Insects feeding on diseased plants ingested less food and assimilated less on diseased plants than on healthy plants. Although there was no significant difference in the number of nymphs that became adults on infected and healthy plants, the growth period was prolonged on diseased plants and a significant reduction in adult lifespan, fecundity, egg hatch and population growth was observed. Survival of adult N. virescens was examined in the presence or absence of food plants (rice, var. TNI) in test tubes by Rahman e t a l . (1986). When deprived of both food and water, 75% of adults died within 6 h and all died within 24 h. When only water was provided, 71% of adults survived the first 24 h and 37% survived for up to 48 h. When both food and water were available, all adults survived at least one day and 59% survived seven days. In the conditions used to maintain GLH in the present study (rice var. TNI at a temperature of 25±1*C, relative humidity (rh) of 90- 100% and a 16 h photoperiod) Hywel-jones (1986) found that the ratio of males to females for adult GLH was 36:100 and the mean longevity was 12.7±0.04 days with 50% mortality occurring by the 15th day. 3. The use of insecticides and the rice pest problem There are several references in the scientific literature about the increasing problem of rice pests caused by injudicious and inefficient use of insecticides. Metcalf (1984) reviewed rice pest control in Japan. During the 1950s DDT and yHCH were applied to control the rice stem borer Chilo suppressalis and the paddy borer Tryporyza incertulas. These broad-spectrum insecticides decimated natural enemies of leafhoppers and planthoppers, specially Lycosa spp, spiders and hymenopteran parasites such as Trichogram m a . The green leafhopper N. cinctinceps and the planthoppers N. lugens, Sogatella f u r c i f e r a (white backed plant hopper, WBPH) and L a o d e l p h a x striatellu s became important secondary pests, especially as vectors of rice virus diseases. Parathion, methyl parathion and y^CH were introduced for their control and increasing numbers of applications were made until yHCH and parathion were banned by the Japanese. The problem worsened with the introduction of the new high-yielding japonica rice varieties of the "green revolution", which were highly fertilizer dependent and lacked the insect pest resistance genes of the indica varieties. Use of these varieties often increased insect pest attacks and required increased insecticide applications. As a result during 1950-74 the use of insecticides increased 33 fold, but yield rose only 15 fold despite the new varieties and high fertilization. The green leafhopper and planthoppers developed very high levels of multiple resistance to almost all available organophosphate and carbamate insecticides. From four to seven applications of insecticides were required during 1969-71 compared to only one or two in 1965. In the late 1970's multiresistant leafhopper and brown planthopper were almost uncontrollable in much of south east Asia (Kesitani, 1979). Even today these insects can be difficult to control. Insecticide resistance in rice pests has been demonstrated in 13 species from 5 orders. Heinrichs (1979) and Heinrichs and Mochida (1984) indicated that despite the introduction of new insecticides and agronomic techniques in tropical rice, insecticides often create pest problems rather than prevent them. Resurgence of brown plant hopper (BPH) following pesticide use is the best known example. Rombach (1987) pointed out that outbreaks of GLH and BPH can often be traced back to resurgence effects caused by pesticide applications, mainly because broad spectrum insecticides kill beneficial predators and parasites. In rice, secondary, or induced pests, can rarely occur as key pests. BPH is very rarely a key pest in the vast rice growing area near Cairns, Northern Australia and only occurred as a key pest in a restricted area, and then only in one year out of six. Some outbreaks of BPH in tropical Asia occur due to massive migrations from the temperate regions of Korea and Japan, where BPH was an important pest even before the use of insecticides. Problems of pest resurgence and decimated populations of natural enemies have been found in rice in the Solomon islands and Java. Insecticide applications are often made on a calendar basis and therefore applied when not needed. These examples illustrate that chemical pesticides alone are not the panacea for rice pests. However, specific and effective insecticides such as moulting inhibitors for use against feeding lepidoptera (diflubenzuron) or plant and leafhoppers (bufoprezin) are now available in some countries and can be included in insect pest - 37 - management programs. The consequences of pesticide resistance, environmental pollution and increasing costs of chemical pesticides are well known, the importance of biological control, within an IPM framework, cannot be overstated. 4. Historical highlights of Insect control by fungal pathogens Fungal diseases of insects have been known for a long time. In 1835 the Italian Agostino Bassi (Burges and Hussey, 1971) elucidated the fungal nature of the white muscardine disease of silkworms and later, the idea of using fungi as biological control agents was suggested independently by Pasteur and the American Le Conte in 1874 (Hall and Papierok 1982). However, the first two scientists to suggest the mass production of entomogenous fungi were Metchnikoff (1879) and Krassilstchik (1888) who mass produced the green muscardine M etarhizium anisopliae and tested preparations for control of the wheat cockchafer, Anisoplia austriaca and the sugarbeet curculionid Cleonus punctiventris (Steinhaus, 1949). At about the same time, large-scale field experiments with fungi were conducted in Europe (Ignoffo, 1981). Since that time, there have been many attempts to use fungi to control agricultural pests. In the past 10-15 years there has been a resurgence of interest, because chemical pesticides sometimes fail to control target pests owing to the development of resistance, and the problems associated with mammalian toxicity and environmental safety. The proliferation of resistance, caused by Intense selection pressure, has resulted in a demand for both greater quantities and new types of insecticide. In addition, indiscriminate use of chemicals has created further problems, such as adverse ecological events on beneficial fauna and the accumulation of residues in the environment, which can affect wildlife outside the surrounding ecosystem of the target pest. These factors, together with greater public awareness of the environment, dictate a new approach to insect pest management in which biological control has an increasingly important role. Fungi are unique among insect pathogens in that they can directly penetrate the cuticular barrier and do not have to be ingested. Given favourable environmental conditions, fungi can reduce some insect populations. However, epizootics generally only occur when pest populations are very high. The aim of using fungi is to prevent the build up of pest populations to damaging levels by artificial application of fungal propagules. It is very rare for a fungus to exert permanent, economically-acceptable, control of a pest population, but this can sometimes occur where monoculture prevails, e.g. control of citrus scale by Aschersonia aleyrodis in Florida (Fawcett, 1907, 1944). There have been numerous attempts to exploit fungi as biological control agents but most have ended in failure and resulted in the conclusion that fungi have little potential in this area. In fact, many of these failures can be attributed to a lack of basic knowledge of these agents as well as the ecology of both fungus and target pest. Some mycoinsecticides have been launched commercially and then withdrawn, e.g. My car, (.H irsute 11a thom psonil; 1980-1983), Mycotal and Vertalec, (V ert id Ilium lecanii ; 1983-1986). However, fungi are still used for pest control, e.g. B e a u v e r i a b a s s i a n a is used in China to control the corn borer O s t r i n i a f u r n a c a l i s and also for control of rice pests (Hussey, 1981). In the _ 3 9 _ USSR, B. bassiana is used to control the Colorado beetle (Ferron, 1981), and M etarhiztum antsopliae is used in Brazil to control pests of rice, maize, potato and sugarcane (Aquino, 1975). 5.. Taxonomic classification of entomogenous fungi Entomogenous fungi are found amongst the Deuteromycetes, Zygomycetes, Oomycetes, Chytridiomycetes and Trichomycetes. The class Deuteromycetes contains around 150 species pathogenic to insects (Samson, 1981). A few have received much attention and their biocontrol potential is relatively well understood. Many others have simply never been studied with a view to biocontrol. As a group, they attack virtually all species of insects and arachnids (Hall, 1982). Genera can be highly specific, e.g. the Deuteromycete Aschersonia infects only scale insects and whitefly, and M a s s o s p o r a (Entomophthoraceae) only cicadas, while others have been recorded from many orders of insects, e.g. B e a u v e r i a and M etarhizium . Some Deuteromycetes are now known to have teleomorphs or sexual stages, e.g. Torrubiella was recently described as the teleomorph of V. l e c a n i i (Evans and Samson, 1986) and Cordyceps spp. as the teleomorph of H irsutella spp. (McOwen, 1963; Tubaki, 1981) In the class Zygomycetes, most arthropod pathogens occur in the Entomophthorales. Entomopathogenic species in other orders are known, e.g. Sporodiniella um bellata in the Mucorales (Evans and Samson, 1977) but have been little studied. In the Entomophthorales, apart from a few species parasitic on algae, ferns, nematodes and tardigrades, there are c.a. 150 species, belonging to the genera M assospora, Entomophthora, N eozygites, Con tdiobolus, Zoophthora, Erynia and Tabanom yces, which are parasitic on virtually all orders of insects and mites (Couch e t a l . , 1979; Remaudiere and Keller, 1980). Conidiospores of the Entomophthorales are forcibly discharged and may produce secondary, tertiary or even quaternary conidia of progressively smaller size before penetrating insect cuticle. The class Oomycetes contains few entomopathogens. The most important of these is Lagenidium giganteum (Lagenidiales), a pathogen of mosquitoes (Glen and Chapman, 1978) with a wide geographical distribution (Federici, 1981). Only one genus of the order Blastocladiales (class Chy tridiomycetes) contains entomopathogenic species. The genus Coelomomyces contains 39 described species of obligate aquatic pathogens parasitic on Diptera-Culicidae, Chironomidae, Simuliidae and Heteroptera (Notonectidae) (Couch and Umphlett, 1963; Roberts, 1974; Bland e t a l . t 1981). Coelomomyces spp occur widely and have been recorded from many countries. The class Trichomycetes contains entomopathogenic fungi which are often associated with aquatic dipteran larvae. Several species of S m i t t i u m (Eccrinales) have been isolated from mosquitoes, blackflies and midges (Farr and Lichtwardt, 1967; Williams and Nagel, 1980). 6.. Class Deuteromvcetes - Biology and life cycle descriptions of important species Many species of fungi in the class Deuteromycetes have been superficially studied with respect to nutritional requirements; in general, Deuteromycetes are easily cultured on basal-dextrose-salts and inorganic nitrogen media. However, there are a few exceptions e.g. certain H irsutella spp (Macleod, 1960) and G i b e l l u l a (Samson and Evans, 1973). In nature, the infective unit is the conidiospore which is usually formed externally on dead insects, though V . l e c a n i i is unusual in that it can sporulate on live aphids (Hall, 1976a). The infection cycle is uncomplicated, conidiospores germinate on host cuticle and penetrate to the haemolymph, where often a yeast-like phase predominates and growth occurs as hyphal bodies. Germ tubes may penetrate directly, as in V . l e c a n i i (Hughes and » Gillespie, 1985) and B b a s s i a n a (Hilber and Gillespie, unpublished), or after formation of an appresorium which produces a penetrant peg, e.g. M. anisopliae (Zacharuck, 1970, a,b,c). For Deuteromycetes, the critical water activity (aw) for germination and mycelial growth is about 0.93, equivalent to a relative humidity (rh) of 93%. Thus, conidia of B. bassiana, M. anisopliae, Paecilom yces spp. and V . l e c a n i i all germinated at 0.94 aw but not at 0.92 aw (Gillespie and Crawford, 1986). Furthermore, rates of germination and mycelial growth were maximal at 0.99, 0.98 and 0.97 aw and markedly reduced at 0.96 and 0.94 aw. In one isolate of M. anisopliae, growth was morphologically abnormal at 0.96 aw. Similar data, demonstrating the importance of moisture to aphid infection by V. lecanii, was obtained with living aphids. Disease transmission was maximal in the presence of free water, less at 100% rh. still less at 97% rh and absent at 93% rh (Milner and Lutton, 1986). Clearly, water availability is of vital importance for fungal growth and small differences in the relative humidity levels occurring after spore application can determine whether or not the fungus can successfully 42 - control pests. Penetration of Insect cuticle by the germinating spore has long been considered as due to a combination of enzymatic degradation of the cuticle and mechanical pressure by the germ tube. Strains of B. bassiana, M. anisopliae, Paecilom yces spp and V . l e c a n l i all produced large quantities of protease and chitinase in liquid culture (St. Leger e t a l . t 1986a). Production of protease, lipase, and chitinase on insect cuticle has also been demonstrated with M. anisopliae by enzyme specific staining and recovery of enzymes from fly wings, previously inoculated with conidia (St. Leger e t a l , 1987). In a number of B. bassiana and M. anisopliae strains the key enzyme is an endoprotease which dissolves the protein matrix masking cuticular chitin (Smith e t a l . t 1981; St Leger e t a l . , 1987). Chitinase production occurs later in the infection process. Once through the cuticle, the fungus must overcome the host defence system before it can enter the haemolymph and spread throughout the insect. Possibly in some fungi, toxins are involved (Roberts, 1981). A crude, partially purified extract from M. anisopliae culture filtrate containing the cyclic depsipeptides, destruxins, inhibited prophenol oxidase production by insect haeraocytes, thus suggesting that destruxins may suppress insect immune responses (Huxham e t a l . , 1986). Probably death eventually results from a combination of mechanical damage, causing water loss, nutrient exhaustion and toxicosis. After death, provided sufficient moisture is present, the fungus penetrates outwards through the cuticle and sporulates on the cadaver providing inoculum for the infection of further insects. If conditions are unfavourable, the fungus remains inside the insect, -43 where it can survive for several months, eventually producing spores when favourable conditions return. Erynia neoaphid is survived inside aphid cadavers for 32 weeks at 0*C and 20 or 50% rh, and for 8 weeks at 20*C and 20% rh (Wilding, 1973). Such mycelial persistence in cadavers is likely to be an important mechanism of fungal survival, particularly in those species unable to produce resting spores. Conidia of some species are able to survive in soil (Milner and Lutton, 1976; Bell and Hamalle, 1970) and so may ensure the persistence of the fungus when conditions are unfavourable or when the host is scarce. Considering this, and the saprophytic potential of Deuteromycetes, it is assumed that the soil is the major reservoir for Deuteromycete pathogens of terrestrial pests, although some species may be permanent residents of the phylloplane flora (Hall and Papierok, 1982). Deuteromycetes probably overwinter as conidia or raycelia on cadavers, as specialised resting spores are generally absent, though thick walled resting spores were reported by Zacharuk (in Ferron, 1981). Conidia can survive in soil for long periods, e.g. B. bassiana * conidia had a half life of 276 days at 10*C (Lingg and Donaldson, 1981) and M. anisopliae conidia survived for at least 21 months at 19*C (Fargues and Robert, 1985). M etarhizium and B e a u v e r i a are the best known genera amongst Deuteromycetes, they have a wide geographical distribution and host range, including many insects of economic and to a lesser extent, of medical importance. B e a u v e r i a contains two important insect pathogenic species, B. bassiana and B. brongniartii, for which almost 500 host species are known (Charles, 1941; Gosswald, 1938; Muller- Kogler, 1965). These are mainly parasitic on Lepldoptera and 4 4 _ Coleoptera although species of other orders are sometimes attacked e.g. mosquitoes (Diptera) (Kalvish and Kukharchuk, 1974; Balaraman e t a l , 1979 ) and assassin bugs (Heteroptera) (Parameswaran and Sankaran, 1977). Tulloch <1976), recognised only two species in the genus Metarhizium: M. anlsopliae and M. flavoviride and considered M. b r u n e u m and M . a l b u m to be synonymous with M. anisopliae. However Rombach e t a l . , (1987) restored the name M etarhizium album for a pathogen of Asiatic cicadellids and also described a new variety: M etarhizium flavoviride var m i n u s (Rombach e t a l ., 1986). Two subspecies of M. anisopliae are recognized, M. anisopliae var. a n i s o p l i a e and M. anisopliae var. m a j u s (Johnston) Tulloch, 1976; Rombach, 1987). M. anisopliae is by far the most important species, described from more than 200 hosts, with records mainly from the orders: Coleoptera, Lepidoptera, Orthoptera and Hemiptera (Muller-Kogler, 1965; Veen, 1968) but it has also been recorded from Homoptera (Foster, 1975) and Diptera (Saubenova, 1976; Shcherbak and Kadryova, 1980). Other well studied genera are: Paecilom yces which is widespread and contains 14 entomogenous species parasitic upon several insect orders (Samson, 1974), H i r s u t e l l a , which is a large genus and contains about 40 species infecting most orders of insects (Samson, 1981). The most studied species is the eriophyid mite pathogen H i r s u t e l l a t h o m p s o n i i ( McCoy, 1981). V. lecanii i-Cephalosporium lecanii; Gams, 1971) has been extensively studied as a pathogen of aphids and whitefly in glasshouses (Hall, 1981). It mainly attacks insects in the order Hemiptera, although occasional isolates are made from other orders of - 4 5 insects e.g. Coleoptera, Lepidoptera (Barson, 1976; Lipa, 1975). V. l e c a n l l has also been reported as hyperparas i tic upon plant pathogenic fungi, e.g. Uromyces dianthi (Spencer, 1980). Some Deuteromycetes have also been reported from mosquitoes, e.g. Culicinom yces clavisporus and Tolypocladtum cylindrosporum (Russell e t a l . , 1979; Soares e t a l . , 1979). 1 _. Fungal pathogens of rice pests Among the organisms available for biological control in rice, fungi are the most promising. It has long been recognised that water is essential for spore germination and high atmospheric humidities are known to favour development of fungal epizootics. Rice growing in water in a tropical climate with temperatures from 25 to 30 *C provides an ideal environment for entomogenous fungi. M. anisopliae occurs most frequently on planthoppers, leafhoppers and rice black bugs ( Scotinophara spp.) while M. flavoviride has been collected from plant hoppers in the Philippines and in the Solomon islands. According to Ferron (1981), M. anisopliae is used as a biological control agent in several crops, in some with great success, e.g. the control of spittlebugs in sugarcane in Brazil (Aquino e t a l . , 1977). However, few studies have been made of the potential of M. anisopliae to control rice pests. In Colombia, total control of Sogatodes oryzicola was obtained 12 days after an application of M. anisopliae (Albornoz and Parada, 1984). M. anisopliae and Paecilom yces ltlacinus gave prolonged control of Scotinophara l u r i d a in Japan (Morimoto, 1959), with mortality rates of 60-100% being observed up to 46 days after treatment. Rombach e t a l . (1986a) achieved significant control of BPH populations in rice with conidia and dry mycelium applications of M. anisopliae. Population reductions of S. coarctata were obtained after applications of B. bassiana, M. a n i s o p l i a e and P. lilacinus in field experiments on Palawan, Philippines (Rombach et al., 1986b). Rombach and Shepard (1987) observed H irsutella ctbriform is infecting BPH and reported significant mortality in several places in Asia. B. bassiana has also been reported infecting rice pests. Aguda e t a l . (1984), tested several isolates for virulence against BPH, GLH and WBPH and observed intra-specific variation in pathogenicity. Larvae of the leaf folder M arasmia patnalis were also susceptible to B . b a s s i a n a (Aguda, 1985). Nomuraea rileyi was described infecting plant and leaf hoppers in China (Hongke, 1983), though previously this fungus has only been reported from Lepidoptera (Getzin, 1961; Ignoffo, 1981). Paecilom yces f a r i n o s u s was reported by Hirashima e t a l . , (1979) infecting GLH in Thailand, as well as BPH in Japan (Aoki, 1957). Pusarium spp are generally regarded as opportunistic pathogens (Teetor-Barsch and Roberts, 1983). However, it has been reported infecting WBPH in India (Patel, 1971; Varma et al., 1979) as well as GLH (Balasubramanian and Mariappan, 1983). Several Zygomycetes have also been reported f r o m hoppers. Entom ophthora sphaerosperm a and E . f u m o s a on BPH in Japan and India (Shimazu, 1979; Sakai, 1932). Entom ophthora sp. from N. clnctinceps in Taiwan (Yen and Tsai, 1970), WBPH and BPH in Fiji (Hinckley, 1963), BPH In India and Philippines (Manjunath e t a L , 1978b), GLH in India and China (Manjunath e t a l . , 1978b; Hsieh, L.Y. 1975). E r y n l a 47 (Entomophthora) delphacis is an obligate pathogen of plant and leafhoppers in Asia. Shimazu (1976, 1977) studied growth and germination of this fungus. Field experiments with E. delphacis to control BPH have been conducted without much success (Holdom e t a l . t 1987). Contdiobolus coronatus is the most common pathogen of plant and leafhoppers of rice (Rombach, 1987) In insect cultures with dense insect populations it can be an important mortality factor. Padua and Gabriel (1975) developed a suitable medium for the mass production of C. coronatus based on coconut milk. However, King (1979) found that C. c o r o n a t u s caused mycosis in horses and man, and this fungus is unlikely to be considered for pest control. 8. Development of mvcoinsecticides Selection of pathogens Strain selection is of great importance when choosing a fungal isolate for control of a given pest (Gillespie, 1988). In such a selection several important factors must be considered: pathogenicity to the target insect, spreading ability which is partially dependent on sporulation from dead insects, ease of production on suitable media and survival of infective propagules. Fungal strains can vary widely in all these aspects, specially in pathogenicity. Papierok and Wilding (1981) studied the pathogenicity of Conidiobolus obscurus isolates to aphids and found that virulence could be classified in two levels as defined by Remaudiere e t a l . (1979), and suggested that C. obscurus occurred as two distinct biological races. Holdora e t a l . , (1988) assayed 48 isolates from Erynla delphacls, Erynia radtcans and an undescribed species of Entom ophaga against adult Sogatodes oryzicola and N . l u g e n s and found a wide variations in virulence. Variation in virulence of four strains of B. bassiana to N. cinctlnceps, N. lugens and Chilo suppressalis was found by Li (1987), and Gillespie (unpublished) found strains of M. anisopllae differed widely in their pathogenicity to N ilaparvata lugens. It is perhaps best to consider each strain as an individual entity in biological terras, and not generalize its characteristics within a species. Samsinakova and Kalalova (1983) suggested that a wild type isolate comprises a number of different genotypes which can vary markedly in characteristics such as virulence. Thus, selection of suitable strains should perhaps be made of both wild type strains and single spore isolates. Over the last 30 years, researchers in the Soviet Union have attempted to develop strains with increased virulence. The methods used included mutagenesis (Andrew e t a l . , 1972; Kirsanova and Usenko, 1974; Eulakhova and Martens, 1968; Levitin, 1971; Nikolaev and Ainkina, 1974; Usenko e t a l . , 1973), and hybridization or production of heterokaryons (Tinline, 1971 a, b; Yurchenko e t a l , 1974). However, the strains selected by these methods did not result in increased pathogenicity. This could be, at least in part, due to reduced pathogenicity of auxotrophic mutants as shown for M. anisopliae by Isaac (1988). Nevertheless, the development of improved isolates should be pursued, in the longer term it may be possible to use recombinant DNA technology to produce strains with increased virulence, though a much greater understanding of the mechanisms of pathogenicity will be required before significant progress can be made. B_i Virulence and bloassay systems The most important characteristic of a candidate microorganism for biological control is its virulence to a target insect. A fungal pathogen must possess a high and stable level of virulence and the only way to measure this parameter is by laboratory bioassay; this allows the comparison of isolates under standardized conditions and assessment of the stability of the virulence trait. In industrial production, bioassays are essential for checking the potency of products before dispatch to customers. Of all groups of microorganisms, entomogenous fungi pose the greatest difficulties in designing bioassays. The delivery of infectious propagules to the assay host in a standardized manner is often difficult, since the usual route of fungal penetration is through the cuticle and techniques which achieve this are often laborious and time consuming. Generally, batches of insects are treated with serial dilutions of pathogens and a response, usually death, is measured. Several assay systems have bec-.n developed, although more research is needed. Hall (1976b) treated batches of aphids by immersion in standardized spore suspensions of V. lecanii, removed suspensions by suction in a Buchner funnel, and used chrysanthemum leaf discs placed in high humidity assay cells to maintain treated aphids. Wilding (1976) and Papierok and Wilding (1979) developed a technique of showering conidia from Entom ophthora spp. cultures 5 0 - directly onto batches of aphids. An adaptation of the same technique was used by Milner and Soper (1979) to bioassay Entom ophthora spp. against the Lepidopteran Choristoneura fum tferana , the dose being calculated by averaging estimates of the concentration of spores falling on water agar dishes before and after insect exposure. A similar technique of showering spores was used by Milner and Soper (1981) to bioassay Entom ophthora spp. against the aphid Therioaphis t r i f o l i i f. m a c u l a t a . In this case the dose was estimated by counting the number of primary spores landing on a 22-mm-diameter coverslip placed beneath the aphids. In a bioassay system, mortality of untreated insects must be low, preferably less than 10%. The method of insect maintenance can markedly affect insect survival. Aguda e t a l . (1985), studied different cage types for maintenance of N . l u g e n s and found that the use of potted plants gave the highest survival with more than 80% of insects alive after 6 days. Using probit analysis (Finney, 1971), dose/mortality data can be transformed to estimate LC50 or LD50 values, i.e. the concentration or dose needed to kill 50% of the test insects. The fiducial limits are used to measure the reliability of such estimates; essentially these show the estimated limits within which the parameter would fall with a chosen probability if the assay was repeated a large number of times. Usually the chosen probability is 95%; fiducial limits at 50% mortality are less than at higher or lower mortalities, so the "50% mortality limits" are the most useful expressions of experimental variability to use when comparing the results of different assays. An additional parameter which can be estimated is the LT50, which is the time taken to kill 50% of treated insects at selected dose(s). - 5 1 Another important factor is the slope of the dose/ mortality regression line which reflects the variability within a batch of insects and the variability within the batch of pathogens. The slope will be constant in successive assays only if there are no such variation. Burges (1967) and Burges and Thompson (1971) proposed the use of a standard to reduce the effects of variation between batches of insects used in consecutive assays and of variation caused by differences in assay technique. The standard must store well and be stable; i.e. suffer no loss of virulence over a period of time. Since fungi do not normally store well, the use of such a standard is impossible. However, in practice it is possible to use a given strain, produced in a standard way in each assay. After a suitable bioassay system has been developed, it can be used to study interactions between insects and fungi, e.g. it is possible to determine changes in pathogenicity during subculturing i n v i t r o and i n v i v o . Hall (1980c) showed that the virulence of single and multispore isolates of V . l e c a n i i remained stable during subculture and passage through an insect host. Sweeney (1981a) found that the pathogenicity of Conidiobolus obscurus remained stable after many subcultures, and Shaerffenberg (1964) maintained that spores of B. b a s s i a n a retained their virulence unchanged until the 16 th subculturing provided that they were grown on an optimal medium, but further subcultures decreased virulence considerably. In contrast, Fargues and Robert (1983) demonstrated the virulence of two pathotypes of M. anisopliae changed after subculture on agar or passage through the host insect. These observations suggesting changes in virulence after i n v i t r o or i n v i v o passages, have also been proposed by other researchers (Muller-Kogler 1965, Aizawa 1971). Increased virulence following i n v i v o passage has been reported for A spergillus flavus (Lepesme, 1938), B. bassiana (Kawakani, 1960; Schaerffenberg, 1964; Timonin, 1974; Wasti and Hartmann, 1975), M. a n i s o p l i a e (Timonin, 1974) and P . f a r i n o s u s (Kerner, 1959). Samsinakova e t a l . (1981) and Champlin et al. (1981), found evidence of attenuation of virulence in single or multispore strains of B. b a s s i a n a after i n v i t r o subculture. However Samsinakova e t a l . (1981) also found single spore isolates, considered mutants, which enhanced their virulence after i n v i t r o subculture. Hall and Papierok (1982) considered the usual parameters measured in bioassays, LC50, LD50 and LT50, as insufficient to obtain results which are meaningful with respect to control in the field. It must be taken into account that relative humidity (rh) varies greatly between the laboratory, where bioassays are conducted, and the field. Generally fungi that provide effective pest control, multiply and spread after infection creating an epizootic. Thus, the epizootic potential, ie. the ability to spread, should be determined. Hall (1982) developed a system (suitable for rapidly reproducing insects only) which is useful for screening isolates of V . l e c a n i i for their likely ability to spread. Papierok and Latge (1980) introduced the concept of inoculum multiplication ability, and considered the most effective strains were those possessing a low LT50, a high sporulation capacity and short infection cycle. 