Functional response of the coccinellid predator <Emphasis Type="Italic">Adalia fasciatopunctata revelierei</Emphasis> to walnut aphid (<Emphasis Type="Italic">Callaphis juglandis</Emphasis>)

The functional response types and parameters of 3rd and 4th instar larvae, and adult females and males of a coccinellid predator, Adalia fasciatopunctata revelierei (Mulsant) (Col.: Coccinellidae), were evaluated at five different densities of Callaphis juglandis (Goeze) (Hemiptera: Aphididae) in order to understand their role for the aphid’s biological control. Experiments were carried out in petri dishes at 25 ± 1°C, 60 ± 10% r.h. and 16L:8D photoperiod in a controlled temperature room. All tested stages exhibited a Type II response determined by a logistic regression model. The attack rate (α) and handling time (T _h ) coefficients of a Type II response were estimated by fitting a “random-predator” equation to the data. Although the estimates of α for all stages of A. fasciatopunctata revelierei tested were similar, the longest T _h was obtained for 3rd instar larva because of the lower consumption rate at densities above 40 prey/day. Results indicated that the adult female has the highest predation of C. juglandis followed by 4th instar larvae, adult males and 3rd instar larvae. However, further field-based studies are needed to draw firm conclusions.     

Biological traits and prey consumption of <Emphasis Type="Italic">Anthocoris minki</Emphasis> fed on <Emphasis Type="Italic">Agonoscena pistaciae</Emphasis> and <Emphasis Type="Italic">Brachycaudus (Thuleaphis) amygdalinus</Emphasis>

The predatory insect Anthocoris minki Dohrn (Heteroptera: Anthocoridae) is an indigenous Anthocoris species for the biological control of pests in pistachio orchards. The pistachio psylla Agonoscena pistaciae Burckhardt and Lauterer (Homoptera: Psyllidae) is an important insect pest in pistachio trees in Turkey. Similarly, Brachycaudus (Thuleaphis) amygdalinus (Schouteden) (Hemiptera: Aphididae) is a pest of almond trees that is considered as alternative prey for A. minki when pistachio psylla are not available in early spring on pistachio trees. The development time, survival percentage of immature stages, longevity, fecundity, prey consumption, and life table parameters of A. minki fed on A. pistaciae and B. amygdalinus nymphs were determined at 25 ± 1°C, 70 ± 5% r.h., and a 16 h:8 h (L:D) photoperiod under laboratory conditions. The nymphal survival rate was significantly higher when nymphs were fed on A. pistaciae (an average of 96.7%) than on B. amygdalinus (an average of 71.4%). The development time of A. minki was significantly shorter when nymphs were fed on B. amygdalinus (10.3 days) as opposed to A. pistaciae (11.0 days). No significant differences among prey species were found for longevity and fecundity. The total female longevity and fecundity of A. minki was 38.0 days and 247.2 eggs, respectively, when nymphs were fed on A. pistaciae; and 35.4 days and 233.0 eggs, respectively, when nymphs were fed on B. amygdalinus. On average, 104.4 A. pistaciae and 77.7 B. amygdalinus nymphs were consumed during the nymphal development time for A. minki. Adults of A. minki consumed significantly more psyllids than aphids throughout their life span. The greater difference did not significantly inpact the longevity and fecundity of A. minki. Females of A. minki consumed an average of 631.0 A. pistaciae and an average of 273.3 B. amygdalinus nymphs, while female predators consumed significantly more prey than males. The intrinsic rate of increase (r _m ) of A. minki fed on A. pistaciae (0.174) was significantly greater than those fed on B. amygdalinus (0.148). The successful development and reproduction of both A. pistaciae and B. amygdalinus indicates that they are suitable prey for A. minki.     