5 3 - Cj. Storage Storage of fungal propagules is another important parameter to consider when developing fungi for pest control, and some attempts to mass produce fungi have been abandoned because of poor spore survival (Bajan et al, 1975; Klochko, 1973). Researchers generally agree that bio insec tic ides should be capable of storage for up to 18 months, unless special rapid distribution facilities are arranged (Angus and Luthy, 1971; Couch and Ignoffo, 1981). In countries where spores are applied in the field within a short time of production, storage may not be a major problem, e.g. B. basslana in China (Hussey and Tinsley, 1981) and M. anisopliae in Brazil (Aquino, 1975). Several methods used for storage of fungal cultures are impractical for mass storage of infective propagules, e.g. use of agar slants under oil (Heseltine e t a l . , 1960; Heseltine and Haynes, 1974), freezing in liquid nitrogen (Simione e t a l . , 1977; Cox, 1968) or in the "deep freeze" (Carmichael, 1956; Mugletton, 1963). At present no fungal preparation in widespread use or produced commercially can be stored satisfactorily at room temperature. . Steinhaus (1960) found that conidia of B. basslana survived considerably longer at 4 than at 23 or 38*C. Daoust e t a l . (1983) found that spores of M. anisopliae died more rapidly at 20 than at 4*C. These findings are consistent with the ones obtained by several Investigators, who have observed that lower temperatures generally favour spore survival (Hart, 1926b; Groom and Panisset, 1933; Schaef fenberg, 1964; Walstad e t a l . , 1970). Fargues e t a l . (1979) lyophilized B e a u v e r l a blastospores in skimmed milk and glycerol and found that preparations stored well at 5 but poorly at 20 and 30*C. Heckley e t a l . (1958) considered that estimation of spore germination alone was not satisfactory as a measure of stability, since some microorganisms may survive but lose their infectivity. Therefore the stability of a stored strain can only be evaluated by measuring both spore viability and virulence. Spore viability is generally estimated by germination on agar (Clerk and Madelin, 1965) or on microscope slides (Muller-Kogler, 1967; Muller-Kogler and Samsinakova, 1969). Muller-Kogler (1967) showed that virulence declined more rapidly than viability. Similarly, changes in conidial viability did not indicate changes in virulence for H. thom psonii (Couch, 1978); a similar result was obtained by Hall and Matewele (unpublished observations) with Tolypocladium cylindrosporum . Di Mass production The production of entomogenous fungi as mycoinsecticides, requires the mass production of a particular infectious propagule and thus is distinct from processes producing secondary products, e.g. antibiotics. There are two traditional and well known methods of mass producing entomogenous fungi: semi-solid substrate or liquid fermentation. Semi-solid substrate techniques are mostly designed to yield infective conidia, this method can be traced back to Krassilstchik (1888) who used Metchnikoff's idea and produced M. antsopliae conidia on grains and other organic materials. Cereal grains are the most attractive substrate, being cheap, nutritious, easily sterilized, and if processed carefully, they become friable afterwards. Trays of media may be inoculated with fungus (inoculum being produced in liquid fermentation) and incubated in a - 5 5 - sterile room, or the need for such facilities may be overcome by culturing in autoclavable polypropylene bags, as has been done in Brazil (Aquino e t a l . , 1975, 1977). The major problem of producing fungi on an industrial scale, is the possibility of contamination with other microorganisms, so quality control is a basic requirement. Very little work has been directed towards enhancing spore production on grain substrates. Matewele (1986) demonstrated increased yields of Tolypocladium cylindrosporum and Culicinomyces clavisporus, when vegetable oil or vegetable oil components were added to cereal grains. In addition, vegetable oils increased the friability of broken cereal grain used in fermentation. Mass production on semi-solid substrate has been extensively used in Brazil with M. antsopllae (Guagliumi e t a l . , 1974; Aquino e t a l , 1975; Moura Costa and de Dessa Magalhaes, 1974; Moura Costa e t a l . t 1974) and China with B. basslana (Hussey and Tinsley, 1981). Other fungi which have been produced by this method are: Entom ophthora thaxteriana (Soper e t a l . , 1975), E. vJrulenta (Matanmi and Libby, 1976), Nomuraea rileyi (Riba and Glandard, 1980; Ignoffo, 1981) and H. t h o m p s o n i i (McCoy e t a l . , 1978). In the case of the last two species, as well as those Deuteromycetes which do not sporulate easily in liquid culture, semi-solid substrate culture is the only suitable method available at present for mass production. A further advantage of semi-solid fermentation is that aerial conidia, the final product, are considered to be more robust than blastospores produced in liquid culture (Blachere e t a l . , 1973; Ferron, 1978; Fargues e t a l . , 1979) Alternatively, semi-solid substrates may be cultured in bulk in a horizontal rotating drum in which temperature and air flow are controlled. Light can markedly influence spore yields. Hall (1977) demonstrated a 5 fold increase in conidial yields of V . l e c a n i i in light compared to dark. Gillespie (1984) reported increased yields of Paecllom yces fum osoroseus when cultured under fluorescent light. Light can also increase melanization and provide increased resistance to UV light. Osman and Valadon (1981) found that hyphal wall thickness of V erticillium agaraclnum was increased when cultures were grown in near uv light. Since conidia are generally produced in dry hydrophobic masses, they may be harvested by aspiration or sieving, or by agitation with a dilute detergent solution. They can also be harvested by drying the bulk product and milling it to a sprayable powder. However, this process can be harmful to spores. For each species, the speed, conditions and degree of drying must be taken into account, and strains of the same species differ in their ability to resist drying and milling, as was demonstrated in V . l e c a n i i (Lisansky and Hall, 1982). Spore yields on solid substrate can approach 1010 spores/g dry weight. (Hall and Papierok, 1982). Many species of entomogenous fungi can multiply in submerged culture and liquid fermentation has been used to produce both blastospores and conidiospores. The spore type depends on the species, nutrient composition of the media and fermentation conditions. Blastospores can be produced in: B. basslana (Samsinakova, 1966), B. brongnlartli (Blachere e t a l . t 1973; Catroux e t a l . , 1970), M. a n i s o p l i a e (Adamek, 1965), Sorosporella uvella (Misikova, 1967) and V. l e c a n i i (Hall, 1977; Samsinakova and Kalalova, 1976). However, yields vary and liquid culture is not economic for all species. Conidiospores can be produced in liquid culture in certain strains of 57 - B. bassiana (Goral, 1973, 1975; Thomas e t a l . , 1986), C. clavisporus (Sweeney, 1981b), Sporodesm ium sclerotivorum (Ayers and Adams, 1983) and H. thom psonii var. synnematosa (Van Wlnkelhoff and McCoy, 1984). Providing that the final product (blastospores or conidiospores) can be stabilized, liquid culture is the most suitable method for mass production of several Deuteromycetes. Harvesting fruit bodies from these cultures is simple (by continuous centrifugation or filtration) and sterility should not be a problem. Those species or strains which do not readily form blastospores may in future be encouraged to do so by manipulation of nutrients or physical factors (Hall and Latge, 1980; Riba and Glandard, 1980). Despite extensive studies, many aspects still need to be elucidated, e.g. differences in pathogenicity of submerged fruit bodies (blastospores and conidiospores) and aerial conidia, as well as many aspects of the culture process itself. Thomas e t a l . , (1986) developed a method and a medium to produce conidia of B. bassiana under submerged cultivation and made some morphological, physiological and biochemical comparisons of these conidia with aerially produced conidia. In addition they provided evidence which suggested that microcycle conidiation may be involved. There is clearly a need for further research in this area. A combination of liquid and semi-solid fermentation process has been tried for some fungi. In this technique, mycelium is grown in liquid media and used to inoculate a solid medium, or an inert carrier, on which conidia are produced (Ferron, 1981; Soper and Ward, 1981). Spores can also be obtained using cultures floating on media in shallow aerated vessels (Kybal and Ulcek, 1976; Samsinakova et al., 1981), but this method seems unlikely to be economic. 58 - McCabe and Soper (1985) introduced and patented a novel method for the production of Entomophthoraceae called the "Marcescent process", in which, dry mycelium is produced by submerged fermentation, dried and milled. The product can be stored for some time and applied in the field with conventional spray equipment; the mycelium sporulates on the plant, and these conidia subsequently infect insects. Rombach et al. (1988) modified the "Marcescent" process for use with B. bassiana, M. anisopllae, and Paecilom yces l i l a c i n u s and studied the application of dried mycelia. Spore production was high, particularly considering that virtually no media remained in the product to sustain conidiation, and it is possible spore production could be increased by the addition of nutrients. The method chosen for mass production of a given strain, is determined by several factors, such as availability of raw materials, ease of handling and training of personnel. The economics of production overwhelm all factors in the overall analysis. Bartlett and Jaronski (1988), considered sufficient production as the key problem taking into account that effective doses of entomogenous fungi to control pests in the field are above 5x10’2 spores per hectare. When that rate is extrapolated to a commercially attractive area, the amount of production needed can be prodigious. The authors concluded that field effect or efficiency of mass production must be increased by several orders of magnitude to be practical and competitive with others ways of control. The two most significant challenges are (1) submerged fermentation to produce aerial conidia, or a mastery of semi-solid substrate fermentation to efficiently produce the large number of conidia required, and (2) satisfactory shelf-life of preparations at room temperature. - 59 E. Formulation Formulation should enable the selected strain to improve its efficacy when it is applied in the field. Uniform spore distribution, longer contact with target insects, protection from deleterious environmental factors and prolonged spore longevity, are some of the characteristics which can be optimised by adequate formulation. To achieve this, the product may contain additives such as wetters, stickers, humectants, UV protectants and thixotropic agents. Each additive must not adversely effect spore virulence. Incorporation of nutrients into formulations can allow fungi to sporulate "naturally" on foliage, thereby increasing persistence. Hall and Turner (unpublished observations) found that the addition of nutrients to V . l e c a n i i resulted in a 40 fold increase in the number of applied spores. In certain cases, this would provide a way of effectively increasing spore production to achieve an effective dose at an economic rate (Hall and Papierok, 1982). Clark e t a l . (1968) proposed that "vegetable oils" be used for application of B, bassiana for mosquito control. Angus and Luthy (1971) proposed fuel oils as a liquid vehicle for dissemination of microbial agents, but later, Daoust (1983) found that survival of M. a n i s o p l i a e conidia was reduced by both mineral and vegetable oils, when compared with survival of "unformulated" conidia. M. anisopliae, B. bassiana, B. brongnlartii and H. thom psonii conidia survived for three years under mineral oil at 4*C and for a shorter period without the oil (Mietkiewski and Soper, 1978). McCoy and Couch (1978) used oil as an adjuvant in H. thom psonii formulations with no adverse effect. And Blachere e t a l . (1973) used a multicomponent ♦ system including oil and wetting agents to stabilize blastospores of B. basslana. Several researchers have used clay silicates to dilute spores in formulations, mainly because they are relatively inert and easily -available at low cost (Polon, 1973). Clay silicates have also been used to coat spores to minimize desiccation, one of the most important factors causing conidial death (Reisenger e t a l . , 1977). In the soil, clay-coated blastospores of B. basslana survived better than untreated ones, The pH of additives or substrate media can affect survival of fungi; spores of Conldiobolus throm boides survived better in alkaline clay (Mietkiewski and Soper, 1978). Physical properties of the carrier may affect the performance of the pathogen. Daoust e t a l . (1982), working with M. anisopliae against Anopheles Stephensi and Culex plplens, found conidia formulated in an hydrophobic carrier, were more effective than un formula ted spores. However, the potency of the formulation against Aedes aegypti larvae was reduced. The most recent development in the application of mycoinsecticides is the use of electrostatic sprayers which increase spore deposition on foliage by charging spray droplets. In particular, spray coverage on underleaf surfaces is increased. In some cases, carriers with special electrostatic properties are needed. Sopp e t a l (1988) demonstrated improved control of chrysanthemum aphids with low volume electrostatic application of V . l e c a n i i compared to high volume treatments with a conventional hydraulic sprayer. 61- SECTION II FUNGAL BIOLOGY MATERIALS AND METHODS 1. General A. Media 12 Sabouraud dextrose agar Sabouraud dextrose agar (SDA) was obtained from Oxoid Ltd., Basingstoke, UK and had the following composition: mycological peptone lOg dextrose 40g agar technical No 1 15g per litre of distilled water, pH approximately 5.6 11) Yeast extract plus glucose liquid medium The components of Yeast extract plus Glucose (YG) were obtained from Difco Lab. Detroit, Michigan, USA and BDH Ltd., Poole, UK respectively. yeast extract 40g glucose 20g + per litre of distilled water, pH approximately 6.6 lil) Sabouraud liquid medium Sabouraud liquid medium (SL) was obtained from Oxoid Ltd. Basingstoke, UK. and had the following composition: pancreatic digest of casein 5 g • peptic digest of fresh meat 5 g d e x tro s e 20g per litre of distilled water, ph approximately 5.7 lv) TK1 broth defined medium TK1 broth was prepared as described by Thomas and Khachatourians (1986) KN03 10 g II KH2P04 5g MgS04.7H20 2g glucose 50g CaC12.2H20 50mg FeC13.H20 12 mg MnS04.H20 2.5 mg Co (N03)2.6H20 0.25mg Na2Mo04.2H20 20mg ZnS04.7H20 2.5 mg • CuS04.5H20 0.5 mg 6 3 - per litre of distilled water pH approximately 5.7 v) Grains used for semi-solid culture The cereal grains, white rice (WR), whole grain rice (WG), prefluffed rice (PR), Wheat(W) and barley withi husk (B) were combined with sunflower oil and water in different proportions as semi-solid media for fungal culture (IV, 3, B). EL Media and equipment sterilization All liquid and semi-solid media as well as the equipment used for fungal production were sterilized at 121 *C for 20 min. C^ Surfactant, antifoam and buffer Triton X-100 and Tween 80 (BDH Chemicals UK) were used to wet and disperse conidia, at a concentration of up to 0.05%. Polypropylene glycol 1000 (BDH Chemicals, UK) was used as an antifoam in liquid media production at a concentration of 0.1%. Media pH were adjusted occasionally using IN HC1 or 10N K0H. Da Temperature, humidity and pH measurement Temperature was preferentially recorded with a thermograph or spot thermometer readings taken over the time course of experiments. When necessary, records of temperature and humidity were taken with a Squirrel meter/logger (Grant Instruments Ltd., Cambridge, UK). pH was measured with a digital pH/temperature meter (Electronic Instruments Ltd., Kent, England) Ej. Total spore counts Total spore counts were made in an improved Neubauer haeraocytometer. Before use the haemocytometer was cleaned thoroughly with alcohol and dried with tissues. The cover slip was moistened by exhalation, applied to the counting chamber and flattened until "Newtons rings" became visible, thus indicating the correct chamber depth had been obtained. Both chambers were then carefully flooded with the spore suspension and the number of spores in the central, 25 triple lined squares (lmm2), was determined for each chamber. This figure was then multiplied by lO* to give the spore concentration per ml of suspension. Standardization was then made by dilution in suitable volumes of sterilized water plus 0.05% Triton X-100. F\ Assessment of spore viability Spore viability was assessed using the modified method of Hall (1977). "Humid chambers" were prepared by spreading sterile SDA along the surface of glass microscope slides, which were placed individually in Petri dishes (9 cm diara.) lined with moist filter paper. A small amount of spores (c.a. 0.3 mg) was suspended in 5 ml 0.05% Triton X- 100 solution and sonicated (lOp amplitude, 15 seconds; Soniprep 1500 sonicator; MSE, Crawley, UK).Aliquots (c.a. 0.1 ml) of this suspension containing around 10s spores per ml were placed on the SDA and plates incubated for 20-30 hours at 25*C. Slides were examined using 6 5 phase contrast microscope and spore viability determined by counting at least 300 spores per slide. A spore was considered viable if it produced a visible germ tube. G^ Production and harvesting of aerial conldia Conidia were produced on SDA by inoculating Petri plates (9 cm diam) with a suspension <0.5 ml, 107 conidia per ml) of fungal conidia in 0.05% Triton X-100 and incubating plates at 25±1*C for 7-9 days. Conidia were produced on cereal grains by inoculating 250 ml Erlenmeyer flasks with a suspension (1 ml, 107 conidia per ml) of fungal conidia in 0.05% Triton X-100 and incubating flasks at 25±1*C for 10-14 days. Harvesting of conidia was done by flooding plates or Erlenmeyer flasks with 0.05% Triton X-100, agitating with a sterile aluminium rod, filtering suspensions through two layers of muslin, sonicating for 15 seconds (lOp amplitude, 15 seconds) and centrifuging (12.100 G, five minutes). Resulting pellets were resuspended in fresh 0.05% Triton X-100 solution and counted in an improved Neubauer haemocytometer, before being adjusted to the required concentration. Ih Production and harvesting of hvphal bodies and submerged conidia Hyphal bodies and submerged conidia of B. bassiana 268-86 were produced by inoculating conidia into 250 ml Erlenmeyer flasks containing 50ml of YG or SL (for hyphal bodies) or TK1 medium (for submerged conidia), or into 1.6 litre fermenters (IV, 4). Yields were optimal after 120 h. when hyphal bodies and submerged conidia were » harvested by filtration through tissue, centrifuged (12.100 g, five min.) washed three times and resuspended in 0.05% Triton X-100. 2. Source of strains used A total of 29 fungal strains from the genera V erticillium , M etarhizium , Paecilom yces, Beauveria, Trichothecium and Sporothrix, from 20 different hosts and originated from several countries were used in this study (Table 2) 3. Effect of temperature on germination and mycelial growth /L The effect of temperature on spore germination 12 The SPA slide technique Pure cultures of V . l e c a n i i 11-73 and 19-79, P. farinosus 104-82, Trichothecium 213-85, M. flavoviride 259-85, M. anisopliae 83-82, 260- 85 and 275-86, and B. bassiana 110-82, 138-83, 235-85, 261-85, 266- 85, 268-86 and and 269-86 were obtained on SDA after 7-9 days incubation at 25±1*C. Conidia were harvested in 0.05% Triton X-100 as previously described, suspensions adjusted to 5X10e conidia per ml and kept at 5±1*C. SDA slides were then inoculated with single drops of spore suspension, maintained in humid chambers at various temperatures (10-37*0 and examined after 12, 15, 18, 21, 36, 39, 42 and 45 or 9, 12, 15, 18, 21, 24, 36 and 42 hours Conidial germination was determined by direct counts of 300 6 7 - TABLE 2. Origin of fungal isolates Strain Species Host Source 11-73 Verticillium lecanii Unknown UK 19-79 ii ii Whitefly UK 82-82 Metarhiziura anisopliae Recilia dorsalis Philippines 83-82 ii ii Spittlebug Brazil 94-82 it it Unknown Switzerland 104-82 Paecilomyces farinosus Melolontha melolontha Switzerland 109-82 Beauveria bassiana Nephotettix cinctinceps China 110-82 ii it ii ii ti 138-83 ii ii Nilaparvata lugens Philippines 203-84 Metarhizium flavoviride ii it Philippines 206-85 Beauveria bassiana Unknown Russia 208-85 Metarhizium anisopliae Rhinoceros beetle W. Samoa 213-85 Trichothecium sp. Nephotettix virescens China 231-85 Beauveria bassiana ii ii China 235-85 ii ii Nephotettix bipunctatus China 251-85 it m Deois flavopicta Brazil 259-85 Metarhizium flavoviride Nilaparvata lugens Philippines 260-85 Metarhizium anisopliae Scotinophara coartata Philippines 261-85 Beauveria bassiana Nilaparvata lugens China 265-85 ii it Unknown Philippines 266-85 ii ti Leptocorisa spp. Philippines 268-86 ii ii Unknown Thailand 269-86 ti ti ii Thailand 270-86 ti ti it Thailand 275-86 Metarhizium anisopliae Cydia pomonella Germany 276-86 it ii Otiorhynchus sulcatus Germany 298-86 Sporothrix insectorum Leptopharsa gibbicarina Colombia 299-86 B. bassiana Cosmopolites sordidus Colombia 300-86 M. anisopliae Ancognatha sp. Colombia conidia using phase-contrast microscope and data used to estimate GT50 and GT95 values ii) The lactophenol technique Suspensions from 7-9 day old SDA cultures of 10 single spore isolates (SSI) and the multi spore isolate (MSI) of B. basslana 268-86 were prepared in 0.05% Triton X-100, as described previously, and adjusted to 5X10e conidia per ml. Aliquots (5 pi) of each suspension were placed on small SDA petri plates (5.5 cm diam.). The suspensions were then spread and kept at 25±1*C. Three samples of each strain were taken from the incubator after 4, 6, 8, 10, 12, 14, and 16 hours and one drop of lactophenol cotton blue placed on the surface of each sample and covered with a cover slip. Plates were then stored at 5±1*C and conidial germination determined by direct observation of at least 100 conidia per sample. EL Effect of temperature on mycelial growth Pure SDA cultures of V . l e c a n i i 11-73 and 19-79, P. farinosus 104-- 82, Trtchothecium 213-85, Af. flavoviride 203-84, M. anisopliae 83-82, 275-86 and 300-86, and B. bassiana 110-82, 138-83, 235-85, 261-85, 266-85, 268-86, 269-86 and 270-86 were obtained as described previously. Conidia were harvested, suspended in 0.05% sterile Triton X-100, adjusted to 5X107 conidia per ml and 4-5 drops of this suspension were put on SDA plates and spread over the agar surface with a sterile aluminium spreader. Inoculated Petri plates were Incubated at 25±1X for 48 hours, when plugs <6 mm diam.) were removed with a sterile cork borer and after inversion, placed in matching holes in the centre of fresh SDA plates. Plates were maintained at 5, 10, 15, 20, 23, 25, 27, 30 and 35*C and four replicate plates used for each treatment. The diameter of resultant colonies was recorded at seven day intervals, using a ruler, for up to 5 weeks and rates of mycelial growth were expressed as the mean diameter extension (mm per day) 4. Effect of water activity on germination and mycelial growth Influence of water activity on germination Agar plus glycerol (AG) solutions were made to provide water activities (aw) from 0.91-1.00 (Table 3; Dallyn and Fox, 1980) and used to coat microscope slides or poured into 9 cm diam Petri plates. Suspensions of B. bassiana 268-86 and M. anisopliae 275-86 from 7-9 days old SDA cultures were prepared in 0.05% Triton X-100, as described previously, adjusted to 5X10S conidia per ml and sonicated. AG slides were inoculated with single 5 pi drops of spore suspension, placed in filter-paper lined Petri plates and examined after 18, 24, and 36 hours. Conidial germination was determined by direct counts of 300 conidia per slide using phase contrast microscope. Treatments were replicated four times. Ih Influence of water activity on mycelial growth Suspensions of B. bassiana 268-86 and M. anisopliae 275-86 were obtained as described previously and used to inoculate SDA plates TABLE 3. Water activity data for aqueous glycerol solutions* Available Glycerol Mass Volume for total Mass for total water molality* equivalent vol. of 1 1 ( ml) vol. of 1 1 (g) (g) 1.00 0 0 0 0 0.99 0.55 50.6 38.6 48.6 0.98 1.10 101.2 74.3 93.6 0.97 1.65 151.8 107.5 135.4 0.96 2.22 204.2 139.5 175.8 0.95 2.77 254.8 168.3 212.1 0.94 3.33 306.4 195.6 246.5 0.93 3.89 357.9 221.2 278.7 0.92 4.44 408.5 244.8 308.4 0.91 5.00 460.0 267.5 337.1 * Taken from Dallyn and Fox (1980) ** Molality is defined as moles of solute per Kg solvent 71 which were then incubated for 48 hours <11, 3, B). Plugs were then placed in 6 mm diam holes in the centre of 9 cm Petri plates containing agar with aw 's from 0.90-1.00 Petri plates were incubated at 23*C and five replicate plates used for each treatment. Resultant colony diameters were recorded as described previously 5. Shelf life experiments A^, Influence of temperature on survival of dried conidia Groups of ten SDA Petri plates were inoculated with V . l e c a n i i 11- 73 and 19-79, P. farSnosus 104-82, Trichothecium 213-85, M. flavovirtde 259-85, M. anisopliae 83-82 and 260-85, or B. bassiana 261-85 and incubated for 7-9 days at 25+1‘C. Conidia were harvested in sterile 0.05% Triton X-100 as described previously, sonicated (lOp amplitude, 15 seconds) centrifuged (12.100 g for five min.) and the pellet suspended in sterile 0.01% Triton X-100 before centrifuging again (12.100 g for five rain.). The supernatant was removed, the spore pellet resuspended in 5 ml sterile 0.005% Tween 80 solution and spore viabilities determined on SDA slides. Spore suspensions were placed in Petri plates and dried in a vacuum chamber (Townson and Mercer Ltd, Croydon, UK) for 48 hours at room temperature (20 + 2*0. Films of dry spores were lightly ground in a pestle and mortar, the powder divided into portions and placed in small sterile bottles with sealed caps (polystyrene screw cap Bijoux; 7 ml, Sterilin Ltd., Feltham, UK). Immediately after drying, viabilities were assessed by examining spore germination on SDA and bottles stored at 5, 20 and 30* C. Viabilities were further assessed after 1, 2, 4, 8, 12, 20, 32, 40, 43 and 49 weeks with three samples examined at each assessment. Influence of drying method and temperature on germination and pathogenicity of stored conidia Groups of ten SDA Petri plates were inoculated with B. bassiana 110-82, 235-85, 266-86 and 268-86, or M. anisopliae 275-86 and incubated for 7-9 days at 25±1*C when conidia were harvested in 0.05% sterile Triton X-100 and sonicated as described previously. The suspension was centrifuged (12.100 g; five min.) and the pellet suspended in 0.01% sterile Triton X-100 before further centrifugation <12.100g for five min.) and final resuspension in 5 ml aliquots of 0.01% sterile Tween 80 solution. Spore viabilities were then determined on SDA slides. Spore suspensions were divided in two equal volumes, half were vacuum dried and half were freeze dried (lyophilized). Vacuum drying; Suspensions were placed in Petri plates and dried for 48 hours at room temperature <22±1‘C) in a vacuum chamber (760 mm Hg pressure). Films of dry spores were ground in a pestle and mortar, the powder divided into portions and placed in small sterile Bijoux bottles. Bottles with loose caps were left in the vacuum chamber at room temperature for a further 18 hours and viabilities checked by the SDA slide technique. Caps were then tightened and bottles stored at 5, 20 and 30* C. Viabilities were further assessed by checking three independent samples after 28, 95, 180, 285 and 369 days. Freeze drying: Pellets were placed in sterile bottles with sealed caps (polystyrene screw cap universal, 30 ml; Sterilin Ltd, Feltham, UK) pre-frozen at -19±1*C for 24 hours and then left in the freeze drying chamber (Modulyo, Edwards High Vacuum and Industrial products Ltd, Shoreham, UK) for 48 hours at -58±2*. Dried spore powder was lightly ground, divided into portions and placed in small sterile Bijoux. Bottles with loose caps were left in the freeze drying chamber for a further 18 hours and viabilities checked. Caps were then tightened and bottles stored at 5, 20 and 30*C. Viabilities were further assessed as described for vacuum dried samples. To assess the virulence of stored spores, suspensions with 5X107 viable spores per ml were prepared after 180 and 360 days storage and assayed against GLH adult to obtain LC50 values (III, 3, A) C^. Influence of temperature and antibiotic addition on germination and pathogenicity of stored spore suspensions. B . b a s s i a n a 268-86 aerial conidia produced on SDA, (II, 1, G), submerged conidia from TK1 broth (IV, 4) and hyphal bodies from YG and SL (IV, 4) were harvested and standardized as explained previously. Suspensions were centrifuged (12.1000g for five min.), pellets resuspended in 0.01% sterile Triton X-100 recentrifuged and resuspended in 0.005% Triton X-100 before final centrifugation and suspension in 0.005% sterile Tween 80. Antibiotics were then added to half the suspension to provide final concentration of 100 pg per ml chloramphenicol, 50 pg per ml ampicillin, 50 pg per ml gentaraycin and 50 pg per ml tetracycline and viabilities determined by the SDA slide technique. Suspensions were kept in sterile Universal bottles with sealed caps (30ml), and stored at 5 and 20*C. Viabilities were assessed by checking germination on SDA slides after 17, 39, 50, 60, 70, 80, 90, 122 and 180 days storage, three samples were examined at each assessment time. Dj, Influence of vacuum drying on viability of various B. b a s s i a n a spore types B. bassiana 268-86 aerial conidia produced on SDA and wheat (II, 1, G and IV, 3, B), submerged conidia from TK1 broth (IV, 4, A) and hyphal bodies from YG and SL (IV, 4, B) were suspended in 0.005% Tween 80 and dried in a vacuum chamber for 48 h. (23+1*0. Viabilities were assessed before and after drying by the SDA slide technique. 6. Effect of SDA subculturing on production of conidia Multispore isolates (MSI) of B. bassiana 268-86 and M. anisopliae 275-86 together with their respective single spore isolates (SSI's) BbSSI81, BbSSI96, MaSSIl and MaSSI3 obtained as explained in III, 6, C, were inoculated onto SDA Petri plates and incubated for 9 days at 25±1*C. Each culture was then subcultured by streaking a loopful of spores onto fresh SDA plates and the procedure repeated 9 times. - 75 " After the 1st and 10th subculture, the sporulation of each culture was assessed by removing 5 mm diam. plugs from three replicate plates with a cork borer. Plugs were agitated in 10 ml 0.05% Triton X-100 containing 10 glass balls (2-3 mm diara.) for two minutes on a vortex mixer (Gallenkamp, Loughborough, UK; 2.800 rpm). Spore concentrations were determined using an Improved Neubauer haemocytometer and sporulation expressed as mean number of conidia per mm2 of culture. 7. Influence of ultra violet light on germination of B. bassiana conidia. submerged conidia and hvphal bodies Aerial conidia, submerged conidia and hyphal bodies of B. bassiana 268-86 were produced on SDA (II, 1, G), TK1 broth and YG (IV, 4), harvested in 0.05% Triton X-100 filtered through muslin and sonicated (lOp for 15 seconds). Suspensions were adjusted to 10G conidia per ml and 15 ml aliquots poured into Petri plates bases (three replicates) which were then arranged randomly inside a closed box with two lamps of Ultra violet (UV) light (Philips germicidal lamps TUV 40W, wave length 253.7 nm) for 40 minutes. Samples of 100 pi were taken from each suspension after 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, and 25 minutes and germination assessed by the SDA slide technique. Every five minutes the box containing the Petri plates was moved gently forward and backward five times to maintain spores in suspension. 8. Measurement of conldial germination by the optical brlghtener technique When conidia of B. basslana and M. anisopltae were stained with bi-striazinyl amino stilbene (Ciba Geigy, Manchester, UK) and observed under UV light, it was noticed that a proportion of the spores fluoresced brightly while the majority exhibited only limited fluorescence (Plates 2 and 3). It was considered that possibly only non viable conidia were fluorescing brightly and that the method could perhaps be developed as a rapid method for assessing conidial viability. To test this hypothesis, cultures of B, bassiana 268-86 and M. anisopliae 275-86 were produced on SDA Petri plates. Conidia were harvested as described previously and suspensions standardized at 107 conidia per ml. Suspensions were then divided into half and one part autoclaved (2 min.; 121 *C) to kill the conidia. Viable and non viable conidia were then recombined to provide suspensions with nominal viabilities of 100, 80, 60, 40, 20, and 0%. Aliquots of each suspension (5 pi) were then placed onto SDA slides, kept in humid chambers and incubated at 25±1X for 19 hours when the percentage of germinated conidia was recorded by direct observation under phase contrast microscopy. Further aliquots of conidial suspensions were centrifuged (3020g; five minutes), resuspended in 0.05% Tinopal solution (containing bi- striazinyl amino stilbene in 0.05% sterile Triton X-100 left for 30 minutes and then centrifuged again (12.100; five minutes) and suspended in sterile 0.05% Triton X-100. Samples of spore suspension were taken, placed on microscope slides, cover slips applied on the slide and sealed using Glyceel (Gurr Ltd, UK) to avoid drying of the PLATE 2 Aerial conidia of Beauveria bassiana 268-86 stained with bi-striazinyl amino stilbene and observed under UV light (X250). 78 viable conidia non viable conidia PLATE 3 Aerial conidia of Metarhizium anisopliae 275-86 stained with bi-striazinyl amino stilbene and observed under combined UV and normal light (X250) - 79 - sample. Slides were then viewed using a fluorescence microscope (Leitz-Dialux 20 with ultra violet filter: A. 513596; Leitz, FRG) at X250 magnification. The number of fluorescing (dead) and non fluorescing (viable) conidia were recorded in 20 fields of view per sample, i.e. approximately 500 conidia were examined per treatment. Approximately one month old SDA cultures of B. bassiana 110-82, 261-86, 266-86, 268-86 and 269-86, and M. anisopliae 36-79, 83-82, 101-82, 135-82, 137-83, 170-83, 189-83 and 275-86 were taken and conidial suspensions prepared in 0.05% Triton X-100. Conidial germination were then compared using the two methods as described previously. 9. Viability of stored conidia as measured by the SDA slide and the optical brightener techniques. The survival of B. bassiana 110-82, 235-83, 266-85 and 268-86, and M. anisopliae 275-86, after lyophilization and vacuum oven drying (II, 5, B), and storage at 5 and 20*C was examined, initially by germination assessment on SDA, but from 95 days onwards by the additional use of the rapid fluorescence method. After 95, 180, 285, 369 and 451 days, samples were taken, suspended in 0.05% Triton X-100 and sonicated as described previously. Viabilities were then determined on SDA or after Tinopal staining. - 8 0 10. Differentiation ofB, bassiana 268-86 spore types Samples of aerial conidia produced on SDA or semi-solid medium <11, 1, G), hyphal bodies produced in complex media (IV, 4) and submerged conidia produced in defined media (IV, 4) were taken, filtered through a two layers of muslin, washed two times by centrifugation (12,100 g for five minutes) and resuspended in 0.05% Triton X-100. Spore dimensions were then measured under phase microscopy using a graticule eye piece. - 81 - RESULTS 11. Effect of temperature on germination and mycelial growth Temperature was a critical factor; fungal strains differed in their rates of germination and mycelial growth in response to temperatures. Aj. Temperature Influence on germination of fungi V ertlcilllum lecanii strains 11-73 and 19-79 germinated over a wide range of temperatures, and were able to germinate from 5 to 37*C. Germination rates were fastest between 20 and 30’C, but the optima varied from 25*C for 11-73 to 20*C for 19-79 (Table 4). M. anisopliae 83-82, 260-86 and M. flavoviride 259-86 failed to germinate at 5, 10 or 37*C, during the 45 h observation period. For all three germination was most rapid at 30*C, but M. flavoviride was particularly slow and needed more than 45 hours to reach 95% germination at any of the studied temperatures Paecilom yces farinosus 104-82 germinated at temperatures between 10 and 30*C and produced germination tubes most rapidly at 25*C. Trichothecium 213-85 did not germinate at 5*C during 45 hours observation but did at 37*C. Germination rates were the fastest of all studied strains and at 30*C it reached 95% germination within 12 hours. Beauverla basslana 261-85 failed to germinate at 5 or 37*C but spores developed rapidly between 20 to 30*C with optimum at 30*C. TABLE 4. Estimated time needed for 50 or 95% germination of fungal strains on Sabouraud dextrose agar Estimated time to 50 Incubation temperature in °C Strain or 95% ______germination (hours) 5 10 20 25 30 37 Verticillium lecanii GT50* >45 22.4 <12 <12 <12 >45 11-73 GT95** >45 35.3 15.2 12.3 17.9 >45 Verticillium lecanii GT50 >45 28.0 <12 13.1 14.8 >45 t 19-79 GT95 >45 39.1 15.1 18.5 19.2 >45 Metarhizium anisopliae GT50 N N 12.9 12.8 <12 N 83-83 GT95 N N 19.3 17.4 14.1 N Metarhizium anisopliae GT50 N N 22.8 12.0 <12 N 260-85 GT95 N N 32.8 18.3 15.2 N Metarhizium flavoviride GT50 N N >45 34.5 31.4 N 259-85 GT95 NN >45 >45 >45 N Paecilomyces farinosus GT50 N >45 15.7 <12 <12 N 104-82 GT95 N >45 28.0 23.2 25.1 N Trichothecium GT50 N 26.9 <12 <12 <12 >45 213-85 GT95 N 36.0 <12 12.0 <12 >45 Beauveria bassiana GT50 N >45 21.2 15.2 13.8 N 261-85 GT95 N >45 28.2 19.6 17.7 N * Estimated germination of 50% spores ** Estimated germination of 95% spores N No germination during 45 hours observation period 83 Germination rates of strains whith high pathogenicity to GLH and the ability to stick cadavers to rice plants are shown in Table 5. B. bassiana 110-82, 138-83, 235-85, 266-66, 268-86 and 269-86 germinated at temperatures between 10 and 30 *C. All of them germinated most rapidly between 20 and 30*C, with 27*C the optimum for isolates, 110-82, 266-85, 268-86 and 269-86 and 30*C for isolates 138-83 and 235-85. Conidia of M. anisopliae 275-86 failed to germinate at 10*C during 45 hours observation, but germinated rapidly between 27 and 30 *C, reaching 95% germination in less than 12 hours. EL Effect of temperature on mycelial growth Both V . l e c a n i i strains 11-73 and 19-79 were able to grow between 5 and 30*. Highest growth rates for V . l e c a n i i 11-73 occurred between 20 to 27*C with a maximum of 3.80 mm per day at 23*C (Fig. la). V . l e c a n i i 19-79 responded to temperature similarly with maximum growth of 2.20 mm per day at 20*C (Fig. lb). Both M. anisopliae 83-82 and 275-86 grew between 10 to 30*C but not at 5 or 35*C. The optimum temperature for M. anisopliae 83- 82 was 25*C (6.30 mm per day; Fig. lc) and 27*C for M. anisopliae 275-86 (5.30 mm per day; Fig. Id) though in the latter case growth was similar at 23 and 25‘C. M. anisopliae 300-86 grew most quickly at 27 and 30’C with respective growth rates of 5.10 and 5.40 mm per (Fig. 2e) In contrast M. flavoviride 203-84 grew slowly with a maximum rate of 1.70 mm per day at 25*C. It failed to grow at 5, 10 or 35*C (Fig. 2f) 34 - TABLE 5. Estimated time needed for 50 or 95% germination of strains with high pathogenicity to Nephotettix virescens Estimated time to 50 Incubation temperature in °C Strain or 95% germination (hours) 10 15 20 23 25 27 30 Beauveria bassiana 110-82 GT50* >45 36.6 22.0 18.1 16.9 13.7 14.2 GT95** >45 >45 27.8 22.0 20.8 16.5 18.8 - 138-83 GT50 43.1 23.1 20.2 16.0 13.1 <12 Cl2 GT95 >45 30.5 26.4 20.1 17.4 16.0 13.9 235-85 GT50 44.8 22.5 19.5 15.3 14.3 12.9 11.6 GT95 >45 29.5 25.1 19.4 18.6 18.0 15.8 266-85 GT50 44.5 23.3 22.0 15.7 15.1 12.2 12.3 GT95 >45 28.5 28.0 22.3 21.7 15.9 17.3 268-86 GT50 >45 26.1 23.9 18.4 16.6 12.3 12.9 GT95 >45 33.4 30.2 25.3 23.8 16.0 17.4 269-86 GT50 45.0 25.9 24.3 17.9 15.6 12.4 12.9 GT95 >45 33.5 31.7 24.3 21.1 15.9 18.2 Metarhizium anisopliae 275-86 GT50 N 20.0 18.9 12.5 <12 <12 <12 GT95 N 24.4 22.4 17.0 14.3 <12 <12 * Estimated germination of 50% spores ** Estimated germination of 95% spores N No germination during 45 hours observation period 85 Fig. i Effect of temperature on mycelial growth of Verticillium lecanii (a) 11-73 (b) 19-79 , and Metarhi- .zium anisopliae (c) 83-82 and (d) 275-86 on Sabouraud dextrose agar. Points are means of four replicates with 95 °/0 confidence limits. 86 Fig. 2 Effect of temperature on mycelial growth of Meta- rhizium anisopliae (e) 300-86, M. flavoviride (f) 203-84, Paecilomyces farinosus (g) 104-82 and Trichothe- cium sp. (h) 213-85 on Sabouraud dextrose agar. Points are means of four replicates with 95/i confidence limits 87 Paecilom yces farlnosus 104-82 grew between 5 and 30X with highest rates at 20 to 27X and a maximum of 4.60 mm per day at 20*C (Fig. 2g). Trichothecium 213-85 was able to grow between 10 and 35X and grew most rapidly from 23 to 30X. It had the highest growth rates of all the studied strains reaching 10 mm per day at 25X (Fig. 2h). B. bassiana 110-82 and 138-83 showed similar patterns of growth (Figs. 3i and 3j>. They were both able to grow between 10 and 30X with highest rates between 23 and 30X but strain 138-83 grew more quickly with a maximum rate of 2.00 mm per day at 25X(c.f. 110-82 1.82 mm per day). B. bassiana 235-85 grew optimally from 20 to 30X with a maximum rate of 1.55 mm per day at 20X and a range of growth between 10 and 30X (Fig. 3k). B. bassiana 261-85, 266-85, 268-86, 269-86, and 270-86 had higher growth rates between 23 and 27X with maximum growth rates at 25X of 3.70, 3.30, 2.70, 1.90 and 1.80 mm per day respectively, and were able to grow between 5 and 30X (Figs. 31, 4m, 4n, 4o and 4p). Sporothrix insectorum 299-86 grew optimally between 23 and 27X with a maximum of 8.30 mm per day at 25X. Growth occurred from 5 to 30X (Fig. 5q> 12. Effect of water activity on germination and mycelial growth Influence of water activity on fungi germination The germination behaviour of M. anisopliae 275-86 was influenced by the water activity (aw of the media. Between 0.96 and 1.00 a w , * Fig. Mycelial growth (nun per day) Mycelial growth (mm per day) means of four replicates with 952 # Fig. 4 Effect of temperature Effectongrowthofof mycelial 4 Fig. Mycelial growth (min per clay) with 95/o confidence95/olimits.with euei asaa (m) (n) (o)Beauveria 266-85, 268-86, 269-86bassiana n (p)and 270-86. Points arefourreplicatesofmeans 0 9 Fig. 5 Effect of temperature on mycelial growth of Sporothrix insectorum (q) 299-86. Points are means of four replicates with 95% # confidence limits. 9 1 germination reached 100% in 36 hours at 23±1*C (Fig. 6). At 0.95 aw and below, germination decreased rapidly and at 0.92 only 15% germination was recorded while at 0.91 conidia failed to germinate. Conidia of B. bassfana 268-86 germinated optimally at 1.0-0.97 aw and 100% germination was obtained after 18 hours (Fig. 7). However, at 0.96 aw germination was dramatically reduced and after 18 hours only 54% of conidia had formed a visible germ tube (c.f. M. anisopliae 96% germination). Numbers of germinated conidia continued to decline with reducing and at 0.92 only 12% germinated even after 36 hours. At 0.91 and 0.90 aw , small numbers of conidia germinated after 36 hours (max. 7%). No M. anisopliae conidia germinated at these levels. Effect of water activity on mycelial growth M. anisopliae 275-86 grew most quickly between 0.98 and 0.99 aw levels with a maximum of 2.85 mm per day at 0.99 aw compared with 2.65 mm per day at 1.00 aw (Fig. 8). Growth rates decreased rapidly at aw levels below .98 aw ; at .95 aw the grow rate was 0.47 mm per day and at 0.91 aw no growth was recorded (Plate 4). Mycelial growth of B. bassiana 268-86, in response to varying aw was similar to that of M. anisopliae. B. bassiana grew optimally at 0.99 aw (3.5 mm per day) and growth was reduced at 1.0 aw (Fig. 9). Below 0.98 aw growth fell markedly and was 0.54 mm at 0.95 aw . The fungus grew very slowly at 0.92 aw (0.10 mm per day) and 0.91 aw (0.03 mm per day; Plate 5) 100 Water activity Fig. 6 The effect of water activity on conidial germination of Metarhizium anisopliae 275-86 after 18 ( *-- x ) , 24 (o-- o) and 36 (♦--♦ ) hours incubation at 23±1°C. Points are means of four replicates with 9 5 % confidence limits. 93 Fig. 7 The effect of water activity on conidial germination of Beauveria bassiana 268-86 after 18 (*---X), 24 (o--- o) and 36 (♦-- #•) hours incubation at 23^1* C. Points are means of four replicates with 95>s confidence limits. 9 4 Colony extension (mm per day) Fig. 8 The effect of water activity on mycelial growth activityonmycelial of water effect The of Metarhizium anisopliae 275-86 at 23-1° at275-86anisopliae ofMetarhizium Points C. are means of five replicates with 95V 95V confidence fivereplicates with of are means limits. 5 9 0.91 0.92 0. 9? 0.94 0.95 PLATE 4 Mycelial growth of Metarhizium anisopliae 275-86 on SDA after 30 days at 2 3il*C and water activities between 0.91 and 1.00 • 96 Colony extension (mm per day) 3.5 Fig. 9 The effect of water activity on mycelial growth onactivitymycelialof effectwater The 9Fig. 90 .1 .2 0.93 0.92 0.91 means of five replicates with 95 0 91 0,92 0 93 0 94 0.95 PLATE 5 Mycelial growth of Beauveria bassiana 268-86 on SDA after 30 days at 23 - lc and water activities between 0.91 and 1.00. 98 13. Storage experiments A. Influence of temperature on survival of dried aerial conidia Vacuum dried conidia survived poorly at 30*C (Fig. 10) and most strains were non viable after 100 days storage. Only P. farinosus 104-82 was viable after 140 days but less than 10% of spores were able to produce germ tubes. Conidia survived longer at 20*C (Fig. 11) and significant differences between strains were found. Trichothecium 213-85 and B. bassiana 261-85 maintained over 50% viability for more than 200 days and even after 300 days some 15% of spores germinated. At 5*C most strains survived better than at 20 *C (Fig. 12) Trichothecium 213-85 and B. bassiana 261-85 maintained viability well and more than 70% germinated after 200 days storage; V . l e c a n i i 19- 79 and P. farinosus 104-82 were intermediate with around 30% germination after the same storage period. After 360 days storage some 15% of Trichothecium 213-85 spores were viable, but B. bassiana 261-85 retained more than 50% germination after this time. Influence of drying method and temperature on germination and pathogenicity of stored aerial conidia Vacuum dried spores survived better than lyophilized ones at 5, 20, or 30*C. Lyophilized conidia stored at 30*C died quickly, and after 25 days storage all strains examined had less than 10% viable spores (Fig. 13). Vacuum dried conidia stored at 30*C survived a bit longer and after 50 days two strains B. bassiana 268-86 and 266-86, still 9 9 - Time (days) Fig.IQ Survival of vacuum dried conidia of Verticillium lecanii 11-73 (X------X ), _V. lecanii 19-79 (X----- ‘-X) Paecilomyces f arinosus 104-82 ( ©•----- © ) , Trichothecium sp. 213-85( 0- — —0 ) , Metarhizium anisopliae 83-82 ( «#— — —*8» )_M. anisopliae 260-85 ( ----- # ) , M . f lavoviride 259-85 ( X------X ) and Beauveria bassiana 261-85( * ------X ) stored at 30° C,- as indicated by germination on SDA- (points are means of three replicates Bars on the left are approximately the 9 5/< confidence intervals for different levels of mortality. 1 0 0 £ •HO +j £rd £ Di(U i—I rd •H •H £ UO Fig.11 Survival of vacuum dried conidia of VerticiIlium lecanii 11-73 (X------X ) , _V. lecanii 19-79 (X------X) Paecilomyces farinosus 104-82 ( © ------0 )-, Trichothecium sp. 213i-85( ©- — — 0 ) , Metarhizium anisopliae 83-82 ( •*— — — * )_M. anisopliae 260-85 ( ------<%> ) , M. flavoviride 259-85( X------X ) and'• Beauveria bassiana 261-85( X------X ) stored at 20 c, as indicated by germination on SDA- (points are means of three replicates Bars on the left are approximately the 95% confidence’ intervals for different levels of mortality. - 1 0 1 - 0c +> Crd *H 6 Di Fig. 12 Survival of vacuum dried conidia of Verticillium lecanii 11-73 (X------X ), _V. lecanii 19-79 (X------X) Paecilomyces farinosus 104-82 ( ©• — : --0 )•, Trichothecium sp. 213-85( ©- — —& ) , Metarhizium an'isopliae 83-82 ( +- — — * )_M. anisopliae 260-85 ( ------* ) , m . f lavoviride' 259-85 ( X------X ) and Beauveria bassiana 261-85{ X ------¥ ) stored at 5° c, as indicated by germination oh SDA- (points are means of three replicates) Bars on the left are approximately' the 95X confidence' intervals for different levels of mortality. - 1 0 2 - 100 Fig. 13 Survival of lyophilized conidia of Beauveria bassiana 110-82 (X------X ); _B. bassiana 235-85 ( G— — -0 ) , _B. bassiana 266-85 ( ^----- ) , 33. bdssiana 268-86 ( X ------X ) and Metarhizium anisopliae 275-86 ------X ) stored at 30° C, as indicated by germination on SDA (points are means of three replicatesl Bars on the left are approximately the 95% confidence intervals for different levels of mortality. 1 0 3 had more than 50% viability. However, spores then died rapidly and after 100 days all the strains had less than 10% germination. (Fig. 14). Of the studied isolates conidia of B. bassiana 268-86, 235-85 and 266-85 survived longest after lyophilization or vacuum drying when stored at 20*C (Figs. 15 and 16). Lyophilized conidia of B. b a s s i a n a 268-86, 266-86 and 110-82 were the better survivors at 5*C (Fig. 17) all of them survived even longer when were vacuum dried and stored at this temperature (Fig. 18) The strain which survived best after vacuum drying was B. b a s s i a n a 268-86 which maintained more than 50% germination after 369 days storage at 20 or 5*C. This strain also maintained viability well after lyophilization but only 20% of conidia were alive after 369 days at 20 and only 10% at 5*C. In contrast, the strain which survived least well was M. anisopliae 275-86 which failed to germinate after 100 days storage at any of the three studied temperatures irrespective of the drying method. Interestingly, B. bassiana 235-85 survived longer at 20 than at 5*C and its longest survival was obtained after vacuum drying and storage at 20*C (8% after 369 days; Fig. 16). Although fungal strains responded differently to the drying method and storage temperature, and hence should be analysed separately, an overall Anova analysis of germination with time, showed (P<0.01) that storage at 20 *C after vacuum drying was the best treatment to maintain survival. Results of pathogenicity bioassays to GLH adults, carried out before and at 180 and 369 days after storage treatments with strains which showed more than 20% germination are shown in Table 6. Comparison of LC50 values and 95% fiducial limits of B. bassiana 235- CO •H-M rd •H£ 6h tn0 H rd •HTS •H£ UO r Fig. 14 Survival of vacuum dried conidia of Beauveria bassiana 110-82(X------5< ) , _B. bassiana 235-85 ( ©— — -© ) , _B_. bassiana 266-85 ( — --- ) , J3. bdssiana' 268-86( ^ ------^ ) and Metarhizium anisopliae 275-86 ( *------X ) stored at 30° C, as indicated by germination on SDA (points are means of three replicates Bars on the left are approximately the 95% confidence intervals for different levels of mortality. - 1 0 5 - Fig.15 Survival of lyophilized conidia of Beauveria bassiana 110-82 (X-^----- X ), _B. bassiana 235-85 ( 0— — -Q ) , _B. bassiana 266-85 ( *8*----- ) , b . b£ssiana '268-86( X------* ) and Metarhizium anisopliae 275-86(*---- 1— * ) stored at 20° C, as indicated by germination on SDA (points are means of three replicates! Bars on the left are approximately the 95% confidence intervals for different levels of mortality. - 1 0 6 - Fig.] 6 Survival of vacuum dried conidia of Beauveria bassiana 110-82(X------X ) , JB. bassiana 235-85 ( ©— — -£> ) , _B_. bassiana 266-85( *8*------) , J3. bdssiana 268-86( X ------*= ) and Metarhizium anisopliae 275-86(X------X ) stored at 20 c, as indicated by germination on SDA (points are means of three replicates Bars on the left are approximately the 95% confidence intervals for different levels of mortality. - 1 0 7 - F i g . . 17 Survival of lyophilized conidia of Beauveria bassiana 110-82(X------X ) , _B. bassiana 235-85 ( ©— — -O ) , _B. bassiana 266-85 ( ------) , _B_. bdssiana 268-86( Sfc------^ ) and Metarhizium anisopliaa 275-86(*c------) stored at 5# c, as indicated by germination on SDA (points are means of three replicates] Bars on the left are approximately the 95% confidence intervals for different levels of mortality. - 1 0 8 - Fig. 18 Survival of vacuum dried conidia of Beauveria bassiana 110-82 (X------X ), JB. bassiana 235-85 ( ©— — -& ) ' j3. bassiana 266-85( *8-----— •* ) , JB. b&ssiana '268-86 ( Jfc------^ ) and Metarhizium anisopliae 275-86(5K------^ ) stored at 5° c, as indicated by germination on SDA (points are means of three replicates Bars on the left are approximately the 95% confidence intervals for different levels of mortality. - 1 0 9 - TABLE 6. Pathogenicity of fungal strains (aerial conidia) stored in dry conditions to adult Nephotettix virescens LC 95% fiducial 50 l i m i t s Strain Treatments S lo p e C o n id ia / + SE m lx lO Low er U pper Beauveria bassiana 235-85 Initial pathogenicity 1.40 0.52 3 .6 3 0 .7 3 + 0 .1 5 After lyophilization and 180 days storage (20°C) 1 .8 0 0 .7 2 4 .5 3 0 .7 8 + 0 .1 6 B. bassiana 266-85 In itia l pathogenicity 9 .3 1 2 .6 0 8 4 .6 0 0 .4 6 + 0 .1 2 After vacuum drying/180 d/5°C 9 .9 3 2 .2 1 2 3 .6 0 0 .3 8 + 0 .1 1 A fter vacuum drying/180 d/20°C 5 .0 5 1 .3 0 3 8 .4 0 0 .4 3 + 0 .1 2 B. bassiana 268-86 In itia l pathogenicity 0 .6 7 0.26 1.57 0.84+0.17 After lyophilization/180 d/20°C 0 .9 6 0 .3 6 2 .3 7 0 .7 7 + 0 .1 6 A fter vacuum drying/180 d/5°C 0 .9 7 0 .3 9 2.31 0.83+0.17 A fter vacuum drying/180 d/20°C 1 .1 0 0 .4 7 2 .5 1 0 .9 1 + 0 .1 8 After lyophilization/369 d/20°C 1 .4 0 0 .6 0 3 .2 4 0 .9 0 + 0 .1 8 A fter vacuum drying/369 d/20°C 0 .8 6 0 .3 7 1 .9 4 0 .9 4 + 0 .1 9 After vacuum drying/369 d/5°C 0 .5 1 0 .1 9 1.23 0.81+0.17 * M ortalities were determined after six days incubation in optimal relative humidity conditions 85, 266-85 and 268-86 conidia, before and after storage, indicate that pathogenicity to GLH adults was maintained during storage. C;. Influence of vacuum drying on germination of spore types Aerial conidia of B. bassiana 268-86 survived vacuum drying well at room temperature and viability was only reduced by about 10%. In contrast, submerged conidia and hyphal bodies survived vacuum drying poorly and final viabilities were less than 3% (Table 7). IL Influence of temperature and antibiotic addition on germination and pathogenicity of liquid stored spores At both 5 and 20*C, conidia survived longer than hyphal bodies. The inclusion of antibiotics reduced numbers of viable conidia, with the effect being most marked at 20°C. At 5*C; conidia survived well and over 60% of both aerially and liquid produced spores were viable after 180 days (Fig. 19). In contrast, only 20-30% of hyphal bodies were viable at the same time. At 20*C, aerial conidia, in the absence of antibiotics, survived well and 57% were viable after 180 days (Fig. 20). The inclusion of antibiotics reduced viabilities to 35%, and these levels were similar to those of submerged conidia where the detrimental effect of antibiotics was less marked. Hyphal bodies survived poorly at 20'C and no viable spores were observed 180 days after storage. Results of pathogenicity bioassays to GLH adult carried out before and after 180 days storage are shown in Table 8. A comparison of LC50 values and 95% fiducial limits of aerial conidia, submerged - 1 1 1 - TABLE 7. Effect of vacuum drying on survival of Beauveria bassiana 268-86 spores as indicated by germination on Sabouraud dextrose agar Germination before drying Germ, after drying* % + SE % + SE Submerged conidia 97.33 0.66 1.88 0.91 Hyphal bodies 99.00 0.23 2.22 0.64 Aerial conidia 99.11 0.35 91.22 1.95 * Figures are mean of nine replicates - 1 1 2 - % Fig.19 Survival of Beauveria bassiana 268-86 spores stored in water at 5°C, submerged conidia( ©— — -© ), submerged conidia plus antibiotics ( *------X ), aerial conidia (X------X )f aerial conidia plus antibiotics( 0------0 ) hyphal bodies(YG)(X------X), hyphal bodies (YG) plus antibiotics( *------) , hyphal bodies (SL ) ( — -* ) , hyphal bodies (SL ) plus antibiotics ( ------5# ) , as indicated by germination on SDA (points are means of three replicates). Bars are approximately the 95/oconfi dence intervals for different levels of mortality. - 1 1 3 - Fig. 20 Survival of Beauveria bassiana 268-^36 spores stored in water at 20°C, submerged conidia( G- — — 0 ), submer ged conidia plus antibiotics ( x------X )i aerial conidia (X------X ), aerial conidia plus antibiotics (©---- —© ) hyphal bodies(YG)(X------X ), hyphal bodies (YG) plus antibiotics ( #------) , hyphal bodies(SL ) ( — — *8» ) , hyphal bodies (SL ) plus antibiotics ( X------X ), as indicated by germination on SDA (points are means of three.replicates). Bars are approximately the 95^confi- dence intervals for different levels of mortality. - 1 1 4 - » TABLE 8. Pathogenicity of Beauveria bassiana (268-86) spores stored in liquid conditions to adult Nephotettix virescens* LC-fl 95% fiducial ^ limits Spore Treatment Slope type Conidia/ + SE mix 10 Lower Upper Aerial conidia Initial pathogenicity 1.60 0.61 4.10 0.75+0.15 After 180 days storage in water (5°C) 1.10 0.40 2.76 0.74+0.15 After 180 days storage in water (20°C) 1.25 0.54 2.80 0.94+0.19 Submerged conidia Initial pathogenicity 0.50 0.20 1.05 0.95+0.20 After 180 days storage in water (5°C) 0.69 0.32 1.46 1.07+0.22 After 180 days storage in water (20°C) 0.61 0.26 1.33 0.97+0.20 Hyphal bodies Initial pathogenicity 0.53 0.22 1.18 0.96+0.20 After 180 days storage in water (5°C) 0.76 0.30 1.77 0.85+0.17 * Mortalities were determined after six days incubation in optimal relative humidity conditions # - 1 1 5 - # conidia and hyphal bodies demonstrated virulence was maintained during storage. 14. Effect of SPA subculturing on conidial production Continuous subculturing on SDA reduced the conidial production of both B. bassiana 268-86 and M. anlsopllae 275-86. B. bassiana 268-86 SSI 81, 96 and MSI produced 6.96, 6.11 and 5.98X10^* conidia per mm2 respectively, when first cultured on SDA (IMP) after removal from liquid nitrogen. After ten SDA subcultures (10 MP), equivalent spore yields were 2.07, 2.94 and 5.39X10^ conidia per mnr-'respectively (Fig. 21 ). For M. anisopliae 275-86 SSI 1, SSI 3 and MSI, yields after one subculture were 3.86, 2.81 and 5.48X10s conidia per mra2 respectively. After 10 subcultures yields were reduced with only 1.44, 1.94 and 1.17X10s conidia per mm2 being produced (Fig. 21). 15. Influence of ultra violet light on the survival of B. bassiana 268-86 aerial conidia. submerged conidia and hyphal bodies Hyphal bodies and submerged conidia of B. bassiana 268-86 survived ultra violet light irradiation (UV.) better than aerial conidia. After 5 minutes exposure to a UV. source (253.7 nm) only 28% of submerged conidia and 50% of hyphal bodies were viable. Aerial conidia were more sensitive and only 14% retained viability at this exposure time (Fig. 22). After 8 minutes exposure, all aerial conidia - 1 1 6 - Number of conidia per mm (XlO Fig* 21 Effect of oneof Effect Fig*21 Beauveria bassiana 268-86 Metarhizium anisopliae 275-86 anisopliae Metarhizium 268-86 bassiana Beauveria S8 SI6 S SI SI MSI SSI3 SSI1 MSI SSI96 SSI81 obtained from the mean of threereplicates.of the frommean obtained anisopliae 275-86. Bars represent 9 5 % confidence limits confidence % 5 9 represent Bars275-86. anisopliae on conidial production of multi and singlespore and ofmulti production conidial on sltso euei asaa288 and268-86Metarhizium bassiana Beauveria ofisolates -117- SDA subculturesandSDA ten 100 X.o N co •H -P Cd •H ep QJ Di i—I d •HT5 •Hc uo 5 10 15 Time (minutes) Fig. 22 The effect of ultraviolet light on survival of Beauveria bassiana 268-86 spores: submerged conidia (X------X ) aerial conidia (♦------41 ) and hyphal bodies ( 0------0) . Points are means of three repli- ates. Bars are approximately the 95/^confidence intervals for different levels of mortality. -1 1 8 - died while submerged conidia and hyphal bodies still had more than 107* germination. Submerged conidia survived for the longest time although after 10 minutes exposure, spore viability was less than 5%. 16. Measurement of conidial viability by the optical brlghtener technique When conidial suspensions of B. bassiana 268-86 and M. anisopliae 275-86 containing defined numbers of viable conidia were examined by the optical brightener and the SDA slide technique, the two methods gave similar estimates of conidial viability (Figs. 23 and 24) When six B. bassiana strains were examined, the agar method again gave similar results to the fluorescent method (Table 9). This contrasted with the result for M. anisopliae , where both methods gave similar viabilities for the variety a n i s o p l i a e strains but consistently higher results were obtained by the fluorescent method for variety m a j u s isolates (Table 9). 