Prey density-dependent feeding activity and life history of <Emphasis Type="Italic">Scymnus subvillosus</Emphasis>

The functional response, development time, survival and reproduction of the lady beetle Scymnus subvillosus (Goeze) (Coleoptera: Coccinellidae) were evaluated at different densities of the mealy plum aphid Hyalopterus pruni (Geoffroy) (Homoptera: Aphididae). Treatments were carried out at 25 ± 1°C, 60 ± 10% r.h. and 16L:8D photoperiod in a controlled temperature room. The larvae and adults of S. subvillosus were fed with different densities (5, 10, 20, 40 and 80) of H. pruni in petri dishes. The shape of the functional response was determined by logistic regression, and the values of the coefficient of attack rates (α) and handling times (T _h) were estimated by using nonlinear least-squares regression. Behavior of each larval stage and adults matched Holling’s type II functional response. Estimates of α for all stages of S. subvillosus tested were similar, but estimates of T _h varied; it was the shortest for adult females and the longest for third instar larva. Larvae were able to complete their development at each of the five prey densities, but increased prey densities reduced development time and mortality rate. Increased prey consumption did not change longevity, but resulted in a higher intrinsic rate of increase (r), the finite rate of increase (λ), net reproduction rate (R ₀ ), gross reproductive rates (GRR), and shorter mean generation time (T) and doubling time (DT). This research was funded by the Yuzuncu Yil University/foundation of scientific research projects (No. 2002-ZF 044)     

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Effect of botanical extracts on the biological activity of granulosis virus against <Emphasis Type="Italic">Pieris brassicae</Emphasis>

A granulosis virus strain infecting Pieris brassicae (PbGV) was isolated from the dry temperate region of northwestern Himalayas as a potential microbial agent for its management. The effect of different botanicals (having insecticidal action against P. brassicae) on the bioefficacy of PbGV was evaluated under laboratory conditions using leaf disc bioassays on cabbage for improving the insecticidal performance of the PbGV. The synergistic action of different botanical extracts was evident in terms of reduction in LC₅₀ values against different botanical extracts. Among different extracts, petroleum-ether extract of neem seed kernel (NSK) when combined with PbGV resulted in maximum reduction of LC₅₀ value (4.39 × 10² occlusion bodies [OBs] ml⁻¹) followed by methanolic extract (7.38 × 10² OBs ml⁻¹) and aqueous extract (9.36 × 10³ OBs ml⁻¹) as compared with PbGV alone (1.85 × 10⁴ OBs ml⁻¹) for 2nd instar larvae of the test insect. These trends were found analogous in cases of 3rd and 4th instars of P. brassicae with different solvent extracts of NSK. The other botanicals evaluated, viz., Eupatorium and Artemesia, also resulted in reduction of LC₅₀ values for 2nd, 3rd and 4th instars as compared with PbGV alone when different extracts were combined with virus for bioassays. The studies suggest that the PbGV in combination with botanical pesticides could be more useful as a bio-pesticide against cabbage butterfly (P. brassicae) in IPM programs.     

Effect of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> mixed with diatomaceous earth on mortality,mycosis and sporulation of <Emphasis Type="Italic">Rhyzopertha dominica</Emphasis> on stored wheat

A laboratory assay was designed to determine the insecticidal efficacy of Beauveria bassiana (Balsamo) Vuillemin (Hyphomycetes: Moniliales) and diatomaceous earth (Diafil 610) against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). The fungus B. bassiana was applied at 2.23 × 10⁷, 2.23 × 10⁸ and 2.23 × 10⁹ conidia kg⁻¹ of wheat individually as well as mixed with 200 and 400 ppm of Diafil 610. The conditions for the trials were 30 ± 2^oC with 55% r.h. and the counts for mortality were made after 8, 16 and 24 d. All the dead adults were removed after each count and the vials were kept for the next 60 d to assess the emergence of the F₁ generation. The findings from these studies proved that the extended exposure interval and the highest combined dose rate of the entomopathogenic fungus and the diatomaceous earth gave the maximum mortality of the beetles. The emergence of the progeny was also highly suppressed where the maximum dose rate of the synergized treatments was applied. The rate of mycosis and sporulation in the cadavers of R. dominica was maximum where the low dose rates of B. bassiana were applied.     