17. Viability of stored conidia as measured by the fluorescent and the SDA slide methods Both fluorescent and SDA methods gave similar conidial viabilities for four lyophilized or vacuum dried B. bassiana and one M. anisopliae spore powders maintained at 5*C during 451 days storage (Tables 10 and 11). This data suggests that the fluorescent technique may be a reliable method for measuring viabilities of conidia stored at 5*C 1 1 9 20 40 60 80 100 Expected viability ( ° /* ) Fig. 23 Dispersion diagram of expected and observed viabilities of Beauveria bassiana 268-86 using agar Q and fluorescent A techniques. - 1 2 0 - Fig.24 Dispersion diagram of expected and observed viabilities of Metarhizium anisooliae 275-86 using agar □ and fluorescent techniques /\ - 1 2 1 - TABLE 9 Conidial viabilities of Beauveria bassiana and Metarhizium anisopliae using Sabouraud dextrose agar and fluorescent techniques % germination Logit transformation Strain LSD* 4 df Fluorescent SDA Fluorescent SDA Beauveria bassiana 110-82 97.04 90.99 3.341 2.265 1.098 261-86 (fresh culture) 93.68 92.58 2.626 2.465 0.510 261-86 (old culture) 34.90 33.66 -0.616 -0.671 0.262 266-86 91.24 91.73 2.294 2.354 0.905 268-86 98.72 99.00 4.021 4.195 0.417 269-86 92.03 90.71 2.392 2.232 0.258 Metarhizium anisopliae 36-79 87.55 86.45 1.917 1.823 1.092 83-82 92.03 92.14 2.392 2.406 0.405 101-82 90.96 89.75 2.260 2.128 0.756 135-82 var. majus 33.10 0 -0.696 0 0 137-83 var. majus 57.02 11.29 0.280 -2.023 0.2898 170-83 var. majus 60.57 4.31 0.425 -2.995 0.5790 189-83 88.38 79.34 1.993 1.328 0.5332 * Least significant difference between the means (p <^0.05) - 1 2 2 - 0.02 0.10 3DF* 7.0 7.0 7.6 FL FL SDA -2.54 -2.43 0.15 9.6 9.6 9.3 FL FL SDA 369 369 days days 451 SED -2.19 -2.19 -2.22 285 days 285 0 0 0 25.0 25.0 20.0 -1.09 -1.09 -1.36 28.3 -0.92 49.0 -0.03 Percentage Percentage viability after: 180 days 0 0 28.6 49.3 -0.90 -0.02 1.04 0 0.86 8.0 0 74.0 70.6 -2.510 * Standard error of difference of means after lyophilization and storage at at 5°C and storage after lyophilization FL SDA FL SDA FL SDA 1.02 0 0.96 0 73.6 11.0 72.6 -2.060 Logit Logit % Logit Logit Logit Strain days 95 275-86 % 268-86 % 110-82 % 266-86 % 235-85 TABLE 10. TABLE 10. methods agar dextrose (SDA) Sabouraud and using fluorescent (FL) Viabilities conidia fungal of Beauveria bassiana Beauveria Metarhizium anisopliae Metarhizium -123- m 0.04 0.12 0.10 0.11 SED 3DF* days 6.6 6.6 0.3 0 0 451 -2.62 -2.62 -4.9 0.25 0.39 9.0 59.6 -2.26 SDA FL SDA days 0.73 FL 12.0 67.6 -1.95 after: 1.66 0.25 0 56.3 82.6 SDA 1.59 0.28 0 83.3 57.0 2.02 0.32 5.3 0 58.0 88.3 SDA FL -2.80 Percentage Percentage viability 180 days 285 days 369 0 0 2.35 5.6 0.38 0 FL 91.6 59.6 -2.75 4.6 1.21 2.86 1.3 12.6 77.3 95.0 -2.92 -1.90 -3.55 * Standard error of difference of means after vacuum drying and storage at at 5°C drying and storage vacuum after 1.44 5.0 1.3 3.26 FL SDA -2.85 96.6 14.3 81.0 -3.55 -1.76 % Logit % Logit % Logit Logit Logit % Strain days 95 275-86 110-82 % 268-86 235-85 266-86 TABLE TABLE 11. methods agar dextrose (SDA) Sabouraud and using fluorescent (FL) Viabilities conidia fungal of Metarhizium anisopliae Metarhizium Beauveria bassiana Beauveria -124- (II, 16). In contrast to the data obtained at 5*C, when the same isolates were stored at 20*C, after some time (which varied between isolates) the fluorescent method showed a constant viability in successive records, while the SDA method showed a decreasing spore viability (Tables 12 and 13). This data indicates that the fluorescent method should be used with caution with some stored conidia. 18. Differentiation of B. bassiana 268-86 spore-types B. bassiana 268-86 was able to produce 4 types of spore differing in size and morphology dependent on media and cultivation method. Aerial conidia produced in semi-solid culture (cereal grains) or SDA were subsphaerical, regular in size and the smallest of spores examined with a mean diameter of 2.33±0.20 pm (n>20; Plate 6). Submerged conidia produced in defined media (TK1) were also subsphaerical, regular in size, but larger than aerial conidia with a mean diameter of 3.0±0.23 pm (n>20; Plate 7). Hyphal bodies (blastospores) produced in complex media (Yeast plus glucose or Sabouraud liquid) were relatively large, usually cylindrical in shape, and irregular in size with a mean length of 8.10±2.27 pm (n>20; plate 8). A 4th spore type was observed in both complex and defined liquid media, with an size intermediate between submerged conidia and hyphal bodies. The mean diameter was 4.8±0.44 pm (n>20) and the morphology was similar to that of submerged conidia (Plates 7 and 8); these spores were described as "conidia like" spores. -125- SED 3DF* 0.06 0.20 -.05 1.6 1.6 0 451 days 451 0 0 0 0.02 -4.930.02 0.22 20.3 20.3 0 50.6 50.6 0 -3.84 -3.84 -5.30 0.18 -1.34-5.30 0.3 0 0 SDA FL SDA 20.3 -4.93 -5.30 -1.35 -5.30 369 days 369 1.6 1.6 0.04 19.3 51.0 -3.84 -1.09 -3.84 0 0 13.0 days 36.0 -5.30 -0.83 -1.87 -5.30 285 1.3 0.06 16.0 19.3 51.6 -3.47 -1.63 -1.41 0 0 0 Percentage viability viability after: Percentage 40.3 34.3 -3.88 -5.30 -5.30 -0.67 1.3 FL SDA FL SDA FL 0 0.06 41.3 21.0 51.6 -3.49 -1.31 -4.02 9.3 0.70 0.68 0 SDA 66.6 48.0 66.6 -0.08 -5.30 -2.23 95 days95 180 days Standard error of difference of means * after lyophilization and storage at 20°C FL 1.3 8.6 0.77 0.64 68.6 49.6 65.6 -2.31 -0.01 -4.02 % Logit Logit % Logit % Logit % % Logit • • Viabilities of fungal conidia using fluorescent (FL) and Sabouraud dextrose agar (SDA) methods Strain 110-82 275-86 268-86 TABLE 12 Beauveria bassiana Beauveria i i 235-85 1 1 266-86 Metarhizium Metarhizium anisopliae NJ cr> 126h-> 0.07 0.07 0.06 SED 3DF* 0.10 0 0 0 25.0 -1.08 0.16 -5.30 -5.30 days 451 FL SDA 2.26 5.3 0 0.10 91.0 52.6 -2.79 8.6 0 0.40 10.6 56.6 -5.30 -2.31 -2.39 2.43 5.3 0.09 15.6 52.3 92.3 -1.65 -2.79 1.11 0 0 15.6 12.0 SDA FL SDA 75.3 -5.30 -1.66 -1.95 FL 2.42 5.3 0.12 0 15.6 53.0 92.0 -1.65 -2.79 SDA 2.23 4.0 Percentage viability after: Percentage viability 0.06 0 34.6 51.6 90.6 -3.06 -0.67 -5.30 2.43 5.0 4.6 0.20 55.0 92.3 35.0 -2.86 -2.92 -0.61 2.34 5.3 0.91 10.3 71.6 91.6 34.3 -2.12 -0.64 -2.79 95 days95 180 days 285 days 369 days after vacuum drying and storage at 20°C * Standard error of difference of means FL SDA FL 1.05 2.93 5.0 11.3 74.3 95.3 37.0 -2.03 -0.52 -2.85 % % Logit % Logit Logit Logit Logit . . Viabilities of fungal conidia using fluorescent (FL) and Sabouraud dextrose agar (SDA) methods Strain 110-82 % 266-86 275-86 268-86 % TABLE 13 Beauveria bassiana Beauveria Metarhiziura anisopliae Metarhiziura i i 235-85 -127-NJ PLATE 6 Beauveria bassiana 268-86 aerial conidia (Normal light, X250) "Conidia like" spores Plate 7 Beauveria bassiana 268-86 spores produced in submerged culture showing Submerged conidia and "Conidia like" spores, (X2 50). -129- "Conidia like" spores Hyphal bodies PLATE 8 Beauveria bassiana 268-86 spores produced in submer ged culture showing Hyphal bodies and "Conidia like" spores, (X2 50). -130- DISCUSSION As living entities, entomogenous fungi are affected by environmental factors. In their review, Hall and Papierok (1982) described temperature and relative humidity (rh) as major abiotic factors affecting fungal pathogenicity, although other factors such as sunlight, rainfall, wind, pH and water pollution may also be important. Fungal pathogenicity itself is a rather complex biological phenomenon which depends on many factors. In the last five years extensive research has attempted to identify the major biotic factors affecting fungal pathogenicity. This knowledge could be used as a basis for selection and improvement of fungal strains in the laboratory (Drummond, 1986; Samuels, 1986; Isaac, 1988). This discussion will be restricted mainly to the relationship between the main abiotic factors encountered in rice crops and the biological characteristics of fungal strains which showed some potential for the control of N. virescens in the controlled conditions of the laboratory. Influence of temperature on germination and mycelial growth Conidial germination and mycelial growth are affected by temperature and relative humidity. Gillespie (1984) considered that conidial germination rates are of great importance in fungal infection, and rapid germination may allow the fungus to establish infection before conditions become unfavourable. Rapid germination can sometimes be correlated with pathogenicity to a determined target -1 3 1 - pest, e.g. mutants of M. anisopliae exhibiting rapid germination were hypervirulent towards Culex pip tens when compared to the wild type (Al-Aidross and Roberts, 1978). Similarly, most virulent isolates of V. l e c a n i i to M acrosiphonie 11a sanborni germinated rapidly i n v i t r o (Hall, 1984) . Drummond e t a l . , (1987) inoculated whitefly scales with different strains of V. lecanii. After 16 hours at high humidity 095%) scales were then transferred to 70% rh, before being returned to high humidity. Under such a regime one strain (K) was virtually non pathogen ic, but another strain (A) maintained its virulence. The authors suggested that this was possibly due to the higher i n s i t u germination rate of strain A, which enabled it to penetrate rapidly, or that strain A was able to survive the adverse humidity period and continue growing when humidity was increased. Mycelial growth rates may also be correlated with virulence, Samuels e t a l . t (1988) found a significant correlation between rapid radial growth rate and pathogenicity of M. anisopliae strains to N. l u g e n s . However, a similar correlation was not observed in the case of V . l e c a n i i isolates pathogenic to M. sanborni (Jackson e t a l . , 1985) . The relationship between germination, mycelial growth and the pathogenicity of studied fungal strains will be discussed in the section on Insect-fungus interactions. As a result of this study it was found that fungal strains varied greatly with respect to conidial germination rate and mycelial growth at different temperatures (II, 11). V . l e c a n i i 11-73 grew optimally at 23 *C while V . l e c a n i i 19-79 did so at 20*C, and M. anisopliae 83-82, 275-86 and 300-86 grew optimally at 25, 27 and 30*C respectively. Similarly, most B. bassiana strains grew optimally at 25 *C, but B. b a s s i a n a 235-85 showed the fastest growth at 20-23*C. Thus, each strain can respond differently to temperature and fungal strains should be considered as separate entities and generalizations not made about species. The characterization of strains by germination and mycelial growth profiles at different temperatures is an important step in the understanding of their ecological behaviour. Such information allows the selection of isolates capable of rapid growth at the temperatures likely to be encountered in the field. Influence of water activity on germination and mycelial growth Relative humidity (rh) is probably the most critical environmental factor influencing fungi; saturated or near saturated air or a water film is necessary for spore germination in the vast majority of entomogenous fungi (Hall and Papierok, 1982). In a study of eight soil fungi, Magan (1988) found marked differences in the ability to germinate and grow on media with modified osmotic potentials. He also found a strong interaction between temperature and the range of osmotic potential which permitted conidial germination, as shown previously for grain fungi (Magan and Lacey, 1984a). Several researchers have found that for Deuteromycete pathogens of terrestrial insects, the lower limit for spore germination is in the region of 92-93% rh (Walstad e t a l . , 1970; Ferron, 1981; Milner and Lutton (1986) demonstrated the importance of rh to aphid infection by V. lecanii. Disease transmission was maximal in the presence of free water, less at 100%, still less at 97% rh and absent at 93% rh. Gillespie and Crawford (1986) compared germination and mycelial growth of B. bassiana, M. anlsopliae, Paecilom yces spp. and V. -133- l e c a n i i on SDA containing defined concentrations of glycerol, providing water activities (aw) from 0.90 to 1.00 (equivalent to 90- 100% rh). They found the lower limit for germination and mycelial growth was about 0.92 aw . This method is easy to follow and is preferable to the use of saturated salts recommended by other researchers (Scott, 1957; Walstad e t a l . „ 1970). Results from this study indicated that more M. anisopliae 275-86 conidia germinated at 0.93 aw than did those of B. bassiana 268-86. Below this point, the two strains behaved similarly and only small numbers of spores germinated, with a slightly higher number for B. b a s s i a n a 268-86. In contrast, mycelial growth of B. bassiana 268-86 and M . a n l s o p l i a e 275-86 were similar (II, 12, B), with mycelial growth being markedly reduced for both strains below 0.97 a w and small differences of 0.01 aw resulting in a big difference in mycelial growth. To compare the response of fungi to varying water activity it is suggested that strains should be compared on the basis of both germination and mycelial growth. Storage Couch and Ignoffo (1981) recommended that bioinsecticides should be capable of storage up for 18 months to service agricultural markets. Clerk and Madelin (1965) found that conidia of M. anisopliae could be stored at high rh without great loss of viability. However, this is not practical on a large scale because of the likelihood of contamination. Ward and Roberts (1981), stabilized B. bassiana conidia with clays at 26*C and found that 70-78% were still viable after 12 months, in comparison to only around 6% of unformulated conidia. When vacuum dried conidia of B. bassiana, M. anisopliae, M. -1 3 A r flavoviride, Trichothecium sp., V . l e c a n i l and P. farinosus were stored at 5, 20 and 30 *C, high inter and intraspecific variability was observed in spore survival (II, 13, A). This again, emphasises that each fungal strain should be considered separately. In general most strains survived longer at 5 than at 20 or 30*C, and B. bassiana 261- 85 survived best with more than 50% viability after 360 days storage at 5*C. Roberts (1980) found variation in the survival of M. anisopliae strains stored at 37, 26, 19 and 4*C. Strain variation in B. bassiana with respect to pathogenicity (Gillespie, 1984) and sporulation in submerged culture (Gillespie unpublished observations) is well documented but has not been reported for survival of dry stored conidia. In this study, vacuum dried B. bassiana 268-86 conidia survived for 451 days at 20*C (25% germination) while three other B. b a s s i a n a isolates were non viable at this time. It is therefore important that conidial survival is considered when selecting isolates for development as myco insec tic ides as poor storage characteristics are often quoted as a limitation on commercialization (Hall and Papierok, 1982) Lyophilization is often used to preserve microorganisms. Fargues et al., (1979) lyophilized hyphal bodies of B. bassiana in skimmed milk and glycerol and obtained a spore viability above 90%. The preparation stored well at 5*C, but poorly at 20 or 30*C. In the present study (II, 13, B), lyophilization of conidia was compared with vacuum drying at room temperature, and strains stored at 5, 20 and 30 *C. Strains varied both in their ability to survive the drying process and in their subsequent longevity. Survival at 30*C was poor compared to 20 or 5*C and vacuum drying resulted in improved longevity of stored conidia both at 20 and 5 ‘C, compared with lyophilizatlon. Surprisingly one Isolate of B. bassiana (235-85) survived for longer at 20 than at 5*C and 28% of lyophillzed and 32% of vacuum dried conidia were still viable after 180 days storage compared with 0% viability after the same time at 5*0. It is likely that poor conidial survival at low temperature results from the cessation of metabolic activity at 5*C; B. bassiana 235-85 grows well at 20 to 25*C but not at 5*C (11,11). B. bassiana 268-86 was the best strain with respect to storage and more than 60% of vacuum dried conidia were still viable after 369 days storage at 20 or 5*C. The harmful effect of lyophilization compared with vacuum drying, might be because the latter technique is more accord with the real situation and avoids the freezing process; many of the studied isolates originate in the tropics where temperatures below freezing do not occur. It was also found that submerged conidia and hyphal bodies survived the vacuum drying process poorly while aerial conidia only lost around 10% germination (II, 13, C). Thomas e t a l . , (1986) studied the wall composition of these three spore types and observed a warty brittle outer layer in the walls of aerial conidia which was absent in hyphal bodies and present to reduced extent in submerged conidia. Fargues (unpublished observation) observed morphological differences between aerial and submerged conidia as well. It is logical to expect that this extra outer layer confers protection to aerial conidia and makes them more resistant to the dessication occurring in the vacuum drying process, as well as other environmental factors. Thus, it is reasonable to expect that aerial conidia should be more resistant to decaying environmental factors than submerged conidia and those in - 1 3 6 - turn more resistant than hyphal bodies. Submerged and aerial conidia survived longer than hyphal bodies when stored in water suspensions at 5 or 20 *C, and their performance was better at 5 compared with 20 *C, such results agree with the concepts expressed before. Addition of antibiotics to suspensions impaired germination of the three spore types and this effect was more pronounced at 20 than at 5*C, therefore storage of B. bassiana 268-86 spore types could be feasible in water suspensions at 5*C. Roberts (1980) studied both viability and virulence in stored M. a n i s o p l i a e strains, and concluded that a direct correlation did not always exist between high viability and virulence. Some strains retained high viability after storage but did not maintain their virulence, while in others the converse was true. In this study, the virulence of stored B. bassiana conidia was similar to that of newly produced spores (II, 13, B and C), meaning that the methods of storage used did not impair virulence and only the viability of spores decreased along time. Effect of germicidal UV llgt A unexpected result was obtained when spore suspensions of B. b a s s i a n a 268-86, aerial conidia, submerged conidia and hyphal bodies were compared for resistance to germicidal UV light (II, 14), in that submerged conidia and hyphal bodies survived longer than aerial conidia. (Fargues, umpublished observation) also obtained similar results when he compared hyphal bodies and aerial conidia of several fungi. One possible explanation for increased hyphal body and submerged conidia survival is that they might be binucleate. To study this possibility the specific nuclear dye (Hoescht 33258) was used to -B7 stain submerged conidia and hyphal bodies (Kangatharal ingam and Ferguson, 1984), but all spores shown to be uninucleate. Thus, the increased survival of hyphal bodies and submerged conidia is difficult to explain. It would be interesting to determine if these spores also have increased resistance to sunlight and it is suggested that such experiment be performed. Determination of conidial viabilities by the optical brightener technique A different kind of dye was used to differentiate dead and alive fungal spores (II, 16) and the results suggested that the use of bistriazinyl amino stilbene (0.05% Tinopal BOPT) may be a useful method for determining conidial viabilities of entomogenous fungi from fresh cultures, especially in industrial scale production. The correlations obtained between the rapid brightener method and the germination technique when applied to fresh B. bassiana or M. a n i s o p l i a e spores were encouraging. Observations suggest the method is similar to that described by Hutcheson e t a l . (1988), but the authors did not quote the chemical used describing it by its trade name "Viablue". The observations made on M . anisopliae var majus spores were interesting as viability determinations with the brightener technique suggested spores were viable yet they failed to germinate on SDA. It is possible the conidia were dormant and perhaps only germinate in response to cuticular components of the host insect, the Rhinoceros beetle O ryctes rhinoceros, when first produced M. anisopliae var m a j u s conidia are highly viable and some 98% germinate after incubation on SDA (R.A. Hall, unpublished observation). Within a few days, sporulation increases and the ability - 1 3 8 - of spores to germinate falls, there can be little ecological advantage in producing spores which survive only a few days whereas dormant conidia which germinate only in contact with the host cuticle would be benefical to the fungus. Indeed, one strain of M. anisopliae var. m a j u s survived for 21 months in soil at 19*C without loss in pathogenicity (Farges and Robert, 1985). A good correlation was observed between viability assessments obtained with the brightener and germination methods for conidia stored at 5*C (II, 17). However, at 20*C the correlation broke down after 95 days with lyophilized conidia and 180 days with vacuum dried conidia and the brightener method consistently gave higher viabilities. This perhaps indicates the conidia were alive but incapable of germination, possibly due to the exhaustion of nutrient reserves. Thus, the brightener method may overestimate the viability of spores from old cultures. Since spores must germinate to infect insects or to multiply, a spore incapable of germination is of little use. Thus, this overestimation of capacity is a disadvantage of the brightener method. However, where a viability assessment of dormant spores is required, as is possibly the case with M. anisopliae var m a j u s isolates, the technique could be useful to determine viability of fresh cultures and should be tried on other fungi where spore dormancy is a problem. Production of different types of spores A important characteristic of B. bassiana is the possibility of producing different sporetypes by manipulation of the culture media. B. bassiana produce aerial conidia on semi-solid media and hyphal bodies in submerged culture, and some strains conidiate in liquid culture. (Thomas et al, 1986; Bartlett and Jaronski, 1988; Gillespie, unpublished observation). In this study B. bassiana 268-86 was found to produce aerial conidia in semi-solid media, hyphal bodies in complex liquid media and submerged conidia in defined liquid media (II, 18). In addition, a fourth spore type intermediate in size between submerged conidia and hyphal bodies, was observed in defined or complex media. Most of the studies in the available literature have been performed on aerial conidia or hyphal bodies with very few on submerged conidia. According to Farges (unpublished observation) there are subtle differences between aerial and submerged conidia. In bioassays carried out in the present study, which are discussed later submerged conidia proved to be the most pathogenic spore type assayed against adult GLH, therefore it is an exciting possibility to go further in the study of this spore type. - 1 4 0 - SECTION III INSECT-FUNGUS INTERACTIONS MATERIALS AND METHODS 1_. General /L Production of rice plants Rice plants were grown in glasshouses. Seedlings were grown in vermiculite in seed trays (32X22X5 cm) at a minimum of 21 *C. After eight days, when they were about 3 cm high, they were transplanted to 12.7 cm diam polyethylene pots (four plants per pot) placed in dishes (11.0 cm diam.) which were filled with water daily. Potted plants were used to feed insects inside perpex cages. From time to time, dead plants were removed from cultures and cages cleaned with detergent solution. Pots were filled with compost detailed in Table 14. Irrigation of plants was frequent either with deionised water, or tap water acidified with concentrated sulphuric acid (0.11 ml per litre). Addition of acid was needed to lower the pH of the compost as it was found that higher pH's caused chlorosis, probably because at higher pH's iron was unavailable or not absorbed by roots. A solution containing 160 ppm N, 40 ppm P, 160 ppm K, 20 ppm Mg and 4 ppm Fe obtained by dilution of a stock solution (Table 15) was used to water plants from one week after potting. After 6-10 days, TABLE 14. Compost used for rice cultivation Ingredient Rate per bushel Irish sphagnum moss peat 0.037 m3 Ammonium nitrate 13 g Potassium nitrate 28 g Superphosphate of lime (18.5% 55 g Dolomite limestone 50 g Frit WM 255 11 g Ferrous sulphate 1 g pH approx. 4.8 - 1 4 2 - TABLE 15. Composition of rice liquid feed Salt Concentration (g per litre) Potassium dihydrogen orthophosphate 17.6 Potassium nitrate 28.7 Ammonium nitrate 35.6 Magnesium sulphate (hydrated) 20.3 Ferrous sulphate (hydrated)* 2.0 Made as separate solution - 1 4 3 - pots were submerged in a nutrient solution containing 80 ppm N, 20 ppm P, 80 ppm K, 10 ppm Mg, 4 ppm Fe, contained in 18 cm diam. pots and maintained at a minimum temperature of 25*C and 70-90% rh. Insect rearing and maintenance for bioassav Green leaf hoppers, N ephotettlx virescens (GLH) were obtained from Professor Claridge, University College, Cardiff. They were reared on rice, variety TNI. Plants were trimmed to a height of 30 cm, and the compost surface covered with plaster of Paris to reduce insect infection by opportunistic soil fungi such as Conidiobolus coronatus (Gillespie, 1986). Galvanised iron trays (85X52 cm) contained rice plants which were covered by Perpex cages (80X53X48 cm) at 25±1*C, 70-80 rh and a 16 hour photoperiod. Each cage had a front opening (38X30 cm) closed with a "Velcro" edged muslin flap. In the rear of each cage there were two holes (10 cm diam), covered with fine cotton cloth, adpressed to which were two 4.5 cm diam. plastic pipes, carrying an air supply provided by an electric fan. This created a small positive pressure within the cages, which reduce humidity and helped to prevent unwanted insects from contaminating cultures. To produce insects for experiments, adult insects were allowed to oviposit on rice plants for two to three days. Then plants were shaken to remove the insects, and transferred to hew perpex cages and allowed to develop with new plants being provided as necessary. Under these conditions adult insects were produced in five to seven weeks. After treatment with fungal spores, insects were placed on rice plants grown individually in polyethylene pots <7.2 max. diam) containing compost covered with plaster of paris. Plants, which were about 20 cm high after 6-8 weeks, were trimmed to a height of 10 cm, stripped of dead leaves and contained in clear plastic propagators (Stewart plastic Pic, Croydon, UK; size 3) and maintained at 25±1*C. Plastic propagators were adapted to provide three levels of relative humidity Humidity inside each type of cover was assessed using a Squirrel meter/logger (Grant Instruments, Cambridge UK) set to record relative humidity (rh) levels every 15 minutes. Three types of propagators were used to provided varying rh levels (Plate 9): - Closed: without ventilation holes. It provided a constant rh of 100%. - Ventilated: with a 4 cm diam. hole on the propagator top provided with a mesh sleeve which was tied around the stem and leaves of the plant. This type provided a rh of 92- 96% considered suboptimal for the infection process and mimicking the rh conditions of the rice canopy. - Open: with four, 4 cm diam. holes on the walls and one on the top of the propagator which provided rh of 70-87% considered too low for fungi infection Covered plants bearing insects were then maintained in plant saucers and these filled with wat.er daily. -1 4 5 - TABLE 16. Relative humidities provided by closed, ventilated and open covers Type of cover Mean daily rh* Overall mean + SE Closed 100 100 Ventilated 92.06 93.89 92.90 92.30 * 95.75 93.62 0.37 93.60 94.53 93.59 94.00 Open 86.35 77.42 79.18 83.52 84.50 80.19 1.97 • 71.50 70.65 85.50 83.10 Figures are means of 96 records taken at 15 minute intervals - 1 4 6 - PLATE 9 Types of plastic propagators used to provide three levels of rh. -147- C^. Bloassav of fungi to determine pathogenicity to N ephotettix v i r e s c e n s I) Preparation and standardization of spore suspensions Spore (conldtal and hyphal body) suspensions were produced and standardized as described previously (II, 1, E, G and H) and where possible used immediately. Where this was impracticable suspensions were stored In darkness at 5±1 C° for a maximum of two weeks. II) Assay procedures Two methods were employed to assess the pathogenicity of fungal isolates to adult and immature green leafhopper (GLH). a) Direct immersion in spore suspensions This technique was adapted from that described by Hall (1976) and Gillespie (1984). Conidial or hyphal body suspensions were prepared in distilled water containing 0.05% Triton X-100, and standardized. Eatches of 1-3 day old adult, GLH were caught using an aspirator and contained in McCartney bottles. Insects were then immersed in 10 ml aliquots of 0.05% Triton X-100, or similar suspensions containing fungal conidia, for 10 seconds and then poured into a filter paper- lined Buchner funnel (Whatman GFA, 4.7 cm diameter; funnel, 4.7cm diam). Where necessary, insects were anaesthetised by cooling them at 5±1*C for 5 minutes prior to treatment. The suspension was withdrawn from the funnel by means of a filter pump and the insects gently removed using forceps and placed on rice plants var. TNI. Plants were grown in 7.2 cm diam. polyethylene pots containing soil covered with plaster of paris, which made it easier to count dead insects and protect cadavers from saprophytic soil microorganisms. b) Contagion from cadavers This method was used to assess the ability of fungal strains to spread from sporulating cadavers to nymphs. Ten male and ten female, adult GLH were treated with fungi and maintained on potted plants covered with ventilated propagators (92-96% rh) at 25±1*C. Insects were observed daily for up to 28 days and number of adults and nymphs infected with fungi, recorded. Nymphs emerged from eggs laid by the adult insects after approximately 14 days. IX Statistical analysis Bioassay data was generally subjected to probit analysis (Finney, 1971) using the maximum likelihood program (MLP) (Ross, 1980) to calculate the dose, concentration, or time required to kill 50% of test insects: i.e. LD50, LC50 or LT50 values. The program was also used to calculate the time required to obtain 50% germination of conidia,GT50. Further useful statistics are the mean time of death (MTD), the estimated time to kill 50% of test insects (eLT50), and the estimated time for 50% germination (eGT50). i) Problt analysis The MLP program analyses the proportion of insects killed after treatment with different spore concentrations. It fits a straight line to the percentage mortality on a probit scale against the log. of spore concentration and calculates the following parameters: a) LC50, the spore concentration that causes 50% mortality. The program can also calculate any other LC level such as LC95 or LC5. b) The 95% fiducial limits for a specified LC value. c) The slope of the regression line and its standard error. This parameter is mainly used to reflect the precision of the assay as high variability in the reaction of the insects to spore concentration results in a low slope, although it can give a rough estimate of the pathogenicity of the strain. d) The chi-squared statistic for heterogeneity, which is an indication of sample homogeneity. When this statistic is significant, the standard errors are raised by the heterogeneity factor. A goodness of fit chi-squared statistic indicates if the calculated line is an adequate description of the data. e) Analysis of parallelism. When it is intended to combine replicated bioassay date for LD, LC, LT, or GT50 calculation, an analysis of parallelism must be done. The MLP program compares the slopes of regression lines of replicated bioassays using the chi-squared statistic. If the regression lines are parallel the data can be used to calculate the combined value. ii) LT50 or GT50 calculated by MLP MLP can also be used for regression analysis of percentage mortality or percentage germination (on a probit scale) versus time to calculate: a) LT50 or GT50. The time taken to reach 50% of mortality or 50% germination, at a given treatment, with an option to calculate any other LT or GT value, e.g. LT95 or GT95. e) The standard error and 95% fiducial limits for any specified LT or GT value. To enable comparison of treatments by this method, it is necessary to have an independent batch of insects or conidia for each assessment. In the case of germination, it is relatively easy to obtain the high number of spores required, but in the case of insects the large number needed during assessment periods of up to 10 days is difficult. This means the method is costly both in terms of time and insects, and is probably not justified in initial pathogenicity screening treatments. iii) Estimation of LT50 and GT50 by maximum likelihood program. Despite the problem described above, it is possible to use MLP to make an initial approach to the response of an insect population to different treatments in terms of time of death. A single sample of insects is used for each treatment and the number of dead insects at different times is considered as a cumulative response to the treatments. The estimated lethal time to kill 50% of insects (eLT50) obtained from non independent samples , can be reasonably compared with "the mean time of death" as suggested by Fenlon (1989). Both parameters give an idea if the treatments influence the time of death. These prelim inary results can be used to decide if further study is worthwhile. However, eLT50 values and their S.E. from regression analysis, should not be used to construct statistical conf idence limits to compare treatments. It is acceptable, however, to use a t test or Anova to compare mean eLT50 values when bioassays are fully replicated. The same concepts, can also be used to estimate the time needed for 50% germination of a conidial population. # - 1 5 1 - iv) Mean time of death The mean time of death (MTD) is another statistical parameter which can be used to compare the effect of treatments on insect longevity, when the large number of insects required to obtain an LT50 is a limitation. It again uses mortality data provided by repeatedly sampled batches of insects in an statistical program which calculates the mean and standard deviation of time to death based on the mid-time interval. The correlation matrix linking these estimates shows that the eLT50 and the MTD are highly correlated (Fenlon J., unpublished observation). v) Analysis of variance in screening experiments In screening experiments, new strains are frequently added to bioassays before completion of the minimal number of replicates for each strain. As a result the set of assays is usually unbalanced and an analysis of variance of percentage mortality to GLH adults was devised to compare them. At a determined time, a regression model relating all screened strains to the control allows the ranking of the strains. A common s.e.d. can be calculated and using a criterium of, for instance, control mortality + 2 s.e.d. (approximate 95% exceedance) as a qualifier, it is possible to differentiate the fungal treatments which result in insect mortality greater than that in the control population. - 1 5 2 - 2. Initial selection of fungal strains To assess pathogenicity to GLH, batches of 20 adults were Immersed in conId la 1 spore suspensions prepared from the strains detailed in Table 2 and maintained at 25±1*C on potted rice plants covered with 'Ventilated" plastic covers (III, 1, B)). Insect mortality was recorded for 15 days after treatment. In some experiments the number of sporulatlng cadavers and the number of dead Insects attached to the plant were also recorded after 15 days, and the number of dead nymphs assessed after 20 days. In each trial, batches of insects were also treated with 0.05% Triton X-100 as controls and each strain tested at least four times against different batches of insects. 3.Quantification of pathogenicity A. LC50 _i) In suboptimal rh conditions Pathogenic fungal isolates were further tested against adult GLH to determine LC50 values by multiple dosage assays. Groups of 20 adult insects were immersed in 0.05% Triton X-100 or similar suspensions containing 5 x 10A, 5 x 10s,5 x 10G and 5 x 107 conidia per ml. After removal of excess conidial suspension, treated insects were transferred to rice plants in pots covered with ventilated plastic covers, maintained at 25±1*C and observed daily for mortality. Data were analysed using the maximum likelihood program (III, 1, D, - 1 5 3 - i) . Each strain was assayed three times using different batches of insects. ii) In optimal rh conditions B. bassiana 110-82, 268-86, 269-86 and M. anisopliae 275-86 were further tested to determine pathogenicity to GLH adults in multiple dosage bioassays in optimal rh conditions. Batches of 20 adult insects were immersed in 0.05% Triton X-100 solutions, or similar solutions containing serial dilutions from 5xl04 to 5xl07 conidia per ml. Treated insects were contained on rice plants with closed plastic covers at 25±14C and observed daily for mortality. Each strain was assayed three times using different batches of insects. B^ Relationship between spore concentration and number of spores adhering to insects: Determination of LD50 Groups of 30 insects were immersed in conidial suspensions (104, 10s, 10s and 107 per ml) of B. bassiana 261-85 and M. anisopliae 82- 82, as described earlier (III, 1, C). Immediately after removal from the spore suspension, 10 insects from each treatment were taken individually and placed in Bijoux bottles containing 1, 2 or 3 ml of 0.05% Triton X-100 solution and 5-10 glass balls (2-3 mm diam). The Bijoux were agitated for 1 min. using a vortex mixer to remove spores. Aliquots of suspensions were then plated onto SDA containing 50 pg per ml of both streptomycin sulphate and chloramphenicol, to provide approximately 200 colonies per plate. Plates were incubated at 25±1*C for 3-4 days when colony numbers were recorded. Gillespie (1984) showed agitation did not reduce conidial viability of M. anisopliae. Following the same procedure, a second experiment was conducted with B. bassiana 266-86, 268-86, and M. anisopliae 83-82 and 275-86. In the second trial concentrations of conidial suspensions were higher: 5 x 10s, 5 x 10s,5 x 107, and 5 x 10s spores per ml. In order to compare direct counts with plate counts, aliquots of suspensions prepared from insects immersed in 5 x 10a spores per ml were taken and counted in an improved Neubauer haemocytometer. After removal from conidial suspensions, 20 insects per batch were transferred to rice plants and maintained at 25±1*C. Insects treated with B. bassiana 261-85 and M . a n i s o p l i a e 82-82 were contained with "ventilated" plastic covers while those treated with B . b a s s i a n a 266- 86, 268-86, and M. anisopliae 83-82 and 275-86 were contained with closed covers (two different experiments). Insect mortality was recorded for 15 days after treatment and LD50 values calculated by relating spore dose to mortality. 4. Effect of temperature on infection B . b a s s i a n a 110-82 and 268-86, and M. anisopliae 275-86 were studied in this experiment. Suspensions of 5 x 107 spores per ml were prepared as described earlier, batches of 20 insects immersed in spore suspensions and maintained on plants with "closed" covers. For each strain, groups of insects were incubated at 20, 25 and 30*C. Insect mortality was recorded daily for 8 days. - 1 5 5 - * 5. Influence of relative humidity on Infection Ai Infection at optimal and suboptimal relative humidity In order to study the effect of optimal and suboptimal rh on fungal pathogenicity, bioassays were conducted with B. bassiana 110- 82, 268-86 and 269-86, and M. anisopliae 275-86. Insects were immersed in conidial suspensions (5X10T) as described earlier, and maintained on rice plants at 25±1*C and covered either with closed or ventilated covers (III, 1, B). Insect mortality was assessed daily for 9 days. IL Influence of high humidity period on pathogenicity B. bassiana 268-86 and M. anisopliae 275-86 were studied in an attempt to define the period of high humidity needed for infection. Suspensions of 5X107 conidia per ml were prepared and standardized. Batches of 20 insects per treatment were immersed in 0.05% Triton X-100 or similar suspensions containing spores. They were then maintained at high humidity by covering open propagators with polyethylene bags for 24, 48, 72, 96, 120, or 144 hours. After these periods the bags sealing the covers were removed and pots left at low humidity (70-87%) (III, 1, B). Temperature was maintained at 25±1*C throughout the experiment. Mortality was recorded twice daily for up to 8 days. - 1 5 6 - 6. Improvement of pathogenicity A;. Effect of adding nutrients to spore suspensions Adding nutrients to spore suspensions could increase the speed of spore germination and cuticular penetration and thus increase the speed of insect kill. In order to assess this possibility, four different mixtures of nutrients were added to suspensions of B. b a s s i a n a 268-86 and M. anisopliae 275-86 containing 5X107 spores per ml to provide final nutrient concentrations of, 1) 1% glucose and 0 .1% yeast extract 2 ) 0 .1% glucose and 0 .01% yeast extract 3) 1% skimmed milk 4) 0.1% skimmed milk Batches of 20 insects were treated with conidia or conidia and nutrients and insects contained with "ventilated" covers at 25±1*C. Dead insects were recorded for 9 days. IL Effect of pregerminating conidia The effect of pregerrainating conidia on the rate of insect death was studied in this experiment. B. bassiana 268-86 and M. anisopliae 275-86 suspensions were prepared containing 5X107 conidia per ml and aliquots treated as detailed below, 1) Incubated for 6 hours at 25±1’C in 0.05% Triton X- 100 containing 0 .1% glucose and 0 .01% yeast extract, then centrifuged (12.100 G, 5 min.) washed and resuspended in 0.05% Triton X-100. 2) Incubated for 12 hours at 25±1*C in 0.05% Triton X- 100 containing 0 .1% glucose and 0 .01% yeast extract, then centrifuged (12.100 G, 5 min.) washed and resuspended in 0.05% Triton X-100. 3) Incubated for 6 hours at 25±1*C in 0.05% Triton X- 100 4) Incubated for 12 hours at 25±1*C in 0.05% Triton X- 100 5) Incubated for 6 hours at 25±1*C in 0.05% Triton X- 100 containing 0 .1% glucose and 0 .01% yeast extract. 6) Incubated for 12 hours at 25±1*C in 0.05% Triton X- 100 containing 0 .1% glucose and 0 .01% yeast extract. Insects were immersed in suspensions described above or in suspensions containing non germinated conidia and maintained on rice plants in ventilated covers. Insect mortality was recorded twice daily for 9 days. Cj. Experiments with single spore isolates i) Selection of single spore isolates B. bassiana 268-86 was removed from liquid nitrogen storage and subcultured on SDA. Conidia were harvested after 9 days at 25±1*C and used to prepare a suspension containing 5X107 conidia per ml in 0.05% Triton X-100. Adult GLH were immersed in this suspension as described previously and maintained on rice plants with ventilated propagators at 25±1’C. Insects which died first were collected and left in humid chambers (II, 1, F) at 25±1’C until sporulation was complete. Fully sporulated cadavers were collected in universal bottles containing 10 ml 0.05% Triton X-100 and 20 glass balls (2-3 mm diam.) and agitated using a vortex mixer. Suspensions were then sonicated, filtered trough tissue to remove mycelial debris, adjusted to 5X107 conidia per ml and used to treat GLH adults. When insects died they were again removed to humid chambers and resultant conidia harvested in 0.05% Triton X-100. The suspension was adjusted to lO* conidia per ml and suitable amounts to obtain 100 to 300 colonies per Petri dish were plated onto SDA containing 100 pg per ml of kanamycin, streptomycin and chloramphenicol. After 20-24 hours at 25±1*C, conidia with the longest germ tubes were marked by scoring the agar surface adjacent to the spore with a fine needle, and after 36 hours incubation transferred individually to SDA Petri plates and allowed to grow for 9 days at 25±1*C. One hundred single spore isolates (SSI) were taken and nominated SSI 1 to SSI 100. 11) Standardization of suspensions by weight The determination of conidial concentrations using a haemocytometer is time consuming and inconvenient for large numbers of strains. Accordingly the feasibility of standardizing cultures by weight was studied. Nine SSI and the multi spore isolate (MSI) of B. bassiana 268-86 were cultured on SDA for 9 days at 25±1*C. Samples of conidia from each isolate (3 replicates) were taken from the Petri plates with a sterilized spatula and adjusted carefully to 10 mg using an analytical balance (Sartorius, model 2400, Gottingen, Germany). Each sample was suspended in 100 ml 0.05% Triton X-100 and sonicated (lOp amplitude, 15 seconds) before being made up to one litre in a volumetric flask. The concentration of - 1 5 9 - each suspension was then measured in an improved Neubauer haemocytometer. iii) Pathogenicity of single spore isolates to adult N. vlrescens 100 SSI, and the MSI of B. bassiana 268-86 were subcultured on SDA and after nine days conidia were harvested, standardized by weight as described previously and viability assessed. Suspensions were prepared by suspending 10 mg of conidia in 100 ml of 0.05% Triton X-100 to give an approximate concentration of 1X107 conidia per ml. Batches of 10 adult GLH were immersed in those suspensions as previously described (III, 1, C, ) and insects contained in closed covers at 25±1*C. Mortality was recorded twice daily after 2, 3, 4, 5 and 6 days and the number of cadavers with sporulating mycelium and or attached to the rice plant recorded after 10 days. From this first bioassay 31 SSI were chosen and kept in liquid nitrogen (Challen and Elliot, 1986). Following the same bioassay procedure these 31 SSI were again compared with the MSI for virulence to GLH adults using batches of 15 insects. From this second bioassay, 9 SSI were chosen and compared with the MSI for pathogenicity to GLH adults in further experiments using three separate batches of insects. Isolates were subcultured only once on SDA between bioassays. The same procedure, passaging twice through GLH adults, was used to choose 9 SSI’s of M. anisopliae 275-86 which were compared with the MSI for pathogenicity to GLH adults. The experiment was repeated using three separate batches of insects. Dj. Correlation between pathogenicity and rate of conidial germination - 1 6 0 " From the previous experiment, a range of B. basslana SSI's was obtained with higher and lower pathogenicity to GLH than the MSI . To determine if there was a correlation between pathogenicity and conidial germination rate on SDA, 10 SSI's were chosen from this range, with higher and lower pathogenicity to GLH compared with the wild type. Suspensions from 9 day old cultures of the 10 chosen SSI's and MSI were prepared and adjusted to 5 x 10s conidia per ml and 5 pi of each suspension were placed on small SDA Petri plates (5.5 cm diam.). The suspensions were then spread and kept at 25±1*C. Three samples of each strain were taken from the incubator after 4, 6, 8 , 10, 12, 14 and 16 hours and one drop of lactophenol cotton blue placed on the surface of each sample and covered with a cover slip. Plates were then stored at 5±1*C and conidial germination determined by direct observation of at least 100 conidia per sample. 7. The use of the optical brightener bi-striazinvl amino stllbene to study spore germination I n v i v o The optical brightener bi-striazinyl amino stilbene (Tinopal B0PT, Ciba-Geigy, Manchester, UK) was used to stain fungal cells and caused them to fluoresce under UV light, as described by Drummond and Heale (1985) with V. lecanli. A_i The infection process as revealed using the optical brightener B. bassiana 268-86 and M. antsopllae 275-86 were subcultured on SDA at 25±1*C. Conidia were harvested after 9 days and standardized as described earlier(II, 1, E>. Suspensions were adjusted to 5X107 conidia per ml, centrifuged <3.020 g, 5 minutes) and pellets resuspended in 0.05% Tinopal in 0.05% Triton X-100. The suspension was left for 30 minutes, centrifuged again (12.100 g, 5 minutes), resuspended in 0.05% Triton X-100 and sonicated (lOp amplitude, 15 seconds). Aliquots (10 ml) were taken and batches of 40 insects immersed in each suspension and maintained on rice plants covered with closed and ventilated plastic covers at 25+1*C. After 4, 8 , 12, 16, 21, 45, 69, 91, 116, 132, 142, hours incubation, three insects from each treatment were taken and the wings removed carefully. They were then stuck onto slides using double-sided adhesive tape, making sure they were flat on the tape surface. Germination and subsequent mycelial development were recorded by examination under a fluorescence microscope. (Leitz-Dialux 20 with UV filter: A513596; Leitz Wetzlar, W. Germany) Correlation between germination rate of £. b a s s i a n a spores on SPA and insect wings This experiment compared the germination rates on SDA and insect / wings of aerial conidia, submerged conidia and hyphal bodies of B. b a s s i a n a 268-85. Spores were produced on SDA, in TK1 broth or YG (II, 1, G and IV, 4). For germination on SDA, suspensions of 5X10S spores per ml were prepared in 0.05% Triton X-100 and 20 pi of each suspension were spread onto SDA in 5.5 cm diameter Petri plates and incubated at 25±1*C. Three samples for each spore type were taken after four hours and then every 2 hours for 24 hours. Lactophenol cotton blue (II, 3, - 1 6 2 - A, ii) was used to stop spore germination and plates stored at 5±1*C before samples were assessed for germination. To observe germination on insect wings, suspensions of 5X107 spores per ml in 0.05% Triton X-100 were prepared, then centrifuged and stained with Tinopal BOPT as explained previously. Batches of 40 insects were submerged in suspensions of stained spores and left on rice plants covered with closed plastic covers at 25±1#C. After four hours and at two hour intervals for 24 hours, three insects per treatment were taken, the wings removed and adhered to slides with double sided tape. Germination of at least 100 spores per wing was recorded under the UV light microscope. 8 . Assessment of disease spread A feature of some entomogenous fungi is their ability to spread from dead to healthy insects in the field and thus provide prolonged pest control. This series of experiments examined aspects of disease transmission. /u Spore production on insect cadavers Strains studied in this experiment were: M. anisopliae 82-82, 83- 82 and 275-86, B. bassiana 110-82, 138-83, 235-85, 261-65, 266-85, 268-86, 269-86 and 270-86. Batches of 20 GLH adults were immersed in conidial suspensions (5X107 spores per ml), maintained on rice plants in ventilated covers and incubated at 25±1*C. After 12 days, 10 fully sporulated insects for each strain were removed and placed in Bijoux bottles (5 ml) containing 1 ml of 0.05% Triton X-100 and approximately 10 glass balls (0.3 cm diam). The Bijoux were agitated for 1 min. using a vortex mixer which macerated the insects and released spores. Spores numbers were then determined using an improved Neubauer haemocytometer. Sporulation on Insect cadavers and attachment to plants Fungal strains which showed high pathogenicity to GLH were examined in a preliminary experiment. M. anisopliae : 83-82 and 275-86, B. bassiana 110-82, 138-83, 235-83, 261-85, 266-85, 268-86 and 269- 86 were taken from liquid nitrogen and grown on SDA for 9 days at 25±1*C. Conidia were harvested, standardized (II, 1, E) and serial dilutions prepared from 5X10* to 5X107 conidia per ml. Batches of 20 insects (half males and half females) per treatment were treated and maintained on rice plants in closed covers at 25±1*C. Pots were observed daily for dead insects. After 25 days, the number of sporulating cadavers, insects attached to the rice plant and infected nymphs were recorded. Following the same procedure, B. bassiana 110-82, 268-86 and 269- 86, and M. anisopliae 275-86 were studied using closed and ventilated covers (III, 1, B). This experiment was repeated three times with different batches of insects. Correlation between number of dead sporulating insects and disease spread to nymphs B. bassiana 110-82, 268-86 and 269-86, and M. anisopliae 275-86 were used to infect insects as described above (III, 8 , B) and insects maintained in closed or ventilated covers. After 15 days incubation, all the treated insects were dead and most bore sporulating mycelia. After 25 days, live, dead nymphs infected with sporulated fungus and dead nymphs (without any evidence of fungal infection) were recorded. The data was subjected to a correlation analysis to determine if there was any relationship between the number of adults with sporulating mycelia and the number of nymphs infected by fungi. 9. Effect of subculturing and passaging on pathogenicity of strains The effect of repeated passages through the insect host and subculturing on SDA on the virulence of M. anlsopliae 275-86 SSI's 1, 3 and MSI, and B. bassiana 268-86 SSI's 81, 96 and MSI were examined. Samples were taken from liquid nitrogen and cultured on SDA Petri plates (7-9 days at 25±1*C). This was designated the 1st medium subculture (01 MS) and these conidia were used to prepare conidial suspensions (5X107 per ml). Batches of 20 GLH adult insects per strain were immersed in these suspensions as described previously. Insects were maintained on potted rice plants at 25±1*C and contained with "closed" plastic covers until the insects had died and cadavers bore sporulating mycelia. This was called the 1st insect passage (01 IP). Conidia from 01 MS and 01 IP were used to start a series of successive bioassays. Conidia obtained from insects killed with 01 IP conidia were called 02 IP conidia and so on. Simultaneously 01MS conidia were subcultured on SDA to obtain 02MS conidia. The virulence of spores was measured in bioassays by immersing insects in spore suspensions and mortality recorded twice daily. Following this procedure, strains were successively passaged and - 1 6 5 - bioassayed nine times. It was not possible to fully replicate these experiments due to time limitations and a limited supply of insects. However, at the end of subculturing and passaging all strains were removed from liquid nitrogen and bioassayed. 10. Comparison of the pathogenicity of hvphal bodies, aerial and submerged conidia Spores of B. bassiana 268-86: conidia, hyphal bodies and submerged conidia were produced on SDA and wheat grain, glucose/yeast liquid medium or TK1 broth as described previously (II, 1, G; IV, 4). Hyphal bodies and submerged conidia were taken from 5 day old cultures, aerial conidia from 7-9 day old SDA cultures and 10 day old wheat cultures, respectively. Spores were harvested and suspensions standardized as described earlier (II, 1, E) and serial dilutions from 5X10* to 5X10S spores per ml prepared. Batches of 20 adult GLH were immersed in such suspensions or 0.05% Triton X-100 as control. Treated insects were transferred to rice plants with "closed" covers, incubated at 25±1*C and assessed twice daily for mortality. Trials were replicated four times except for the 5X10^ dilution which was replicated only three times. RESULTS 11. Production of plants and insects The method followed allowed the continuous production of rice plants throughout the 3-year study. Plants grew more slowly in winter due to reduced light quantity and quality. Insect rearing on rice plants contained in perpex cages allowed the production of adequate numbers of GLH for the three year- research programme. Numbers of adult GLH obtained per perpex cage varied between 200 to 400 after 5 to 7 weeks. The higher adult populations and shorter development periods were observed during the summer, whilst the lowest numbers and longest development times were observed during the winter. This was surprising as temperature, humidity and photoperiod conditions were constant all the year round. G1H adults contained on whole rice plants with propagators survived well. Plaster of Paris was effective at excluding insect pathogens as in most bioassays more than 90% of untreated insects were still alive after 10 days 12. Selection of fungal Isolates pathogenic to N ephotettlx virescens An analysis of variance of the percentage mortality of GLH adults 9 days after treatment was performed using a regression model relating all strains to the control. This permitted the ranking of the 29 strains examined in order of virulence (Table, 17). Using a - 1 6 7 - TABLE 17. Pathogenicity of 29 strains of fungi to adult Nephotettix virescens* Angular Spreading Strain % mortality** transformed potential (SED = 5.39) Metarhizium flavoviride 259-85 0.49 4.03 Trichothecium sp. 213-85 2.36 8.85 Verticillium lecanii 11-73 2.46 9.03 Metarhizium anisopliae 208-85 2.55 9.20 Verticillium lecanii 19-79 4.20 11.83 Sporothrix insectorum 298-86 5.74 13.87 Paecilomyces farinosus 104-82 9.27 17.73 A Metarhizium flavoviride 203-84 13.21 21.32 M. anisopliae 260-85 14.86 22.68 CONTROL 26.91 31.25 Beauveria bassiana 251-85 27.34 31.53 A M. anisopliae 276-86 32.16 34.55 S B. bassiana 299-86 32.40 34.70 A S M. anisopliae 275-86 35.53 36.59 A S N B. bassiana 231-85 36.53 37.19 A S H 11 265-85 42.88 40.91 A S It II 206-85 43.71 41.39 A M. anisopliae 82-82 44.28 41.72 B. bassiana 109-82 44.53 41.86 m M T D A T M O P T A T T T V -l. o c u n B. bassiana 84-82 52.18 46.25 M. anisopliae 83-82 54.80 47.76 S M. anisopliae 300-86 56.31 48.63 A S B. bassiana 266-85 60.46 51.04 A S N it ii 261-85 62.68 52.35 A S N ii it 270-86 63.81 53.02 A S N it ii 138-83 64.83 53.63 A S N ii ii 269-86 67.80 55.43 A S N ti ii 235-85 77.49 61.28 A S N ti ii 110-82 82.44 65.23 A S N it ii 268-86 85.94 67.98 A S N * Mortalities assessed after 9 days incubation in suboptimal relative humdidity conditions ** Figures are mean of at least four replicates per strain in independent bioassays A More than 2 cadavers adhered to plant leaves after 15 days S More than 2 cadavers sporulated after 15 days N More than 2 nymphs infected after 20 days criterium of control mortality + 2 s.e.d. (approximate 95% exceedance) as a qualifier, eleven strains (2 M. anisopliae and 9 B. bassiana ) gave over 50% mortality and were significantly different from the control. When the mean time of death (Fenlon 1989) was considered, B. b a s s i a n a 110-82, 235-85, 268-86, 138-82 and 269-86, killed insects most rapidly (Table 18). Some strains like Trtchothecium 213-85, V. l e c a n i i 19-79 and 11-73, S. insectorum 298-86, P. farinosus 104-82, M. flavoviride 259-85 and 203-84, and M. anisopliae 208-85 and 260-85 showed very low or zero pathogenicity to GLH. In contrast all B e a u v e r i a strains tested showed at least some virulence. 13. Quantification of pathogenicity A. LC50 D. Under sub-optimal relative humidity The relationship between spore dose and GLH mortality under conditions of suboptimal rh (ventilated covers; III, 1, B) was analysed 9 days after treatment using probit analysis. LC50 values, slopes of regression lines and the Chi2 values of the parallelism analysis are presented in Table 19. Values of parallelism of the three regression lines for each strain were not significant (P=0.05) for B. bassiana 110-82, 138-83, 261-85, 266-85, 268-86, 269-86 and M. a n i s o p l i a e 83-82 and 275-86, indicating parallelism of the dose mortality response in replicate assays. The data was therefore combined in a single probit analysis to obtain a combined LC50 value - 1 6 9 - TABLE 18. Approximate times needed to achieve 50% mortality of adult Nephotettix virescens after treatment with fungi Species Strain Time range of 50% death (days)** Metarhizium flavoviride 259-85 12 Trichothecium sp. 213-85 12 Verticillium lecanii 11-73 12 Metarhizium anisopliae 208-85 12 V. lecanii 19-79 12 Sporothrix insectorum 298-86 12 Paecilomyces farinosus 104-82 10-12 M. flavoviride 203-84 10-12 M. anisopliae 260-85 10-12 Beauveria bassiana 94-82 10-12 B. bassiana 251-85 9-12 M. anisopliae 276-86 9-12 B. bassiana 299-86 9-12 m it 231-85 9-12 ii ii 109-82 9-11 M. anisopliae 300-86 9-11 M. anisopliae 275-86 9-10 B. bassiana 265-85 8-12 M. anisopliae 82-82 8-12 B. bassiana 206-85 8-11 M. anisopliae 83-82 8-10 B. bassiana 261-85 8-10 ii it 270-86 8-9 ii it 266-85 7-10 less than 10 days B. bassiana 269-86 7-9 ti it 138-82 7-9 it ii 268-86 6-9 it it 235-85 6-8 ii it 110-82 5-8 Range are result of at least 4 independent bioassays for each evaluated strain Insects were maintained on rice plants in suboptimal relative humidity conditions - 1 7 0 - dF) (6 3.24 3.24 (NS) 1.17 1.17 (NS) 1.11 1.11 (NS) 1.03 1.03 (NS) Heterogeneity Heterogeneity /Continued..• (2 dF) (2 0.27 0.27 (NS) 9.64** 0.36 0.36 (NS) Chi^ Chi^ values Parallelisra (2 dF) (2 1.96 1.96 (NS) 1.01 (NS) 0.58 0.58 (NS) 4.41 4.41 (NS) Position Position 0.24 0.17 0.20 0.13 0.17 0.15 0.70 2.83 (NS) 0.21 0.22 0.30 0.21 0.48 2.38 (NS) 0.419 (NS) 7.30 (NS) 0.90 : SE : 1.87 + 1.87 0.60 + 0.60 0.41 + 0.41 1.33 + 1.33 0.34 + 0.34 0.60 + 0.60 0.64 + 0.64 0.81 + 0.81 0.47 +0.47 0.18 0.33 + 0.33 0.54 + 0.54 0.96 + 0.96 0.62 + 0.62 0.98 +0.98 0.28 Slope 4 Slope 6 6 (xlO 1.48 1.86 5.03 5.66 8.81 4.17 6.67 6.09 35.78 50 128.58 236.01 + 0.34 conidia/ml) LC 1 3 . 3 4.98 1 2 2 1 3 5.50 2 3 1 3 85.82 2 1 2 3 2.36 number Bioassay • 266-85 261-85 138-85 235-85 110-82 Strain TABLE TABLE 19. conditions* humidity relative virescens Nephotettix in suboptimal adult Pathogenicity fungi to of Beauveria bassiana Beauveria -171- dF) (6 2.83 2.83 (NS) 1.70 1.70 (NS) 1.92 1.92 (NS) Heterogeneity Heterogeneity (2 dF) (2 0.64 0.64 (NS) 1.31 1.31 (NS) 0.13 0.13 (NS) 1.60 (NS) 57.75*** Parallelisra Parallelisra Chi^ values Chi^ (2 dF) (2 2.16 2.16 (NS) 0.24 0.24 (NS) 4.82 4.82 (NS) 1.16 (NS) 3.26 (NS) Position Position 37.78*** 0.11 0.20 + 0.54 + 0.26 0.54 0.56 + 0.17 0.56 + 0.37 0.18 0.18 + 0.18 0.45 + 0.22 0.45 0.28 + 0.28 0.15 0.65 + 0.21 0.65 0.34 + + 0.34 0.21 0.87 + 0.22 0.87 0.68 0.87 + 0.22 0.87 Slope Slope SE responses 6 6 Too Too few (xlO 2.01 2.50 8.54 16.12 21.14 73.95 14.53 28.79 + 0.28 0.47 (NS) 1.39 45.21 84.15 50 100.47+ 0.54 0.43 162.82 conidia/ml) LC 1 2 3 25.87 + 0.16 0.41 3 1 1 2 1 2 3 2.25 2 3 1 3 2 nurabe r nurabe Bioassay Bioassay 275-86 270-86 269-86 268-86 Strain * * conditions + humidity relative in suboptimal at 25 1°C days after nine incubation determined Mortalities ** ** *** ^*0.05) Significant (P Highly (P^O.Ol)significant Metarhizium Metarhizium anisopliae 83-82 Beauveria bassiana Beauveria TABLE 19. TABLE 19. (Continued) -172- for each strain. In the case ofB. bassiana 235-85 there was no strong evidence for parallelism between replicate assays but as the value was close to the limit value and to provide consistency with the other strains, the combined LC50 was taken. For B. bassiana 270- 86, the value was so far from the limit value that combining data was impossible. The LC50 values calculated from the combined data from three bioassays are shown in Table 20. Strains differed in pathogenicity to GLH adults with the most pathogenic isolates, B. bassiana 235-85, 268-86, 110-82, 266-85 and 261-85 with LC50 values of 2.24, 3.01, 4.62, 5.99 and 11.50X10S conidia per ml respectively. The most virulent M. anisopliae isolate was 275-86 with LC50 value of 20.30X10sconidia per ml. ii) Under optimal relative humidity High humidity favoured the pathogenicity of fungal strains to GLH adults, although strains responded differently to this change in humidity. Isolates B. bassiana 110-82, 268-86, 269-86 and M. a n i s o p l i a e 275-86 had respective LC50 values of 0.29, 0.86, 27.60 and 0.70X10S conidia per ml when tested for pathogenicity to GLH adults under optimal rh conditions (Table 21). The data represents 16, 4, 0.1 and 30 fold increases respectively in virulence when rh was maintained at a constant 100% (Table 21). - 1 7 3 - * TABLE 20. Pathogenicity of fungal strains to adult Nephotettix virescens in suboptimal relative humidity conditions; combined data* Fiducial LC50** limits 95% Strain xlO6 Slope + SE Chi2*** conidia/ml Lower Upper Beauveria bassiana 110-82 4.62 1.48 8.39 1.01 + 0.25 13.71 138-85 148.00 33.30 260.00 0.42 + 0.15 8.54 235-85 2.24 1.05 4.14 0.82 + 0.13 13.91 261-85 11.50 3.22 51.40 0.39 + 0.09 8.20 266-85 5.99 2.37 13.70 0.60 + 0.11 6.50 268-86 3.01 1.29 6.33 0.64 + 0.10 6.98 269-86 32.30 10.10 176.00 0.49 + 0.14 6.37 Metarhizium anisopliae 83-82 86.80 26.90 1540.00 0.50 + 0.16 1.87 275-86 20.30 6.67 69.50 0.53 + 0.14 5.10 * Mortalities were determined after nine days incubation at 25 + 1°C in ventilated covers ** Figures are combined results of three different multiple dosage bioassays *** Heterogeneity about regression line with 12 degrees of freedom; all not significant (P > 0.05) - 1 7 4 - * TABLE 21. Pathogenicity of fungal strains to adult Nephotettix virescens in optimal relative humidity conditions* LC50g** _____ Fiducial limits 95% Strain xlO Slope + SE Chi2*** conidia/ml Lower Upper Beauveria bassiana 110-82 0.29 0.13 0.55 0.54 + 0.07 18.39 268-86 0.86 0.48 1.48 0.68 + 0.08 15.17 269-86 27.60 11.20 119.00 0.42 + 0.07 2.78 Metarhizium anisopliae 275-6 0.70 0.40 1.18 0.72 + 0.09 5.50 * Mortalities were determined after six days incubation in closed covers ** Figures are combined results of three different multiple dosage bioassays *** Heterogeneity about regression line with 10 degrees of freedom; all not significant (P > 0.05) - 1 7 5 - Bj. Relationship between spore concentration and number of spores adhering to insects. Determination of LD50 When adult GLH were immersed in conidial suspensions of different fungi, the dose received varied markedly and ranged from 0.05% of the spore concentration per ml with B. bassiana 261-85 to less than 0.01% with M. anisopliae 83-82 (Table 22). A chi-squared test of regression lines of log concentration related to conidia adhering to insects gave significant (P<0.05) goodness of fit values for all the strains tested, illustrating the good relationship between concentration and dose. Probit analysis of mortality data 9 days after conidial application and maintenance of insects at 89.3-94.5% (suboptimal) rh, gave LD5, 50, and 95 values of 1, 1352 and 169000 conidia for B. bassiana 261-85 and 33, 2914 and 259000 for M. antsopliae 82-82, illustrating the greater pathogenicity of B. bassiana 261-85 under these conditions (Table 23). In a second experiment, analysis of mortality data 5 days after conidial application and maintenance of insects at 100% rh gave LD 5, 50 and 95 values as 29, 854, and 25300 conidia for B. bassiana 268- 86; 29, 1822 and 115000 conidia for M. anisopliae 275-86; 22, 3404 and 535000 for J3. b a s s i a n a 266-85 and 5, 5371 and 6290000 for M. a n i s o p l i a e 83-82 (Table 23). The method of removing conidia from insects by agitation and plating them in order to estimate the number of conidia adhering onto the insect, gave very similar results to haemocytometer counts (Fig. 0.2 0.8 6 + SE 13 41 52 + + + Mean xlO Mean xlO Continued... 13 13 13 12 40 41 44 41 41 43 58 insects xlO insects adhering to_^ adhering to_^ % no. % no. conidia/ml 10 1.72 2.72 2.60 2.05 3.82 2.73 67 4.32 0.81 i i log 0.20 2.24 0.22 error + standard + standard 10 1.78 1.63 2.80 1.67 3.81 3.34 4.81 2.31 4.30 2.76 3.83 5.31 0.61 - log insects insects adhering to adhering to Mean no. Mean no. conidia 10 after immersion in spore suspensions* spore in immersion after 5.00 7.00 7.00 5.00 6.00 6.00 8.00 8.00 5.00 6.00 7.00 4.00 i i log Conidial concentration/ml 268-86 266-85 261-85 Strain TABLE 22. Relationship between spore concentrations and number of conidia adhering to adult Nephotettix virescens Beauveria bassiana Beauveria - I l l - + SE 39 8 26 + 0.4 + 0.2 ±2 Mean xlO Mean xlO 8 8 8 38 39 40 40 21 30 24 32 insects xlO insects adhering to_^ to_^ adhering % no. conidia/ml % no. conidia/ml 1.52 1.80 9 2.47 3.82 2.69 i i 10 2.47 0.89 log error _+ standard standard _+ 1.31 3.30 4.29 2.87 2.67 5.27 4.59 3.78 2.50 1.58 3.48 log insets adhering to adhering to Mean no. Mean no. conidia 5.00 6.00 7.00 6.00 7.00 3.60 7.00 8.00 i i 10 5.00 1.62 8.00 5.00 1.39 0.82 6.00 log 4.00 0.32 -0.39 * Figures are mean of mean insects replicate are of ten * Figures concentration/ml 275-86 83-82 Strain Conidial Metarhizium anisopliae 82-82 anisopliae Metarhizium TABLE 22. 22. TABLE (Continued) -178- 1 .8 4 0 .1 0 0 .6 2 0 .3 9 50 0 .2 0 .1 0 .7 4 0 .1 0 .2 + + + + 1 .0 0 .8 0 .7 + 0 .2 Slope + SE* CHi2** 105 ) x 104- 2 3 . 3 x (34.4x10^-588x108) 0.5 ( 3 .0 2 LD95 w ith 95% fiducial lim its 1 .1 5 x l0 5 6 2 .9 x l0 5 1.69xl05 (8.26x10^-154x108) 0.5 + 0.1 0.56 5.35xl05 (9.15x10^-344.3x108) 0.7 2.53x105 (0.82x10^-3.39x105) (0.9-129.9) (1.2-108.3) ( 0 .0 - 4 9 .4 ) LD5 w it h 95% fiducial lim its 2 8 .8 1.1 (0.01-10.5) 21.6 (0.4-114.7) 4 .6 32.7 (0.3-135.9) 2.59xl05 (2.75x10^-6.36x108) (6 7 1 -4 2 8 3 ) (1576-34904) (326-1.843) 28.8 fiducial lim its 1352 (380-11.465) 3403 (1255-10.045) 2914 (1066-27.315) 2 7 5 -8 6 1821 268 -8 6 854 Strain LD50 with 95% Standard error of slope Heterogeneity about regression line with 2 degrees of freedom. A ll not significant (P > 0.05) TABLE 23. Virulence of fungi to adult Nephotettix virescens at optimal and suboptimal relative humidity. LD 9 9 days after conidial application and suboptimal rh _5 _5 days after conidial application and optimal rh * ** B. bassiana 261-85 M. anisopliae 82-82 M. anisopliae 83-82 5371 i i B. bassiana 266-85 -179-VQ 300 Fig. 2 5 'Strain comparison of haemocytometer \/ / A and ■ Petri plate'l I I I counts to estimate number of conidia from Beauveria bassiana and Metarhizium anisopliae adhering to Nephotettix virescens adults. Bars represent 95 % confidence limits obtained from the mean of ten replicates. - 1 8 0 - 14. Effect of temperature on mortality Adult GLH treated with B. bassiana or M. anisopliae conidia died at rates dependent on the incubation temperature. B. bassiana 110-82, and 268-86 killed insects more rapidly at 25* C than at 20 or 30*C, while M. anisopliae 275-86 caused insect death most quickly at 30*C (Table 24). This data agrees well with i n v i t r o growth data obtained on agar, where the two B. bassiana isolates grew optimally at 25*C and M. a n i s o p l i a e 275-86 at 27*C (II, 11, B) 15. Influence of relative humidity on infection Infection at optimal and suboptimal relative humidity Relative humidity greatly influenced the pathogenicity of fungi to GLH adults. Analysis of variance of the factors which influence GLH mortality showed differences (P< 0.001) in mortalities at the two rh levels provided by ventilated (92-96%; suboptimal rh) and closed (100%, optimal rh) covers (Table 25). There were also differences (P<0.001) in the pathogenicity of the four strains examined. When insect death was related to time, the response observed was linear rather than quadratic (Fig. 26). Fungi killed insects most quickly in optimal humidity conditions, and all four isolates killed over 80% of insects within 150 hours (Fig. 26). B. bassiana 110-82 was the fastest killing isolate with eLT50 values of 85 h. at optimal rh and 130 h. under conditions of -181- TABLE 24. Effect of temperature on pathogenicity of fungi to Nephotettix virescens adults as measured by estimated L T ^ q* Incubation temperature S t r a in 20°C 25°C 30°C E s tim a te d L T ^ q (h o u rs ) Beauveria bassiana 110-82 126.1 8 2 .5 9 1 .8 268-86 134.9 7 8 .1 8 1 .3 Metarhizium anisopliae 275-86 139.6 8 7 .1 6 6 .6 M ortalities were determined in optimal relative humidity c o n d itio n s -182- - k kkk kk kk *** kkk - - - - - kkk - •kick kkk kkk kkk F Significance 1 .5 3 2 .2 9 1 .4 0 3 .6 5 0 .9 8 4 .6 6 0 .4 9 2 .4 9 0 .8 1 1 6 .8 6 9 2 .1 3 2 7 .6 0 4 1 .3 7 1 8 6 .0 0 4 9 9 .1 4 10 8 5 .2 0 1 .0 4 1 .5 5 2 .4 7 0 .6 6 0 .3 3 0 .5 2 0 .7 8 1 1 .4 4 1 1 .6 2 1 7 .1 4 3 3 8 .6 3 1 .6 4 0 .5 4 1 .0 4 7 .4 3 8 .0 0 6 .2 1 2 .1 1 3 .9 9 9 .9 3 .1 6 1 7 .0 8 0 .9 4 18.72 1 8 .7 2 7 5 .9 8 0 .6 7 6 2 .5 0 6 2 .5 0 3 0 .4 5 1 .7 0 6 9 .7 6 736.23 736.23 3 3 8 .6 3 757.06 126.17 1 4 0 7 .5 0 Sum Sum of squares Mean square < 0.05; - P > 0.05 (i.e. NS) 1 1 3 1 4 3 1 1 3 6 4 6 3 3 4 .3 2 3 84.19 28.07 12 12 18 18 112 167 ** P < 0.01; * P *** P < 0.001; Source of variation Degrees of freedom conidial applications with four fungal strains and incubation at optimal and suboptimal relative humidity conditions TABLE 25. Analysis of variance of several factors which influence the m ortality of Nephotettix virescens adults after T o ta l Strain x humidity x linear response Strain x humidity x quadratic response R e s id u a l Strain x humidity of incubation Strain x time of incubation Strain x humidity x time of incubation D e v ia tio n s D e v ia tio n s Time of incubation Linear response D e v ia tio n s Strain x quadratic response Humidity x linear response S t r a in D e v ia tio n s Humidity x time of incubation Humidity x quadratic response Humidity of incubation Quadratic response -183- Mortality Fig. 26 Mortality of adult Nephotettix virescens under virescens adultNephotettix of Fig. Mortality 26 conidial application of Beauveria bassiana 110-82(X ),110-82(X bassiana Beauveria ofapplication conidial optimal ( intervals for different levels oflevelsmortality. fordifferentintervals 275-86(-3K). 95% confidence the are approximately Bars )269-86( and268-86(O 4* anisopliae), andMetarhizium ------)suboptimal(------) and rh, after -184- ♦ suboptimal rh. M. anisopliae 275-86 killed insects rapidly at optimal rh (eLT50 105 h.) but performed poorly at suboptimal rh (eLT50 205 h.) and was inferior to B. bassiana 269-86. EL Influence of high humidity period on pathogenicity of fungi to N ephotettix virescens After 72 hours at optimal rh, adult GLH responded similarly to treatments with B . b a s s i a n a 268-86, and M. anisopliae 275-86. In contrast, B. bassiana killed insects more rapidly than M. anisopliae when the optimal humidity period was restricted to 24 or 48 h. (Fig. 27). Analysis of variance of the log10 eLT50 indicated the mortality response to increasing high humidity period was linear (P<0.001) for both isolates; i.e. an increase in the period of high humidity gave a proportional increase in insect mortality. 16. Improvement of pathogenicity * Aj. Effect of adding nutrients to spore suspensions M. anisopliae 275-86 had similar eLT50 values irrespective of the nutrient added to spore suspensions though there was some indication that high nutrient levels delayed infection (Table 26). In contrast, B. bassiana 268-86 showed increased pathogenicity in response to the addition of nutrients, and adding 1% skimmed milk reduced the eLT50 value from 118 h. to 94.8 h. The experiments should -185- 250 4-> -H 100 C O hlT tO lS . C&HTiNi/OUf JL H 1 9 H HunioiTi 50 0 0 20 40 €0 t o ~ m 120 HO 160 160 Period of high humidity(hours) Fig. 2 7 The effect of varying periods of high humidity on the virulence of Beauveria bassiana 268-86(•---- ) and Metarhizium anisopliae 275-86(------) to adult Nephotettix virescens. Bars represent 95X confiden ce limits obtained from the mean of three replicates -186- # TABLE 26. Effect of adding nutrients to spore suspensions on the virulence of fungi to adult Nephotettix virescens* Estimated LT^q (hours) Treatment Beauveria Metarhizium bassiana anisopliae 268-86 275-86 A - 5x10^ conidia/ml in 0.05% Triton X-100 118.8 95.9 B - Treatment A + yeast 0.1% + glucose 1% 107.1 101.2 C - Treatment A + yeast 0.01% + glucose 0.1% 115.6 99.0 D - Treatment A + skimmed milk 1% 94.8 109.4 E - Treatment A + skimmed milk 0.1% 105.0 97.2 F - untreated >200 >200 * Mortalities were determined in optimal relative humidity -187- be repeated to confirm the observations but unfortunately time precluded this during the present study. B j, Effect of con id la 1 pregermination Adding nutrients to conidial suspensions of B. bassiana 268-86, and allowing germination for 6 or 12 hours before insect treatment, reduced eLT50 values compared to those obtained after treatment with non pre-germinated conidia (Table 27). Lowest eLT50 values were obtained when nutrients were not removed from suspensions applied to * insects. Pregermination of spores in the absence of nutrients reduced pathogenicity. In contrast, pregermination of M. anisopliae 275-86 conidia for 12 hours in the absence of nutrients increased pathogenicity. Pregermination of conidia and nutrient removal had little effect on mortality, while pregermination and conidial treatment with nutrients decreased pathogenicity markedly. Thus, the strains of B. bassiana and M. anisopliae studied, responded very differently to nutrient addition and pregermination. Further studies should be undertaken to confirm these observations. (L Selection of single spore isolates (SSI) i) Standardization of single spore isolates by weight Conidial numbers observed per mg in nine SSI's and MSI of B. b a s s i a n a 268-86 were similar and ranged from 6.63 to 7.80X107 with an overall mean of 7.01X107. An analysis of variance of the means showed no significant differences between SSI's ( P<0.05; Fig. 28). The results suggested the preparation of conidial suspensions of -188- 90.7 122.8 108.7 125.5 110.1 (hours) 275-86 q >200 anisopliae Metarhizium LT^- 97.7 105.1 131.2 109.2 100.9 106.6 131.2 117.1 106.1 268-86 > 20 0 bassiana Beauveria Estimated Estimated Nephotettix virescens Nephotettix hours hours germination hours germination + nutrients nutrients + germination hours hours germination + nutrients + washing + nutrients washing + hours germination 6 6 6 * Mortalities were determined in optimal relative humidity Mortalities were relative optimal determined in * Treatment TABLE 27. Effect of pregerminating conidia on the virulence of fungi to adult F - Treatment + A Treatment - F G - Treatment A + 12 hours germination + nutrients nutrients + + hours germination A Treatment - G 12 E - Treatment A + 12 hours + hours germination A Treatment - E 12 B - Treatment + A- Treatment B H - - H Untreated D - Treatment + A Treatment D - C - Treatment A + 12 hours germination + nutrients + washing + washing nutrients + + hours germination - A Treatment C 12 A - 5x10^ conidia/ml in 0.05% X-100 Triton 0.05% conidia/ml in - A 5x10^ -189- Fig. 2 8 Number of conidia per mg found in nine single spore isolates(SSI) and multi spore isolate (MSI) of Beauveria bassiana 268-86. Bar represent least significant difference between strains (P - 1 9 0 - SSI's from the same MSI, by suspending standard weights of conidia in 0.05% Triton X-100 can be used to prepare suspensions with similar spore concentrations. ii) Pathogenicity of Beauveria bassiana 268-86 single spore isolates to adult N ephotettix virescens When one hundred SSI's from B. bassiana 268-86 were examined for pathogenicity to adult GLH, 45 were less pathogenic than the MSI and 55 showed activity similar to the MSI (Table 28). Thirty one SSI's with variable pathogenicities were then examined further and four were similar to the MSI, two less pathogenic and the remaining 25 showed increased virulence (Table 29). Nine SSI's were examined further in replicated experiments and the data used to calculate eLT50 values. An analysis of variance of the eLT50 established significant differences between the SSI's (P<0.01). Single spore isolates 28 and 96 had reduced eLT50's compared with the MSI and isolate 81 an increased value, while six SSI's were similar (1, 8 , 18, 97, 98, and 99; Table 30). When nine SSI's of M. anisopliae 275-86 were examined for pathogenicity to adult GLH, one (SSI 5) proved more pathogenic and one (SSI 9) less pathogenic than MSI (P<0.01, Table 30). Dj, Correlation between pathogenicity and rate of con id la 1 germination on SPA The time required to reach 50 and 95% conidial germination (GT50 and GT95) on SDA at 25±1*C, and the eLT50 to GLH adults in optimal rh conditions were calculated by MLP for MSI and nine SSI's of B. TABLE 28. The effect of single (SSI) and multi spore isolates (MSI) of Beauveria bassiana 268-86 on Nephotettix virescens* No of No of Mortality of cadavers Mortality of cadavers GLH adults after 10 d GLH adults after 10 d SSI after 5 days SSI after 5 days (%) 0 0 Stuck Sporulated Stuck Spor 1 100 0 6 26 70 2 6 2 40 1 6 27 40 3 6 3 30 2 3 28 100 4 6 4 100 3 3 29 60 0 1 5 40 4 5 30 80 3 7 6 20 1 4 31 80 3 5 7 100 2 6 32 100 4 7 8 100 3 8 33 450 4 7 9 100 2 5 34 50 2 7 10 100 5 8 35 80 3 4 11 30 2 4 36 70 1 3 12 50 4 8 37 100 3 8 13 80 2 6 38 100 1 5 14 90 4 8 39 30 2 4 15 50 2 8 40 90 1 6 16 90 0 4 41 80 2 5 17 80 2 8 42 70 2 7 18 80 2 10 43 70 0 6 19 100 3 6 44 100 4 8 20 50 4 7 45 80 3 8 21 100 3 6 46 70 0 4 22 80 5 8 47 80 1 1 23 100 2 7 48 80 3 5 24 70 2 5 49 70 1 5 25 100 2 9 50 100 0 1 /Continued... TABLE 28. (Continued) No of No of Mortality of cadavers Mortality of cadavers GLH adults after 10 d GLH adults after 10 d SSI after 5 days SSI after 5 days (%> (%) Stuck Sporulated Stuck Spor. 51 100 3 6 76 90 0 3 52 100 2 5 77 100 1 6 53 100 1 1 78 90 3 7 54 60 1 4 79 100 3 5 55 80 3 6 80 40 0 3 56 100 4 8 81 70 4 9 57 100 0 2 82 100 2 5 58 90 2 7 83 100 5 0 59 100 2 10 84 100 3 7 60 90 5 7 85 100 1 4 61 100 3 7 86 100 0 1 62 100 3 8 87 100 3 / 63 80 3 6 88 100 3 3 64 100 1 4 89 100 3 5 65 100 4 7 90 80 1 4 66 50 0 0 91 100 1 2 67 100 1 2 92 100 3 7 68 100 4 5 93 100 0 2 69 100 1 4 94 100 1 4 70 100 2 4 95 100 0 9 71 100 2 7 96 100 0 9 72 100 1 2 97 100 4 7 73 100 3 4 98 100 4 6 74 100 4 7 99 100 3 6 75 100 2 0 100 100 0 10 MSI 100 2 8 * Mortalities were determined in optimal relative humidity conditions * TABLE 29. Virulence of Beauveria bassiana multi and selected single spore isolates to adult Nephotettix virescens* Isolates ranked for % cumulative mortality pathogenicity to GLH after 4 days** SSI 1 100 II 96 93 M 28 80 If 51 66 It 92 60 It 74 53 II 99 53 It 06 46 II 08 46 It 54 46 II 61 46 II 69 46 II 57 40 If 60 40 II 83 40 It 10 33 It 53 33 It 26 26 It 65 26 It 66 26 It 68 26 If 71 26 It 97 26 It 98 26 It 32 20 It 21 13 It 38 13 It 75 13 It 84 13 MSI 13 SSI 18 6 It 81 0 * Mortalities were determined in optimal relative humidity conditions ** Figures are results of a single bioassay - 1 9 4 - TABLE 30. Virulence of Beauveria bassiana and Metarhizium anisopliae single and multi spore isolates to adult Nephotettix virescens Mean green leaf- Mean estimated hopper cadavers Strain LT50 (hours)* with sporulating mycelium after 10 days + SE Beauveria bassiana 268-86 SSI 1 78.3 c** 3.0 + 0.6 ii 08 92.0 c 12.0 + 2.5 h 18 89.6 c 9.6 + 3.5 ti 28 77.6 a 7.6 + 2.0 it 81 104.2 b 11.3 + 0.8 it 96 77.8 a 11.6 + 3.4 ii 97 87.8 c 10.6 + 0.6 it 98 85.3 c 19.3 + 2.3 it 99 88.2 c 15.0 + 1.5 MSI 90.3 c 7.0 + 3.2 SED = 6.05 Metarhizium ,anisopliae 275-86 SSI 1 102.8 c 8.0 + 1.5 ii 2 113.4 c 6.6 + 2.6 ti 3 119.6 c 9.8 + 3.4 ii 4 105.0 c 2.3 + 0.8 ti 5 97.9 a 1.6 + 0.3 ii 6 102.1 c 3.0 + 0.5 ii 7 116.8 c 6.3 + 2.3 ii 8 113.7 c 7.6 + 2.4 ii 9 124.6 b 5.0 + 1.7 MSI 111.3 c 6.0 + 1.5 SED 6.55 * Figures are mean of three relicated bioassays with different batches of insects in optimal relative humidity conditions ** Strains followed by a: Shorter estimated LT50 than MSI b: Longer estimated LT50 than MSI c: Same estimated LT50 as MSI (P <0.05) - 1 9 5 - bassiana 268-866 (Table 31) GT50 and 95 values were then related to eLT50 values and correlation coefficients of -0.321 and -0.421 respectively calculated (Fig.29). Those values Indicated that there was no correlation between conldial germination rates and pathogenicity as calculated using eLT50 values 17. The use of the optical brightener bl-striazinvl amino stilbene to study spore germination i n v i v o /L Con id la 1 germination i n v i v o under optimal and suboptlmal relative humidity. At a constant 100% rh a small percentage of both M. anisopliae 275-86 and B. bassiana 268-86 spores started germinating on adult GLH wings after 8 h. Germination only increases slowly and both strains approached their maximum germination after 69 h. (Table 32). For both strains the highest percentage germination observed was approximately 70%. Post germination mycelial development proceeded similarly for both strains although in M. anisopliae 275-86 conidiation was first observed after 69 h. compared to 93 h. for B. bassiana 268-86. Under sub-optimal rh conditions (92-96%) both B. bassiana and M. a n i s o p l i a e germinated more slowly than at optimal rh and both strains approached their highest germination (c.a. 60%) after 116 h. Compared with optimal rh conditions, conidiation was delayed for both strains, but B. bassiana produced conidia more rapidly (after 116 " 1 9 6 " TABLE 31. Comparison of GT50 and 95 values with estimated LT50s obtained from multi and single spore isolates of Beauveria bassiana 268-86 Estimated Isolate GT50 (hours) GT95 (hours) LT50 (hours) + SE + SE + SE % SSI 1 14.9 + 0.1 19.8 + 0 .2* 78.5 + 5.33** " 8 14.8 + 0.1 19.1 + 0.2 92.0 + 7.8 " 18 13.1 + 0.1 16.6 + 0.1 89.6 + 3.3 " 28 15.3 + 0.1 19.4 + 0.2 77.6 + 3.0 " 81 14.6 + 0.1 18.3 + 0.1 104.2 + 4.4 " 96 15.0 + 0.1 20.3 + 0.2 77.8 + 5.9 " 97 14.7 + 0.1 19.5 + 0.2 87.8 + 5.4 " 98 16.0 + 0.1 21.0 + 0.3 85.3 + 5.5 " 99 14.8 + 0.1 19.9 + 0.2 88.2 + 3.2 MSI 14.8 + 0.1 18.4 + 0.1 90.3 + 1.3 * Figures are mean of nine replicates ** Figures are mean of three replicates - 1 9 7 - 41 Fig. 29 Relationship between germination rate and time of death in ten isolates of Beauveria bassiana 268-86(GT50 related to LT50 + ; GT95 related to LT50 o ). -198- C D E C B B B B A A A growth** Mycelial Complete .2 0.8 + 6 Suboptimal Suboptimal rh . + SE % germ. germ. % 12 58.1+11.6 59.3+7.3 50.6+13.5 1.5+0 35.6+8.5 19.3+1.4 30.0+9.8 0 0 0 conidia; conidia; C: E E E C D E B B B A growth** Mycelial Metarhizium anisopliae Metarhizium anisopliae 275-86 .8 Optimal Optimal rh + SE % germ. germ. % 71.0+3.4 E 64.1+6.3 69.0+9.5 67.3+7.5 64.3+3.4 1.3+0.3 61.3+6.3 61•0+6.3 5.6+0 25.0+2.8 0 D E E C E C B B A A A growth** Mycelial ; ; B: Tubes diameter of longer than Suboptimal rh Suboptimal + SE % germ. germ. % 58.0+6.8 57.6+8.3 10.3+4.3 53.3+8.8 35.0+11.5 1.3+0.3 42.3+11.5 24.3+7.3 0 0 0 # E D E E C E C B B B A growth** Mycelial Beauveria bassiana bassiana 268-86 Beauveria .6 Optimal Optimal rh 0.6 + 0 + SE . % germ. germ. % on adult Nephotettix virescens at optimal and suboptimal relative humidities relative optimal and at suboptimal virescens Nephotettix adult on 66.3+5.2 67.3+5.3 62.8+4.5 59.3+6.3 59.3+6.3 46.6+6.1 3.6+0 0 growth key:growth A: No germination tubes colonization over cuticle; cuticle; over colonization under cuticle; colonization D: Noticeable E: Production new conidia of 8 2 4 69 16 7.0+2.0 91 12 21 45 50.0+6.3 142 132 116 (hours) period TABLE 32. TABLE 32. Metarhizium anisopliae 275—86 and bassiana Beauveria 268-86 of growth mycelial and Germination ** ** Mycelial * * Figures mean are of replicates three Incubation -199- h.) than M. anisopliae (142 h). A sequence of conidial germination and hyphal development for both B. bassiana and M. anisopliae at optimal rh is shown in Plates 10 to 13. B_j. Correlation between germination I n v l t r o and i n v i v o There was a significant, positive correlation (r=0.863; P<0.05) between the germination of B. bassiana 268-86 spore types (submerged conidia, hyphal bodies and aerial conidia) on SDA and N. virescens wings (Fig. 30). Hyphal bodies germinated more rapidly than the other spore types with GT50 values of 6.8 hours i n v i t r o and 13.4 hours i n v i v o , compared to 7.3 and 14.6 h for submerged conidia and 15.3 and 22.3 h for aerial conidia (Table 33). Maximum germination levels observed i n v i t r o were generally higher than those i n v i v o . Hence i n v i t r o , at least 95% of hyphal bodies, submerged and aerial conidia germinated, compared to 93, 90 and 60% respectively i n v i v o . 18. Assessment of disease spread /L Spore production on Insect cadavers Fungal strains varied in the number of spores produced on dead insects. Three M. anisopliae strains were studied and produced between 1.1 and 2.5X107 conidia per insect, while B. bassiana strains produced from 1.3 to 8.7X107 conidia per insect (Fig. 31). B. bassiana - 2 0 0 - a) Beauveria bassiana 268-86 germinating conidia b) Metarhizium anisopliae 275-86 germinating conidi PLATE 10 Germination of aerial conidia on GLH wings after 12 hours at 25^*1° C and optimal rh (X150) a a) Beauveria bassiana 268-86 b) Metarhizium anisopliae 275-86 PLATE 11 Germination tubes growing on GLH wings after 14 hours at 25±1°C and optimal rh (X2 50) a b 202 a) Beauveria bassiana 268-86 b) Metarhizium anisopliae 275-86 PLATE 12 External colonization of GLH wings by mycelia after 40 hours at 2 5drl°C and optimal rh (X250) a i - 203- a) Beauveria bassiana 268-86 b) Metarhizium anisopliae 275-86 PLATE 13 Mycelial colonization within GLH wings, after 60 hours at 25^t 1 C and optimal rh (X150) a b 204 # 5-1w Fig.30 Relationship between germination rates in vitro and in vivo for Beauveria bassiana 268-86 submerged conidia((?) ) , aerial conidia(ffl) and hyphal bodies( A). - 2 0 5 - * TABLE 33. Relationship between germination of Beauveria bassiana 268-86 spores on Sabouraud dextrose agar and insect wings* Time to 50 Time needed for 50 or 95% germina or 95% tion with 95% fiducial limits Spore type germination (hours) on; (hours) SDA* Insect wings*** Submerged conidia GT50 7.3 (7.2-7.5) 14.6 (14.2-15.1) GT95 12.1 (11.9-12.4) 29.3 (28.1-30.4) Hyphal bodies GT50 6.8 (6 .7-6.9) 13.4 (13.1-13.9) GT95 10.10 (9.7-10.5) 28.2 (27.1-29.4) Aerial conidia GT50 15.3 (15.2-15.4) 22.3 (21.5-23.2) GT95 20.1 (19.8-20.3) 43.4 (41.0-46.6) * Germination trials were carried out at 25 +_ 1°C and optimal humidity conditions ^ ** Figures are mean of nine replicates with different batches of conidia *** Figures are mean of three replicates with different batches of conidia - 2 0 6 - # Fungal isolate Fig. 31 Spore production on adult Nephotettix virescens infected with Beauveria bassiana(Bb) 110-82, 138-83, 235-85, 261-85, 266-85, 268-86 269-86 . and 270-86 or Metarhizium anisopliae(Ma) 82-82, 83-82 and 275-86. Bars represent 95^< confidence limits obtained from the mean of ten replicates. - 2 0 7 - strains: 261-85, 268-86, 269-86 and 270-86 produced most spores with means of 8.7, 7.7, 5.4 and 5.3X107” conidia per insect respectively. EL Selection of strains by number of dead insects producing conidia and infection of nymphs Disease spread from cadavers to healthy insects and adhesion of cadavers to rice plants varied between strains. B. bassiana 268-86, 269-86, 110-82 and 261-85, and M. anisopliae 275-86 stuck more than 20% of dead insects to the rice plant (Table 34). B. bassiana 268- 86, 269-86, 110-82 and 138-83, and M. anisopliae 275-86 sporulated on a high proportion of dead insects. The highest disease transmission from dead adults to nymphs, with percentages of 68.38, 52.50, 44.34 and 35.00 occurred with B. bassiana 268-86 (Plate 14) 110-82, 269-86 and 138-83, respectively. M. a n i s o p l i a e 275-86 was the better of the two strains of this species examined, although the proportion of infected nymphs was very low at (2.01%; Plate 15). In order to further assess disease spread, B. bassiana 268-86, 269- 86 and 110-82, and M. anisopliae 275-86 were evaluated in replicated trials under optimal (100%) and suboptimal (92-96%) rh. In B. bassiana isolates, similar numbers, ranging from 10 to 60% of adult insects, became attached to the rice plant irrespective of rh conditions (Fig. 32). Slightly more M. anisopliae treated insects were stuck to the plant in optimal rh conditions and B. bassiana 268-86 stuck significantly (P<0.001) more dead insects to the plants (50- 60%) than the other strains. When strains were compared with respect to the number of # TABLE 34. Disease transmission from infected adult Nephotettix virescens to nymphs in Metarhizium anisopliae and Beauveria bassiana* Nephotettix virescens cadavers Strain % infected nymphs*** % stuck on % with sporulating plant mycelia** Metarhizium anisopliae 83-82 1.25 12.50 0 275-86 27.50 45.00 2.01 Beauveria bassiana 110-82 23.75 56.25 52.50 138-82 18.75 55.0 35.00 235-85 8.75 41.25 10.11 261-85 22.50 48.75 11.75 266-85 18.75 42.50 4.74 268-86 51.25 70.00 68.38 269-86 45.00 57.50 44.34 Bioassay was carried out at 2 5 1°C and optimal relative humidity conditions ** Stuck and with sporulating mycelia cadavers recorded at 12 days after infection *** Infected nymphs recorded at 20 days after infection * - 2 0 9 - PLATE 14 GLH cadavers attached to a rice plant with sporu lating mycelia of Beauveria bassiana (268-86). The arrows indicate GLH nymphs that have also be come infected. (X6) -210- PLATE 15 GLH cadaver attached to a rice plant with sporulating mycelia of Metarhizium anisopliae (275-86). (X7) -211- Fungal isolate Fig. 32 Adhesion of adult Nephotettix virescens to rice plants after treatment with Beauveria bassiana (Bb) 110-82, 268-86 and 269-86 or Metarhizium anisopliae(Ma) 275-86 and maintenance at optimal ( ) or suboptimal( 11 I I I I ~1 ) relative humi dity. Bars represent 9 5 ^ confidence limits ob tained from three replicates. - 2 1 2 - sporulating GLH cadavers, similar numbers were observed for each B. b a s s i a n a strain under optimal or suboptimal rh conditions (Fig. 33). M. anisopllae 275-86 sporulated on more cadavers under optimal compared to suboptimal rh conditions. The highest number of sporulating cadavers 080%) was observed in B. bassiana 268-86 under optimal rh conditions. In contrast there were no differences between B. bassiana strains under suboptimal rh conditions (P<0.001). Spread of mycosis from B. bassiana- treated, adult, GLH to nymphs was similar under optimal or suboptimal rh conditions. In M. anisopllae, the number of infected nymphs was slightly higher under optimal than suboptimal rh conditions (Fig. 34 ). B. bassiana 268-86 infected most nymphs both under optimal or suboptimal rh (P<0.001) with some 70 and 60% infection, respectively. C^. Correlation between number of sporulating and stuck GLH cadavers and disease spread to nymphs A correlation analysis between the number of sporulating GLH cadavers infected with B. bassiana 110-82, 268-86 and 269-86 and infection of nymphs under optimal rh conditions, gave a correlation coefficient (r) of 0.856 (PC0.01) (Fig. 35). A similar analysis of data obtained under suboptimal rh conditions, gave a r value of 0.806 (P<0.01) (Fig. 36). The number of GLH cadavers attached to rice plants was highly correlated (P<0.01) with the number of infected nymphs; with r values of 0.939 at optimal rh and 0.804 at suboptimal rh (Figs. 37 and 38).). - 2 1 3 - # 110-82 268-85 269-86 275-86 Fungal isolate * Fig.33 Sporulation of Beauveria bassiana(Bb) 110-82, 268-86, and 269-86 or Metarhizium aniso- pliae (Ma) 275-86 on adult Nephotettix virescens after maintenance at optimal ( Y / / A ) or suboptimal ( II I II (1 ) relative humidity. Bars represent 9 5 % confidence limits obtained from the mean of three replicates. - 2 1 4 - Bb Bb Bb Ma 110-82 268-S6 269-86 275-86 Fungal isolate Fig,34 Spread of mycoses on nymphs after treatment of adult Nephotettix virescens with Beauveria bassiana(Bb) 110-82, 268-86 and 269-86 or Metarhizium anisopliae 275-86 and maintenance at optimal( K 7 7 ^ ) or suboptimal( FI I I I II) rela tive humidity. Bars represent 9 5 % confidence limits obtained from the mean of three replicates. - 2 1 5 - Fig. 35 Relationship between number of adultofnumberbetweenNepho- Relationship Fig.35 Number of._J.nfected_pymphs 0 tettix virescens cadavers with sporulating cadavers mycelium with virescens tettix mal relativehumidity.mal ln, ihsra fmcss toofnymphsopti underspreadmycoses plant, with fBavrabsin (o) andattachedricetothe Beauveriaof bassiana 5 T 15 ~To Cadavers attached to the rice plant rice the to attached Cadavers - 6 1 2 - 20 5 0 35 30 25 G Fig. 36 Relationship between number of adult Nepho- tettix virescens cadavers with sporulating mycelium of Beauveria bds'siana (o) and attached to the rice plant, with spread of mycoses to nymphs under sub- optimal relative humidity. - 2 1 7 - » _J______1______I______1______L. 35 40 45 50 55 Sporulating cadavers + Fig. 37 Relationship between number of adult Nepho- tettix virescens cadavers with sporulating mycelium of Beauveria bassiana (0), and spread of mycoses to nymphs under optimal relative humidity. - 2 1 8 - # 450 - o 400 - » 0 35 40 45 50 55 Sporulating cadavers + Fig. 33 Relationship between number of adult Nepho- tettix virescens cadavers with sporulating mycelium of Beauveria bassiana (©), and spread of mycoses to nymphs under suboptimal relative humidity. - 2 1 9 - 19. Effect of subculturing and passaging on pathogenicity of Beauveria bassiana and M etarhizium anisopliae to adult N ephotettix v l r e s c e n s Passaging B. bassiana 268-86 SSI81 and SSI96 through GLH adults or subculturing on SDA caused changes in pathogenicity to N. v l r e s c e n s , as indicated by changes in their Mean time of death (MTD) (Figure 39). Samples taken from liquid nitrogen (LN) at the beginning and at the end of the experiment showed no differences in their virulence to GLH. Both B. basslana multi spore (MSI) and single spore isolates (SSI) had increased virulence to GLH after passage through insects, with the effect most pronounced after two passages. Surprisingly subsequent passages failed to maintain the increased virulence and the MTD increased until after four to five passages, virulence was similar to the initial level. Spores from insects always gave a lower MTD response than spores from SDA and in most cases this difference was significant. With both B. bassiana MSI and SSI's pathogenicity stabilized after five insect passages to a level similar to that observed for liquid nitrogen. Similarly, virulence stabilized for SSI 96 and the MSI after five SDA subcultures but not for SSI 81 which steadily continued to exhibit reduced virulence until it became virtually non pathogenic (MTD>160 h). Throughout the experiment B. bassiana SSI 96 was more pathogenic than the MSI, whereas SSI 81 was similar to the MSI during insect passage and less virulent during SDA subculture. After a single passage through insects, M. anisopliae 275-86 and t “ 2 2 0 " 170 w pi 50 &o & o • t5J130 4-1 O o o 70 50 I l I I ! • ' i i i : ! I I I ! Ln 2 4 6 8 Ln Ln 2 4 6 8 Ln Ln 2 4 6 8 SSI95 MSI SSI81 Number of passages or subcultures * Fig. 39 Effect of subculturing on Sabouraud dextrose agar( o ) and passaging through insects( • ), on the pathogenicity of two single spore isolates(SSI) and multi spore isolate(MSI) of Beauveria bassiana 268-86 to adult Nephotettix virescens, compared with MSI maintained in liquid nitrogen(Ln, ©). Bars represent least significant difference between points (P<0.05). # - 2 2 1 - single spore isolates SSI 1 and SSI 3 failed to produce sufficient spores to allow treatment of further insects. Thus, it was only possible to examine the effect of SDA subculturing on pathogenicity (Fig. 40). In M. anisopliae increased virulence was observed after a single passage through GLH. Virulence decreased after subculturing on SDA though the effect was less marked than with B. bassiana ; SSI1 was always more virulent than the MSI or SSI3 20. Pathogenicity of Beauveria bassiana 268-86 hvphal bodies, aerial ^ and submerged conidia to adult N ephotettix vlrescens Beauveria bassiana 268-86 spores produced in submerged culture were more pathogenic than those produced on SDA or cracked wheat. Submerged conidia and hyphal bodies had LC50 values of 1.15 and 2.59X10e spores per ml respectively, while corresponding values for aerial conidia were 14.10 (wheat) and 10.00X10e (SDA). Liquid produced spores also killed GLH adults more rapidly than those produced on solid or semi-solid media with eLT50 values approximately 24 hours * less for submerged spores than for aerially produced conidia. (Table 35). - 2 2 2 - 1 4 0 to 2u &O A-P fd 120 □ Q) □ A o □ A A I____ L Ln 2 4' 6 8 Ln Number of passages or subcultures Fig. 40 Effect of subculturing on Saburaud dextrose agar (open symbols) and passaging through insects (closed symbols), on virulence of single spore isolate 1(0), 3(D) and multi spore isolate (A) of Metarhizium anisopliae 275-86, compared with isolates maintained in liquid nitrogen (Ln , @ , 0 , ^ ) . Bar repre sent least significant difference between points (P(0.05), - 2 2 3 - 3.55 SED 96.9 120.2 (hours) LT5 0* * * 0* LT5 Estimated Estimated conditions 15.08 15.97 127.1 27.06 99.1 humidity Chi2*** relative 0.16 0.15 0.2 ■ SE ■ 0.80 + 0.80 Slope 4 Slope in in optimal Upper limits 25 + 25 1°C 6.26 24.60 5.56 16.00 + 0.94 Lower Fiducial Nephotettix virescens* Nephotettix 2.59 1.58 3.97+ 1.03 LC50** 14.10 10.00 Conidia/ral x 10^ Conidia/ral after five days at days incubation after five on on SDA determined Spore Spore type Figures are combined results of 4 different multiple dosage multiple bioassays dosage different 4 of Figures results are combined Heterogeneity about regression line with 20 degrees of freedom; all not significant (P > 0.05) all (P not significant with degrees 20 of freedom; line Heterogeneity about regression TABLE 35. 35. TABLE adult to culture or submerged produced semi-solid in spores bassiana (268-86) Beauveria of Pathogenicity Submerged conidiaSubmerged 1.15 0.75 2.63 + 0.71 0.09 23.04 * * were Mortalities Hyphal Hyphal bodies Aerial produced Aerial conidia Aerial producedAerial conidia wheat on -224- DISCUSSION Bloassay system The most important characteristic of a fungal pathogen is its virulence to a determined target insect and this parameter can be only measured by laboratory bioassay. Several assay systems for entomogenous fungi have been developed but more research is needed (Yendol and Rosario, 1972; Wilding, 1976; Hall, 1976b; Sweeney 1976; Papierok and Wilding, 1979; Vandenberg and Soper, 1979; Fargues 1981; Milner and Soper, 1981). Hall and Papierok (1982) considered that of all the groups of microorganisms, entomogenous fungi pose the greatest difficulties in bioassay design as the fungal propagules must be delivered to the surface of the host in a standardized manner. Rahman e t a l . (1986) showed that the presence of rice plants is necessary for survival of N. virescens after treatment. In the present study, the bioassay systems followed by Gillespie (1984) and Aguda e t a l . , (1985) to assay entomogenous fungi against BPH were modified to provide a suitable assay system for N. virescens. Potted rice plants were used as a food source and plastic covers with variable numbers of muslin covered holes used to obtain three levels of rh and to contain insects on the plants (III, 1, B). To reduce the likelihood of insects shedding the inoculum with the cast skins during ecdysis (Fargues and Vey, 1974), one to three day old adults GLH were used in all bioassays. The use of two batches of control insects allowed a better understanding of the causes of mortality. The. untreated batch allowed the identification of mortality factors characteristic of the insects # e.g. age, quality of food, and the Triton- treated batch allowed the identification of the effect of the treatment procedure. Mortality observed in controls of around 25% during the preliminary screening was reduced to below 10% in the subsequent multiple dosage bioassays. Immersing insects in conidial suspensions proved to be a good method of applying spores to adult GLH in a standardized manner. High correlations between concentrations of spore suspensions and mean of conidia adhering to insects were obtained for all the strains assayed (Table 22). These data agree with that obtained by Gillespie (1984) who assayed M. anisopliae and P. fum osoroseus strains against the glasshouse leafhopper H auptidia maroccana and found similar high correlations. Nevertheless, another important feature emerged from the data obtained; strains varied in the proportion of spores adhering to GLH cuticle indicating another variable characteristic between strains which must influence their virulence. Mean percentages adhering were 0.052, 0.041 and 0.013 respectively, for B. b a s s i a n a 261-85, 268-86 and 266-85, and 0.039, 0.026 and 0.08, for M. a n i s o p l i a e 275-86, 82-82 and 83-82. The importance of adhesion was shown by Al-Aidroos and Roberts (1978) in the infection of mosquitoes by M. anisopliae. Later its genetic nature was better understood when Al-Aidroos and Bergeron (1981) indicated that a gene determining specific adhesion in M . a n i s o p l i a e was linked to a gene for brown spore colour. Fletcher e t a l . , (1980) divided adhesion into three successive stages: -Adsorption of the spore to the insect cuticle -Attachment or consolidation of the interface between pregerminating propagules and cuticle. - 2 2 6 - -Germination and growth of the fungus on the cuticular surface until the emission of penetrant pegs. Fargues (1984) studied physical and chemical aspects of adhesion and Hall and Papierok (1982) suggested adhesion of conidia to insect cuticle as one factor affecting virulence of fungal strains. A detailed electron microscopy study may reveal the cause of the observed differences. Pathogenicity measurement The comparison of fungal strains in screening bioassays, showed inter and intra-specific variations in pathogenicity to GLH (Table 17) as has been reported by many researchers (Hall, 1977; Papierok and Wilding, 1981; Ignoffo, 1981; Gillespie 1984. Strains of V. lecanii, Trichothecium sp., S. Insectorum, M. flavoviride and P. farinosus failed to infect significant numbers of GLH adults. In contrast, most but not all, strains of M. anisopliae and B. bassiana were pathogenic. P. farinosus, B. bassiana and M. a n i s o p l i a e have been previously recorded infecting GLH in rice crops (Rombach, 1987) and intra-specific variation of B. bassiana in pathogenicity to GLH adults has been shown in field trials ( Aguda, 1984; Li, 1986). Several B. bassiana strains and M. anisopliae 275-86, were able to stick GLH cadavers onto the rice leaves and this permitted disease spread to nymphs. This characteristic should be of great importance in the field and has been highlighted by several authors (Papierok and Wilding, 1981; Hall, 1977; Gillespie, 1984; Matewele, 1986). Multiple dosage bioassays in sub-optimal rh conditions allowed the comparison of candidate strains by calculating LC50 values in - 2 2 7 - conditions which partially mimicked those of the rice canopy. For pathogens, such as bacteria, where a standard preparation can be maintained, differences between strains can be evaluated by the ratios between the LC50's of test and standard products in each assay, providing the lines are parallel. It is difficult however to prepare fungal standars as spores do not survive indefinitely and differences are indicated by a lack of overlap between the fiducial limits of the LC50 of one strain to another (Burges and Thompson, 1971). Based on this consideration, five strains of B. bassiana had similar virulence in suboptimal rh conditions, with strains 235-85, 268- 86, 110-82, 266-85 and 261-85 all having LC50 values between 2 and 12X10G conidia per ml (III, 12, A, i). Relative humidity alone can influence markedly the virulence of a candidate strain to a target insect (Gillespie, 1988), and in this study strains responded differently to changes in relative humidity. When multiple dose bioassays were conducted under optimal rh conditions using closed covers (100% rh), B. bassiana 110-82, 268-86, 269- 86 and M. anisopliae 275-86 increased their pathogenicity to GLH adults by 16, 4, 1.2 and 30 fold respectively, compared to pathogenicity in suboptimal rh conditions (III, 12, A, ii). The most striking increase in pathogenicity was observed in M. anisopliae 275- 86 which was highly virulent to GLH adults at optimal rh but only moderately active at sub-optimal rh. The relative humidity also influenced the speed at which adult GLH died (Fig. 26) and once again the contrasting behaviour of M. anisopliae 275-86 under suboptimal and optimal rh was noticeable. Thus, it can be concluded that humidity plays an important role in pathogenicity of fungal strains, and B. bassiana 110-82 and 268-86 -228- were highly pathogenic to adult GLH under optimal or suboptimal rh conditions. The influence of rh on germination and mycelial growth of fungal strains was discussed previously and can partially explain the influence of rh on pathogenicity (Gillespie, 1984). However, B. bassiana 268-86 and M. anisopliae 275-86 had similar virulence under optimal rh conditions but behaved quite differently in suboptimal rh (fig.26 ) but had similar mycelial growth profiles at different aw . Therefore other factors, apart from the effect of aw on spore germination and mycelial growth must have a role to play in the expression of virulence. The differences found perhaps suggest that survival of germinated and ungerminated spores may also be of importance in a situation of fluctuating relative humidity. Spores attached to insect cuticle must survive periods of rh, too low for growth until rh increases (Drummond et ai., 1987). It may be that B. b a s s i a n a survived small reductions in rh, occurring in the ventilated covers (table, 16) better than M. anisopliae. In fact B. bassiana 268- 86 survived longer than M. anisopliae 275-86 when both strains were stored in dry conditions at 5 or 20*C (II, 13, B). The observation that B. bassiana 268-86 was more virulent than M. a n i s o p l i a e 275-86 when the period of high rh was restricted possibly indicates that B. bassiana 268-86 conidia are able to penetrate and establish infection more quickly than M. anisopliae 275-86 . Such a characteristic is obviously of great importance. Influence of temperature in virulence Fungal strains responded differently to temperature. B. bassiana 110-82 and 268-86 killed insects more rapidly at 25 than at 20 or 30 *C, while M. anisopliae 275-86 was more virulent at 30*C (Table 24). This data seems to correlate well with i n v i t r o growth data obtained on SDA where B . b a s s i a n a isolates grew optimally at 25'C and M. a n i s o p l i a e 275-86 at 27*C (II, 11, B). Spore germination and limited mycelial growth must precede penetration and there should be a close correlation between temperatures optimal for growth on SDA and infection. In this study, no correlation was found between germination rates on SDA and pathogenicity of SSI's and MSI of B. bassiana 268-86 (III, 15, D). Conidial germination occurs early in the infection process (after the adherence of conidia to insect cuticle), and is just one component of a complex interaction. The speed of conidial germination could therefore be outweighed by other factors such as the ability of a strain to overcome host defence mechanisms. However mycelial growth rates i n v i t r o can not alone be taken as a reliable indication of pathogenicity as was found when B. b a s s i a n a 110-82, 138-83, 235-85, 261-85, 266-85, 268-86, 269-86 and 270-86 were compared for pathogenicity to GLH adult at 25±1*C (III, 12). The most virulent strains were not the ones with higher mycelial growth rates at this temperature (Figs. 2, 3, 4). Penetration of insect cuticle is at least partly dependent on enzymes (Brobyn and Wilding, 1977; Lambiase and Yendol, 1977; Grula e t a l . , 1978; St.Leger e t a l , 1986a)., thus optimum temperatures for conidial germination and mycelial growth i n v i t r o , may not be directly applicable i n v i v o , as the optimum temperatures for conidial germination and mycelial growth may differ from those for enzyme production and/or activity. w The estimation of LT50 as a measurement of fungal pathogenicity The virulence of a fungal strain to a target Insect must be assessed by bioassay and the usual parameters measured are LC50, LD50 and LT50- i.e. the concentration dose or time needed to kill 50% of test insects (Burges and Thompson, 1971). Referring to the practicalities of bioassay techniques, the use of multiple dosage bioassays to calculate LC50 or LD50's is more economical in number of batches of insects and less time consuming than a bioassay using a determined dose to obtain a proper LT50. Each mortality assessment must be made on separate groups of insects increasing the total # number employed. A feasible technique proposed and used in the present study is the use of only one batch of insects per treatment and the recording of dead insects at different times as a cumulative response to the treatment. Data obtained can be used to estimate the time necessary to kill 50% of treated insects (LT50) and can be reasonably compared with the "mean time of death" (Fenlon, 1989). The result will give an indication of whether the treatments are affecting the time of death and enable the selection of pathogenic strains. When it is intended to compare several treatments by their * estimated LT50's on a target insect, a t test or Anova can be used if bioassays are fully replicated. The availability of such a technique to compare fungal isolates for virulence to a given insect, using a small number of insects could be of great importance in microbiological control projects, particulary where resources are limited. The importance of insect uniformity and the size of the colony for bioassays has been discussed by Felton e t a l ., (1987). # - 2 3 1 - Augmentation of virulence After selection of virulent strains their efficacy in the field can be increased by several techniques, eg. formulation (Hall and Papierok, 1982). Data obtained in this study (III, 15, A) showed that M. anisopliae 275-86 did not . increase its virulence to GLH adults when nutrients were added to spore suspensions and there were some indication that high nutrient levels delayed infection. In contrast, B. b a s s i a n a , 268-86 increased its pathogenicity in response to the addition of nutrients. Skimmed milk at a concentration of 1% reduced the estimated LT50 value by almost 24 hours (Table 26). When the effect of adding nutrients and pregermination were studied simultaneously on these strains, the results confirmed that B. b a s s i a n a 268-86 showed enhanced virulence in the presence of nutrients, while N. anisopliae 275-86 responded to the pregermination treatment only (Table 27). M. anisopliae conidia are bigger than those of B. bassiana and this difference in size could explain these results. Matewele (1986) explained the higher pathogenicity of Tolypocladium cylindrosporum hyphal bodies compared with conidia as a consequence of the larger size of blastospores. Last (1960) suggested that the endogenous nutrient levels of spores required for infection needed to be greater than that required for germination. He concluded that blastospores had more nutrient reserves available for germination and penetration than conidia, which resulted in increased blastospore virulence. Results obtained by Hunt e t a l . , (1984) tend to support this hypothesis, as he found that conidial germination of B. b a s s i a n a on the cuticle of the beetle Dendronocus ponderosae was greatly increased by the presence of haemolymph on the cuticle. Another technique to improve virulence is the selection of single -2 32 - spore isolates (SSI), with increased virulence compared with the multispore isolate (MSI) parent. Samsinakova and Kalalova (1983) selected SSI's from a MSI strain of B. bassiana and found "spontaneous mutants" which surpassed the "mother strain" in pathogenicity. In further experiments, it was found that these "newly formed mutants" retained their high pathogenicity after repeated subculturing or storage on SDA. In fact these SSI's were probably not mutants but simply different genotypes already present in the MSI. In the present study, SSI's of B. bassiana and M. anisopliae selected from MSI showed increased, similar or reduced virulence to GLH when compared to the parent strains (III, 15, C). When SSI of M. a n i s o p l i a e and B. bassiana with increased or reduced pathogenicity and the parent strains were subcultured on SDA or passaged through GLH, changes in virulence were observed in all strains (III, 19). From this experiment it was concluded that: -Single or MSI of B. bassiana and M. anisopliae were not stable in their pathogenicity to GLH and virulence tended to decrease with successive subculturing on SDA. -Single or MSI of B. bassiana increased their pathogenicity markedly after one or two passages through the host but this increase in virulence was not stable and pathogenicity tended to revert to the initial level after successive passages. There are several researchers' reports of fungal isolates retaining virulence during repeated i n v i t r o subculture (Hall, 1980, Verticillium lecanii ; Sweeney, 1981a, Conidiobolus obscurus; Samsinakova and Kalalova, 1983, Beauveria bassiana ). However there are also many reports which demonstrate changes in pathogenicity after i n v i v o or i n v i t r o passage (Muller-Kogler 1965, Aizawa, 1971; Champlain e t a l . 1981; Fargues and Robert, 1983). Results obtained in this study support the latter point of view and agree with the observations made by Fargues and Robert (1983), who found a significant increase in virulence of M. anisopliae after one or two insect passages and also a rapid loss in virulence after i n v i t r o subculture. The authors suggested that changes in virulence were due to phenotypic responses indicative of inducible enzymes. The reduction in pathogenicity of B. bassiana observed after the peak reached in the second host-passage (Fig. 39), can perhaps be interpreted as an ecological adaptation of the fungal strains. Deuteromycete fungi are facultative pathogens which survive as saprophytes in the absence of hosts, and it is reasonable to suppose that contact with insects triggers an increase in virulence allowing the fungi to kill large number of insects and create an epizootic. However, many insect outbreaks are of limited duration and the maintenance of high virulence could mean that the fungus is poorly adapted to return to a saprophytic mode of growth when the insects disappear. A high correlation was found between germination of B. bassiana hyphal bodies, submerged and aerial conidia i n v i v o and i n v i t r o (III, 16, B). Germination rates i n v i v o were about half of those i n v i t r o for hyphal bodies and submerged conidia and even less for aerial conidia. Germination rates are most likely reduced i n v i v o due to limitations in the supply of nutrients. In this study, submerged conidia and hyphal bodies of B. bassiana 268-86 had similar pathogenicity to GLH adults but were more active than aerial conidia (> 5 fold). The results showing the increased virulence of submerged conidia are encouraging, as liquid fermentation is the most suitable method for production of B. b a s s i a n a . Conidiation in liquid culture has been previously reported (Goral, 1973; Thomas e t al., 1986) but no indications of spore virulence were presented. The pathogenicity of B. bassiana hyphal bodies has been previously reported by other researchers, Keller, (1982) used high volume field applications of B. brongniartii hyphal bodies to successfully control the may beetle M elolontha m elolontha in Switzerland, while Fargues e t a l . t (1979) used B. bassiana hyphal bodies to control the Colorado potato beetle Leptinotarsa decem lineata in Normandy. Hall (1977) found V . l e c a n i i hyphal bodies and aerial conidia had similar pathogenicity to M acros ipbonie 11a sanborni, while Gillespie (1984) found a similar result with M. anisopliae against H auptidia ma.-occana. In contrast, hyphal bodies of Nomuraea rileyi were not pathogenic to H eliothis zea and Bombyx m ori (Bell, 1975; Riba and Glandard, 1980). Staining spores with the optical brightener bi-striaziny 1 amino stilbene (0.05% Tinopal BOPT) has been confirmed as a good method for observing germination and mycelial growth of entomogenous Deuteromycetes i n v i v o (III, 16). In this study the method was used with spores of M. anisopliae and B. bassiana on GLH wings. Drummond and Heale (1985) used the same vital staining technique to visualize V . l e c a n i i spores on aphid cuticle and Matewele (1986) also used the technique with Tolypocladium cylindrosporum on Aedes aegypti. The technique is simple, does not impair germination or mycelial growth and allows visualization of i n v i v o germination without resorting to the electron microscope. The intensity and mode of sporulation have been regarded as determinant factors in the subsequent spread of disease in the field (Papierok and Wilding, 1981). In this study, most B. bassiana strains sporulated better on GLH cadavers than did M. anisopliae strains (III, 17, A). .A good correlation was found between the infection of nymphs and the number of GLH cadavers with sporulating mycelium attaching the dead insects to rice leaves, (III, 17). This criterium was used in the selection of strains, and B. bassiana 268-86, 269-86 and 110-82 and M . a n i s o p l i a e 275-86 were selected as the strains with the best epizootic potential. Besides the variation of strains with respect to epizootic potential, B. bassiana strains sporulated profusely at both high and suboptimal humidities, while sporulation of M. anisopliae was reduced below 100% rh. Variation in sporulation has been regarded as a strain characteristic. Papierok and Wilding (1981) considered the two types of C. obscurus, as defined by Remaudiere e t a l . , (1979) were two different biological races. Criteria examined included cultural characteristics i n v i t r o , pathogenicity toward aphids, duration of incubation and differences in sporulation on the cadavers. The influence of high humidity on sporulation of entomogenous fungi was also shown by Gibson e t a l . t (1979), who found that sporulation of H. thom psonii on mite cadavers only occurred when rh was 98% or above. In contrast, Wilding (1981a) reported sporulation of E . m u s c a e at 20% rh and Kramer (1980) reported sporulation of the same species at a nominal 0% rh. The importance of the characteristics discussed above in the selection of strains is supported by Papierok and Latge (1980) who introduced the concept of inoculum multiplication ability.They considered the most effective fungal isolates were those possessing a low LC50, a high degree of sporulation and a short infection cycle. In this study, the ability of certain strains to attach dead -2 3 6 - Insects to rice leaves and sporulate on cadavers were considered of great importance in the selection of strains for possible exploitation as mycoinsecticides against GLH. Strains that fail to attach dead insects to the rice plant are likely to be less effective control agents because nymphs emerging from eggs laid within the leaf sheat will tend to scape infection. SECTION IV PRODUCTION OF FUNGI MATERIALS AND METHODS 1. General Descriptions of media, equipment, sterilization procedures, surfactants, antifoam, buffer, temperature and humidity measurement, pH measurement, total spore counts and spore viability assessments are given in II, 1 2. Preparation of inoculum Inocula were prepared by harvesting SDA Petri plates of B. bassiana 268-86 or M. anisopliae 275-86 in 0.05% Triton X-100 as described previously (II, 1, G). Inoculations, unless otherwise indicated, were made at the rate of one ml of 1X10-7 conidia per ml for 25 g of semi-solid medium or one ml of 1x10s conidia per ml for each 50 ml of liquid medium % -238 - 3. Production on semi-solid medium A) General Standard weights of 25 g of grains and different amounts of sunflower oil (Gateway Foodmarkets Ltd, Bristol, UK) and water were mixed in 250 ml Erlenmeyer flasks. Water and oil were mixed first . Necks were plugged with cotton wool, flasks autoclaved (121*C for 20 min) and while hot, shaken vigorously to break clumps of cereal grain. In some cases a sterile glass rod was used to break up clumps. Flasks were inoculated with one ml aliquots of M. anisopliae or B. b a s s t a n a conidia and incubated at 25±1*C. Flasks were shaken vigorously after inoculation to distribute spores and briefly agitated on each of the next two days to maintain grain friability. Samples of grain (1 g) were removed with a sterile spatula and placed in universal bottles containing 10 ml of 0.05% Triton X-100 and ten to fifteen glass balls (0.5 mm diam) and shaken for two minutes to remove conidia from the cereal grains. Further agitation failed to increase conidial numbers showing that two min shaking removed all the spores from the grain. Resultant suspensions were suitably diluted and conidial numbers estimated using a improved Neubauer haemocytometer. A picture of M. anisopliae and B. bassiana production on cereal grains, using 250 ml Erlenmeyer flasks is shown in Plate 16 41 " 2 3 9 " PLATE 16 Production of Beauveria bassiana 268-86 and Metarhizium anisopliae 275-86 on semisolid media using 250 ml Erlenmeyer flasks. * - 240 - IL, Effect of Increasing inocula on spore yields Standard weights of twenty five g of Pre-treated rice (Tilda Rice Ltd, Leicester, UK) and aliquots of 0,5 ml sunflower oil and 25 ml water per 250 ml Erlenmeyer flasks, were used to prepare semi-solid medium as explained previously. Aliquots of 0.5, 1.0 and 5.0 ml of a suspension containing 1X107 conidia per ml of B. bassiana 268-86 and M. anlsopllae 275-86 were used to inoculate four replicate flasks per treatment which were then incubated at 25±1*C Conidia production was estimated after 7, 14 and 21 days and expressed as conidia per g of dry cereal grain. C, Production of B. bassiana on different cereal grains Cereal grains, white rice (WR; Gateway foodmarkets Ltd, Bristol, UK) whole grain rice (WG, Dornay Foods, King's Lynn, UK), Pre-treated riceCPR, Tilda Rice Ltd, Leicester, UK) wheat (W) and barley with husk (B) (from a local grain merchant) were compared as substrates for B. b a s s i a n a 268-86. Flasks were prepared with 25 g of each cereal and mixed with 25 or 30 ml of water and aliquots of 2.5, 5,0 or 10,0 ml of sunflower oil. Flasks were inoculated, and incubated at 25±1*C. Yields were then estimated after seven, 15, 21 and 28 days and expressed as the number of conidia per g of dry cereal. % - 2 4 1 " Effect of shaking on production Beauveria of bassiana con Id la Flasks were prepared with 25 g of wheat and 25 ml of water and aliquots of zero, three, five, six and seven ml of sunflower oil per flask were added. Flasks were sterilized, Inoculated with B. bassiana conldia and incubated at 25±1*C. Three replicate samples of each media were shaken every two days throughout the experiment or only shaken every two days during the first six days. Yields were then estimated after 28 days and expressed as conidia per g of dry cereal E. Effect of oil on yields of Beauveria bassiana Wheat or pre-treated rice (25 g) contained in 250 ml Erlenmeyer flasks was mixed with 25 ml water and zero, one, two, three, four, five, six, seven or eight ml of sunflower oil. Flasks were inoculated with conidia of B. bassiana and incubated at 25±1"C. Yields were estimated after 14, 21, 28 and 35 days and expressed as conidia per g of dry cereal grain. F\ Effect of adding perlite on yields of Beauveria bassiana Wheat contained in 250 ml Erlenmeyer flasks was mixed with 25 ml aliquots of water and three, six or nine g of perlite were added to each of three flasks (three replications). Flasks were inoculated with B. bassiana 268-86 conidia incubated at 25±1*C and yields estimated after 14, 21 and 28 days. - 2 4 2 - 4. Production ofBeauveria bassiana spore types In submerged culture In submerged culture in a defined medium (TKI broth, II, 1, A,) the fungus B. bassiana produced "submerged conidia", but in a complex medium (II, 1, A) it produced only hyphal bodies (blastospores, Thomas e t a l . t 1986). Experiments were carried out to compare the production of B. bassiana 268-86 in defined and complex media using Erlenmeyer flasks and 1.6-litre fermenters A- Production using Erlenmeyer flasks Aliquots of media (50 ml) were contained in 250 ml Erlenmeyer flasks plugged with loose-fitting non absorbent, cotton wool and the flask neck wrapped in aluminium foil before autoclaving (121 *0 for 20 minutes). Flasks were cooled, inoculated aseptically with 1 ml of a suspension of B. bassiana 268-86 conidia in 0.05% Triton X-100 and incubated in a rotary shaker (25±1*C; 250 rpm). Spore numbers were determined by removing 1 ml aliquots of media with an adjustable micropipette (Gilson, Anachem Ltd, Luton, UK), fitted with wide bore tips (min diam 2,5 mm) and counting suitable dilutions using a haemocytometer. To determine biomass, 10 ml samples were removed using glass pipettes with widened tips (min diam 3 mm), placed on 4.7 cm diam glass fibre filter paper (GFA grade *c*, Whatman Co. Ltd) and vacuum - 2 4 3 - IL Medium scale production using 1.6 litre fermenters Fermenters (Plate 17) comprised stainless steel impeller assemblies (Biolaffite S.A. France), 1.6 litre capacity glass pods (Hampshire Glass Co. Ltd. England), electric motors (125 watt, Parvalux Co. Ltd. England) and speed controllers (M.R Supplies Co. Ltd., London, England). Temperature was regulated by water circulated through a temperature controller (Churchill Co. Ltd. England) and maintained at 2 5 ± 1 *C. After placement of media into glass pods, the apparatus was assembled and ports for inoculation, sampling, air inlet and exhaust were fitted with autoclavable polyethylene tubes. An air filter (Gamma 12, grade 12-03 filter tube. Whatman Co. Ltd.) was fitted to the air inlet and all tubes plugged with non absorbent cotton wool. All tubes except the air exhaust, were then clamped and the apparatus autoclaved (121 *C for 20 minutes) and allowed to cool. Fermenters were inoculated with B. bassiana 268-86 conidia. Cultures were agitated at 450 rpm and aerated with compressed air (100 litres per hour) Culture samples for assessment of spore numbers and biomass were obtained by clamping the exhaust tubing to create sufficient pressure for the sample to be expelled through the sampling port. Assessments were carried out as explained previously (IV, 4, A) - 2 4 4 - PLATE 17 Submerged fermentation of Beauveria bassiana 268-86 using a 1.6 litre fermenter. - 245- RESULTS 5. Production of conidia on semisolid medium Aj. Effect of increasing inoculum on conidial yields After seven days incubation, highest conidial yields of both B. b a s s i a n a 268-86 and M. anisopliae 275-86 were obtained from flasks inoculated with 1 or 5 ml of suspension compared to those inoculated with 0.1 ml. (P<0.07), (Fig, 41). After 14 days however no differences in yields were observed between any of the three inoculum levels. Yields of B. bassiana were generally higher than those obtained with M. anisopliae though after 21 days yields were similar, B. b a s s i a n a 1.66X1010 and M. anisopliae 1.06X1010 conidia per g). Production of B. bassiana on cereal grains Yields of B. bassiana 268-86 conidia increased steadily after seven days incubation on all the tested grains. Highest yields were obtained on wheat with 1.7X1010 conidia per g or on pre-treated rice (1.6X1010 after 28 days (Table 36). The optimum amount of sunflower oil was 2.5 ml for all the grains examined. " 2 4 6 ~ 17,5 15------ —12,5 ------] n> O (HX & 10 f o a •Hm T3 7,5------•Hc o u 5- 2.5 7 14 21 Incubation period (days) Fig. 43. Effect of 0.1 ( \/A )< 1*0( LLil ) and 5.0( ) ml inocula on semisolid media yields of Beauveria bassiana(Bb) 268-86 and Metarhizium anisopliae(Ma) 275-86. Bars represent 9 5 % confi- (5bnee limits obtained from the mean of three re— plic ates. - 2 4 7 - TABLE 36. Production of Beauveria bassiana on different cereal grains Incubation period (days) Media 7 14 21 28 Water Oil (ml) (ml) Spore yield ^ (conidia/g x 10 ) 25 g white rice 25 2.5 1.25 1.80 2.20 2.63 it ii 25 5.0 0.93 1.60 2.40 2.10 m ii 25 10.0 0.80 0.38 1.20 1.50 II ii 30 2.5 0.10 0.19 2.10 2.50 it ti 30 5.0 0.30 0.50 0.50 0.64 it it 30 10.0 0.02 0.13 1.30 1.46 25 g whole grain rice 25 2.5 0.84 2.40 2.90 2.95 it ii ii 25 5.0 0.72 2.32 2.70 2.80 II ii ti 25 10.0 0.34 1.43 1.80 1.85 it ti it 30 2.5 0.75 2.43 3.90 4.12 it ii ii 30 5.0 0.70 2.40 3.40 3.51 it ii it 30 10.0 0.23 1.12 1.81 2.46 25 g pretreated rice 25 2.5 2.22 9.54 15.20 16.75 it II 25 5.0 2.00 5.40 12.20 13.50 it ti 25 10.0 1.18 4.11 6.25 8.92 II ii 30 2.5 1.73 9.34 14.80 16.00 II ii 30 5.0 1.72 4.18 12.35 14.50 ii it 30 10.0 1.34 2.42 6.10 8.37 25 g wheat 25 2.5 5.40 9.80 17.30 17.50 it 25 5.0 4.20 6.50 14.90 15.00 II 25 10.0 2.50 3.30 8.50 9.30 ii 30 2.5 3.50 8.20 17.20 17.30 it 30 5.0 3.20 8.00 12.50 12.80 it 30 10.0 2.10 2.50 8.80 9.00 25 g barley 25 2.5 1.42 2.50 3.52 4.00 ii 25 5.0 1.33 2.42 2.80 3.10 If 25 10.0 1.20 2.24 2.42 2.52 it 30 2.5 1.23 2.40 3.21 3.43 it 30 5.0 1.01 2.00 3.73 3.20 ii 30 10.0 0.35 1.48 2.05 2.20 - 2 4 8 - Cj, Effect of shaking onB. bassiana y ie ld s Shaking flasks every two days after inoculation had a deletereous effect on conidial yields of B. bassiana 268-86, (Fig. 42) Increasing the proportion of sunflower oil from zero to eight ml reduced conidial yields in both continuous and restricted shaking treatments. Effect of adding oil on B. b a s s i a n a yields Wheat and rice responded differently to the addition of sunflower oil when B. bassiana 268-86 yields were assessed over time (P<0.01). Maximum yields of 1.84 to 1.87X1010 conidia per g were obtained after 28 days when zero to two ml sunflower oil were added to wheat, while 1.69 to 1.82X1010 conidia per g were obtained when zero to four ml sunflower oil ware added to rice. Higher volumes of oil gave significant yield reductions (P<0.05) with both grains. Yields obtained after the addition of zero to two ml of oil to wheat, or # zero to four ml of oil to pre-treated rice, increased steadily up to 28 days after incubation but decreased after that for both grains (Figs. 43 and 44). Ej. Effect of adding Perlite on yields of Beauveria bassiana After 14 days incubation at 25±1*C, no significant differences (P<0.05) were observed in conidial yields irrespective of the presence of perlite (Fig. 45). However after 21 and 28 days incubation, significantly less (P<0.05) conidia were produced where nine g of * - 2 4 9 - Fig. 42 Yields of Beauveria bassiana 268-86 from static ( y: ) and shaken ( ) cultures of wheat with different amounts of oil. Bars re present 95% confidence limits obtained from the mean of three replicates. - 2 5 0 - # * Fig.43 Effect of adding sunflower oil on yields of Beauveria bassiana 268-86 on wheat( £2 ) or rice( CD ), after (a) 14 or (b) 21 days. Bars represent 95^% con fidence limits obtained from the mean of three replicates. - 2 5 1 - * 70 CTl o r—I ft ^ 1S ft0) fU •H •H §10 CJ IH O uo 2:o I i 20 4 - 1 ft U15 d fto rtf •H 'O n4l I 10 i 11 u 4-1 1 ¥ o i I a) ! i 1-| - i 1 1 2 5 1 5 ^ 7 * Amount of oil per flask (ml) Fig. 44 Effect of adding sunflower oil on yields of Beauveria bassiana 268-86 on wheat ( £2 ) or rice( [£) ), after (c) 28 or (d) 35 days. Bars represent 95,% con fidence limits obtained from the mean of three replicates. - 2 5 2 - * Fig.45 Effect of adding perlite on yields of Beauveria bassiana 268-86 obtained on semisolid media at 14( /// ), 2 1( 111) ) and 28 days( ) fermentation. Bars represent 9 5 % confidence limits obtained from the mean of three replicates, - 2 5 3 - * perlite were added to flask than in the presence of six, three or zero perlite. 6 . Production of spores in liquid culture Three types of B. bassiana 268-86 spores were obtained in liquid culture. Submerged conidia were observed in defined medium (TK1), hyphal bodies in complex (yeast plus glucose, YG, or sabouraud liquid media, SL) and one intermediate spore, "conidia-like” in both defined and complex media. A description of the three spores type was provided previously (II, 18). /L Erlenmever flasks After 24, 48, 72, 96 or 120 hours incubation at 25+1°, spore yields were lower in TK1 compared with YG or SL. Highest yields were obtained after 120 hours incubation in YG, though there were no significant differences (P<0.05> in spore yields in YG compared with SL after 96 hours incubation (Fig. 46). After 24 hours incubation spore numbers produced in the three tested media was barely detectable by microscope counts «1X10S spores per ml). After 48 hours a mean of 1.2X10e (93% hyphal bodies, 5% submerged conidia and 2% conidia-like) was produced in TK1, while a mean of 7.5X107 spores per ml (90% hyphal bodies and 10% "conidia like") was produced in YG and 1.9X107 spores per ml was produced in SL (100% hyphal bodies). After 72 hours 7.5X107 (65% hyphal bodies, 25% submerged conidia - 2 5 4 " + * i--- 1--- r 24 48 72 96 120 24 48 72 96 120 24 48 96 120 Fermentation period (hours) TKl YG SL * Fig. 46 Production of Beauveria bassiana 268-86 spores (submerged conidia I I I I I I I , conidia like K / // 7 1 and hyphal bodies K.X20KX] ) in TKl, YG and SL media using 250 ml Erlenmeyer flasks. Bars represent 95X confidence limits obtained from the mean of four replicates. 9 - 2 5 5 - and 10% conidia-like), 6.6X10® (98% hyphal bodies and 2% conidia-like) and 1.7X10® (100% hyphal bodies) spores per ml of medium were produced in TK1, YG, and SL respectively. The type of spore produced in TK1 changed markedly after 96 hours incubation and yields reached lO.SXlO'7 spores per ml and comprised 94% submerged conidia, 4% conidia-like and only 2% hyphal bodies. After the same period a mean of 8.6X10® spores per ml was produced in YG comprised of 86% hyphal bodies and 14% conidia-like while a mean of 5.1X10® spores per ml was produced in SL (100% hyphal bodies). After 120 hours incubation, 1.7X10® spores per ml were produced in TK1 and all spores were observed to be submerged conidia. In YG yields of 1.3X10® spores per ml were obtained (89% hyphal bodies and 11% conidia-like) while in SL 1.1X10® spores per ml were produced (96% hyphal bodies and 4% conidia-like). 1L Using 1.6 litre fermenters After 24, 48, 72, 96 or 120 hours incubation at 25±1°C, spore yields were again lower in TK1, compared with YG or SL. Highest yields were obtained after 120 hours incubation in YG though there were no significant differences (PC0.05) in the amount of spores per ml produced in YG compared with SL after 96 hours incubation (Fig. 47). As was observed in Erlenmeyer flasks, after 24 hours spore production in any media was low. After 48 hours a mean of 2.7X10® spores per ml (85% hyphal bodies and 15% submerged conidia) was produced in TK1, while a mean of 9.2X107 spores per ml (87% hyphal i— r 48 72 96 120 24 48 72 96 120 24 48 72 96 120 Fermentation period (hours) TKl YG SL Fig. 47 Production of Beauveria bassiana 268-86 spores (submerged conidia f I I II (1 , conidia like V / / and hyphal bodies KJKXXXJ ) in TKl, YG and SL media using 1.6 litre fermenters. Bars represent 95^ - 2 5 7 - bodies and 13% conidia-like) was produced in YG and 2.3X107 spores per ml was produced in SL (100% hyphal bodies). After 72 hours 8.2X107 (85% hyphal bodies and 15% submerged conidia), 7.4X103 (97% hyphal bodies and 3% conidia-like) and 1.9X103 (100% hyphal bodies) spores per ml were produced in TK1, YG and SL respectively. After 96 hours incubation almost all the spores produced in TK1 (1.3X103 spores per ml) were observed to be submerged conidia (99%). After the same period 9.6X103 spores per ml was produced in YG (85% hyphal bodies and 15% "conidia-like") while a mean of 5.9X103 spores per ml was produced in SL (96% hyphal bodies and 4% "conidia like". After 120 hours incubation, 2.7X103 spores per ml were produced in TKl and all were submerged conidia, 1.6X103 spores per ml in YG (90% hyphal bodies and 10% "conidia-like") and 1.2X10-1 spores per ml in SL (94% hyphal bodies and 6% conidia-like). Biomass after 120 ho-.;rs incubation was 4.6, 18.9 and 13.2 mg per ml in TKl, YG and SL respectively, and yields were higher than those obtained in Erlenmeyer flasks (Table 37). TABLE 37. Biomass of Beauveria bassiana (268-86) spores produced in liquid culture* Biomass Culture vessel Medium + SE mg/ml** 250 ml Erlenmeyer flasks TK1 2.9 0.4 Yeast plus glucose 16.0 1.2 Sabouraud liquid 12.6 2.4 1.6 litre fermenters TK1 4.6 0.1 Yeast plus glucose 18.9 1.4 Sabouraud liquid 13.3 5.3 Records were taken after 120 hours fermentation at 25 1°C * Figures are means of three replicates * - 2 5 9 - DISCUSSION In the production of entomogenous fungi the aim is to produce infective propagules rather than metabolites, and this type of mass production can be considered a new technology (Hall and Papierok, 1982) Entomogenous fungi must be produced cheaply enough to compete with existing control agents (Bartlett and Jaronski, 1988). Candidate strains which can be produced easily in a variety of media, are attractive with respect to mass production. The initial inoculum level failed to influence yields of M. a n l s o p l i a e 275-86 or B . b a s s i a n a 268-86 after 15 days incubation. Yields obtained were 1.66 and 1.06X1010 conidia per g for B. bassiana and M. anlsoplisa respectively. Hall and Papierok (1982) considered cereal grains the most attractive semi-solid substrate for mass production of entomogenous fungi. They are easily sterilizable and in some cases, friable afterwards if processed carefully. In the present study, three types of commercial rice, wheat and barley with husk were evaluated for mass production of B. bassiana 268-86. Yields on pretreated rice and wheat were the highest with 1.6 and 1.7X1010 conidia per g respectively, being obtained after 28 days (IV, 5, B). Yields obtained after 21 days incubation were similar to those at 28 days, indicating 21 days as the most suitable time to harvest spores. Wheat has been used widely as a semi-solid substrate for fungal fermentation, Bartlett and Jaronski (1988) mentioned that the high starch content of wheat and the particle size can enhance greatly the production of some entomogenous fungi. The observed results may be more due to the nutrient content of wheat and pretreated rice than their size as the other two types of rice, with similar size grains, yielded considerably less conidia. Shaking inoculated wheat throughout the incubation period, reduced conidial yields of B. bassiana. In contrast, when wheat was shaken three times during the first 6 days, yields from undisturbed flasks were similar to those obtained without agitation (Fig. 42 ). The reduced yields obtained after repeated shaking could be due to the removal and premature germination of conidia when they come into contact with nutrients. This then reduces final conidial yields. This phenomenon has been reported in rotating drum fermentation (Hall and Papierok, 1982). Reduced B. bassiana yields were obtained when more than two ml of sunflower oil was added to 25 g of wheat and 25 ml water or more than 4ml of sunflower oil was added to 25 g of pretreated rice and 25 ml water (IV, 5, D). These results suggest that sunflower oil plays a purely physical role in the mixture, making it more friable and increasing the available surface area for sporulation. These results contrast with those obtained by Matewele (1987) who found that the addition of high levels of vegetable oil to cereal grains dramatically increased yields of Tolypocladium cylindrosporum and Culicinom yces clavisporus. The author, suggested that vegetable oils provided an important nutrient source for the fungi. Addition of perlite increased yields of B. bassiana 268-86 when three or six g were mixed with 25 g of wheat per flask (IV, 5, E). Further addition of perlite (nine g) reduced yields considerably. Perlite has been used as an inert component in semi-solid fermentation to increase friability and available surface area (Bartlett and Jaronski, 1988). One of the problems observed when wheat was tested as a substrate in B. bassiana fermentation was the tendency of the grains to clump after the first week of incubation, perhaps due to the mycelia growing between grains and holding them together . Perlite perhaps decreased this effect by separating wheat grains and thus increased the surface area available for growth. The growth of M. antsopliae 275-86 and B. bassiana 268-85 was examined in submerged culture. M. anisopliae 275-86 formed mycelial balls and produced very few hyphal bodies. No all M. anisopliae strains are amenable to mass production in submerged fermentation and Adamek (1963) and Gillespie (1984) found strains varied greatly in their ability to produce hyphal bodies. B. bassiana 268-86 produced aerial conidia in semi-solid fermentation (IV, 5), hyphal bodies in complex liquid medium (IV, 6) and submerged conidia in defined liquid medium. The ability of B. b a s s i a n a 268-86 to conidiate in submerged culture was fortunate as relatively few strains produce submerged conidia in liquid fermentation( Bartlett and Jaronski, 1988; Gillespie, unpublished observations). In the present study (IV, 6 ) B. bassiana 268-86, produced around 2X10a spores per ml in defined media (TK1) and 2X10* spores per ml in complex media (YG or SL) with biomass of around 4 or 15 mg per ml respectively. In general spore yields and biomass were higher in 1.6 litre fermenters compared with 250 ml Erlenmeyer flasks. This result could be due to the efficient utilization of oxygen in fermenters compared with Erlenmeyer flasks. Tabak and Cooke (1968) discussed * the inhibitory effect of insufficient aeration for numerous fungi. - 2 6 2 " Aeration also helps to remove volatile metabolites generated by growing- fungi, some of' which may inhibit growth (Fries, 1973). Yields of hyphal bodies and submerged conidia obtained in this study approached those reported in the literature (Bartlett and Jaronski, 1988; Hall and Papierok, 1982). Romano (1966), defined fungal "dimorphism" as an environmentally controlled interconversion of yeast and mycelial morphologies. The determinants are poorly understood, with both nutritional and physical parameters being implicated. Kulkarni and Nickerson (1981), observed changes in C eratocystis ulmi morphology in submerged culture when some elements and amino-acids were added or withdrawn from the culture. Mahvi, 1965 observed that H istoplasm a capsulatum grew raycelially at 25 X but developed a "yeast-like form" when the temperature was increased to 37’C. Carbon dioxide, introduced into an anaerobic atmosphere, induced yeast like growth of Mucor rouxii (Bartnick-Garcia and Nickerson, 1962). Gillespie (1984), considered "hyphal bodies" was a better term than "blastospores" as it agreed with the definition: "Hyphal bodies are fragments of hyphae of various sizes and shapes and are produced by budding and division within the host or in an artificial medium" (Prasertphon and Tanada, 1968). The results obtained in the present study support Gillespie's idea and "hyphal bodies" is the most appropriate description for B. b a s s l a n a spores produced in submerged culture. The term "polymorphic" should describe better the biology of B. bassiana 268-86 as it is able to produce conidiospores in semi-solid medium and hyphal bodies (complex media) or submerged conidia (defined media) in submerged % culture. - 2 6 3 - Production of B. bassiana spore types in submerged culture has been well studied in the Soviet Union (Goral 1973, 1975; Kondrayatiev e t a l ., 1971 and Belova, 1978) and more recently in Canada (Thomas e t a l \ 1986). The author described in detail, the morphogenesis of spore types in submerged culture and how the spore type can be changed by alterations in the proportion of nutrients in the media. Further study on B. bassiana 268-86 would be worthwhile and could lead to an economic production methodology, thereby improving the chances for commercialization. - 2 6 4 " SECTION V GENERAL DISCUSSION AND RECOMMENDATIONS This study was carried out to evaluate the potential of entomogenous fungi for control of the rice pest N. virescens. The rice crop, growing in flooded soil with resultant high humidity, is likely to be a suitable environment for the exploitation of fungi as myco insec tic ides (Gillespie, 1984; Rombach, 1987). Perhaps the most important finding is the great variation found within a single species of fungus. In particular, virulence to GLH varied markedly, but other characters such as conidial survival and the adhesion of dead insects to the host plant were also highly variable. Much of the research presented in this thesis wq t q therefore directed towards the selection of fungal isolates with an optimal combination of characters thought to be important for the development of a successful mycoinsecticide. Strain Selection Hall and Papierok (1982) and Gillespie (1988) suggested some properties likely to be important for a successful mycoinsecticide and those can be applied to potential control agents for GLH. 1. High pathogenicity to GLH under conditions encountered in the rice crop. 2. Ability to spread and cause epizootics 3. Stability of virulence 4. Rate of spore germination and mycelial growth at temperatures and humidities occurring in the rice crop. 5. Availability of suitable storage technologies 6 . Ability to grow in different culture systems and produce large numbers of spores. For further development, laboratory selection must be complemented with field trials to assess the performance of selected strains before commercialization could be considered. The compatibility of the fungus with other control measures, safety to mammals and non target insects and formulation development must also be investigated. Of the 29 fungal strains studied, Beauveria bassiana 268-86 possessed the best combination of attributes and is a very good candidate for development as mycoinsecticide. This strain was highly pathogenic to GLH in conditions of humidity and temperature which mimic those found in the rice crop, but its most striking characteristic was its ability to sporulate on GLH cadavers and attach them to rice leaves. In the laboratory, this was shown to correlate with the infection of nymphs; i.e. this strain showed the highest inoculum multiplication potential (Papierok and Latge, 1980). This characteristic is likely to be of great importance with respect to epizootic development amongst field populations of GLH. Other Deuteromycete strains sporulate on insect cadavers but fail to attach them to the rice leaves. Consequently cadavers fall into the water and disappear rapidly; thus the spreading potential is lost. Further experiments to understand the mechanisms involved in this phenomenon are recommended. B. bassiana 268-86 was isolated in Thailand in 1986 on an insect but not GLH, subcultured once on SDA and kept in liquid nitrogen at IHR-Littlehampton. Perhaps this lack of repeated subculture on a defined medium could account for its adaptation and high bioassay pathogenicity towards GLH. The fact that an entomogenous fungal strain can be pathogenic to an insect other than the species for which it was isolated should be taken into account in selection of candidate strains. Improvement of virulence The use of single spore isolates (Samsinakova and Kalalova, 1983) allowed the selection of those with increased virulence to GLH. Virulence was further increased in both single and multispore isolates after two passages through the host insect. Unfortunately this increase in pathogenicity was not maintained on subsequent insect passage or subculturing on SDA, and virulence tended to revert to the level obtained with isolates stored in liquid nitrogen. Based on these results, it is recommended that samples of B. bassiana 268- 8 6 should be maintained in liquid nitrogen and used as cultures to mass-produce the fungus for field application. Thus avoiding repeated medium subculture. One or two passages through the insect host before mass production may increase virulence but how quickly the activity reverts to the initial level after subculture on artificial media is unknown. Further research is obviously needed in this area and stabilization of the increased virulence observed after insect passage should be an important objective. Influence of relative humidity in virulence B. bassiana 268-86 killed adult GLH at both optimal and suboptimal relative humidities and its mycelial growth was maximal at temperatures between 23 to 27*C, temperatures frequently encountered in the rice rice crop. Nevertheless, its mycelial growth at different water activities (aw) showed it to be highly dependent on humidities above 95% for rapid mycelial growth, as was found by Gillespie and Crawford (1986) for several entomogenous Deuteromycetes. In this study, humidity proved to be the main environmental factor restricting fungal pathogenicity. Gillespie (1984) found that a strain of M. anisoplias pathogenic to BPH in the laboratory, was unable to infect the insect in glasshouse conditions. He suggested the reason for the failure was due to an insufficiently high rh in the glasshouse. It means that although B. b a s s i a n a 268-86 is a good candidate for development as a mycoinsecticide, a lot of work should still be done to try and reduce its dependence on high rh. Therefore the development of moisture-retaining formulations which allow fungal growth at suboptimal relative humidities is recommended. Philipp and Hellstern (1986) reported an emulsion of liquid paraffin allowed the fungus Ampelomyces qutsqualis, to parasitize powdery mildew mycelium at relative humidities as low as 80%. Similar formulations should be studied for use with entomogenous fungi Storage B. b a s s i a n a 110-82 and 235-85, which showed similar pathogenicity to GLH adult compared to 268-86 at suboptimal rh, survived poorly after lyophilization or vacuum drying, while 268-86 survived well at 20 or 5*C. The simple method of vacuum drying spores at room temperature, gave better fungal survival than the widely used lyophilization. B. b a s s i a n a 268-86 aerial or submerged conidia could also be stored suspended in water at 5*C. Furthermore, the storage techniques used did not impair fungal virulence and only a small delay in germination " 2 6 8 " was observed. Thus, vacuum drying could be a useful method for maintaining conidial viability of a future mycoinsecticide product. Mass production In view of its desirable characteristics, the feasibility of producing B. bassiana 268-86 was studied. This strain can be produced on cheap grains, e.g. pretreated rice or wheat with yields above 1.5X1010 spores per gram. This means that one kilogram of these cereals can produce around 1X10’ 3 spores, which should be sufficient to treat one hectare of rice crop at the rates suggested by Rombach (1987). If B. bassiana 268-86 were effective in controlling GLH, it presents the possibility for rice farmers or cooperatives in the tropics producing B. bassiana locally. However, the production method of choice for commercialization is fermentation in stirred tank reactors, allowing existing engineering, fermentation and production regimes to be utilized. B. bassiana 268-86 was able to produce submerged conidia or hyphal bodies in fermenters with yields around 2.5X10Q and 1.6X103 spores per ml respectively. The production of submerged conidia in liquid fermentation is an exciting possibility as these spores were some 10 times as pathogenic to adult GLH than aerial conidia produced on wheat. The potential to control GLH in the rice crop Strains of B. bassiana and particulary 268-86 showed promise for control of GLH in the laboratory. However a careful analysis must be made concerning the requirements of a microbial insecticide for control of N. virescens in the field. This pest is a virus vector transmitting tungro virus to healthy rice plants even at low population densities. Therefore a quick acting insecticide is perhaps the only way to obtain the required control and a slow acting mycoinsecticide would seem an unlikely control agent. However, it might be possible to use B. bassiana as a preventative treatment applied before the insects reach damaging levels; field-cage experiments carried out in rice crop, showed high control of GL.H populations with different B. bassiana strains. (Aguda et al., 1984: Li, 1986). The addition of nutrients to the spore spray could allow B. b a s s i a n a to grow and sporulate on the rice plant, thus providing a source of spores for infection of insects. In the present study a preliminary trial indicated that virulence of B. bassiana 268-86 increased when skimmed milk was added to suspensions used to treat GLH. Another possible strategy is the application of fungi together with low doses of insecticides. The insecticide would' give rapid knockdown and !.he fungus could provide prolonged control. Potential of S. b a s s i a n a for the control of other pests The current trend in insect pest control is moving from total reliance on chemical pesticides and total plant resistance towards more complex insect pest management (IPM) systems. These IPM systems should be based on as many control tactics as possible and this provides an opportunity for B. bassiana to be developed for control of other insect pests on several crops. B. bassiana is perhaps the most extensively studied entomogenous fungus and has been isolated from a wide range of insect pest (Roberts et al., 1983). In China, B. b a s s i a n a is produced in communes and it is applied against the european corn borer, O strinia furnacalls (Hussey and Tinsley, 1981). In Europe, it has also shown great potential for control of O strinia nubllalts and a registration is currently being considered for France. Perhaps the more attractive possibility for using B . b a s s i a n a is in the humid environment of tropical crops. The coffee berry borer, Hypothenemus ham pei, is perhaps a good candidate for control with B. b a s s i a n a . This insect spends most of its life cycle inside coffee berries making it very difficult to control with chemical insecticides. 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