Assessment of <Emphasis Type="Italic">Pichia anomala</Emphasis> (strain K) efficacy against blue mould of apples when applied pre- or post-harvest under laboratory conditions and in orchard trials

The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1 × 10⁸ cfu ml⁻¹ than when it was only 1 × 10⁵ cfu ml⁻¹. The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1 × 10⁷ cfu ml⁻¹), β-1,3-glucans (YGT 2 g l⁻¹), and CaCl₂.2H₂0 (20 g l⁻¹), was applied to apples pre- or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the three-component mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1 × 10⁴ cfu cm⁻² of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.     

Effect of apple cultivar and of temperature on the biology and life table parameters of the twospotted spider mite<Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Development duration and reproduction rate of the twospotted spider miteTetranychus urticae Koch (Acarina: Tetranychidae) were studied on five different apple cultivars (‘Amasya’ (local cultivar), ‘Golden Delicious’, ‘Granny Smith’, ‘Starking Delicious’ and ‘Starkrimson Delicious’) at 25°C, 65±10% r.h. and 16:8 L:D. In addition, the same parameters were determined on Golden Delicious leaves at three constant temperatures (20°, 30° and 35°C, 65±10% r.h. and 16:8 L:D) in the laboratory.T. urticae performed better on Granny Smith than on the other cultivars, due mainly to high daily egg production (4.6 eggs/♀/day) and the intrinsic rate of natural increase (r _m , which was 0.243 ♀/♀/day). The lowestr _m was observed on Amasya variety (0.231 ♀/♀/day). Development periods of immature stages ofT. urticae varied from 6.5 to 15.5 days at 35° and 20°C, respectively, for females, and from 5.9 to 14.5 days at 35° and 20°C, respectively, for males. The development thresholds of the eggs and pre-adult stages, respectively, were 10.78° and 8.43°C, and total effective temperatures were 57.80 and 172.41 degree-days. Mean generation time (T _o ) of the population ranged from 9.94 days at 35°C to 25.99 days at 20°C. The net reproduction rate increased from 66.99 ♀/♀ at 20°C to 92.19 ♀/♀ at 25°C, and decreased to 84.34 ♀/♀ at 30°C and to 12.04 ♀/♀ at 35°C. The highest r _m occurred at 30°C (0.302 ♀/♀/day) and the lowest at 20°C (0.161 ♀/♀/day). http://www.phytoparasitica.org posting Dec. 19, 2003.     

Biological control of grapevine crown gall by nonpathogenic <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine (Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato (Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants.     

Effect of temperature,relative humidity,leaf wetness and leaf age on <Emphasis Type="Italic">Spilocaea oleagina</Emphasis> conidium germination on olive leaves

The effects of temperature, relative humidity (RH), leaf wetness and leaf age on conidium germination were investigated for Spilocaea oleagina, the causal organism of olive leaf spot. Detached leaves of five ages (2, 4, 6, 8 and 10 weeks after emergence), six different temperatures (5, 10, 15, 20, 25 and 30°C), eight wetness periods (0, 6, 9, 12, 18, 24, 36 and 48 h), and three RH levels (60, 80 and 100%) were tested. Results showed that percentage germination decreased linearly in proportion to leaf age (P < 0.001), being 58% at 2 weeks and 35% at 10 weeks. A polynomial equation with linear term of leaf age was developed to describe the effect of leaf age on conidium germination. Temperature significantly (P < 0.001) affected frequencies of conidium germination on wet leaves held at 100% RH, with the effective range being 5 to 25°C. The percent germination was 16.1, 23.9, 38.8, 47.8 and 35.5% germination at 5, 10, 15, 20 and 25°C, respectively, after 24 h. Polynomial models adequately described the frequencies of conidium germination at these conditions over the wetness periods. The rate of germ tube elongation followed a similar trend, except that the optimum was 15°C, with final mean lengths of 175, 228, 248, 215 and 135 μm at 5, 10, 15, 20 and 25°C, respectively after 168 h. Polynomial models satisfactorily described the relationships between temperature and germ tube elongation. Formation of appressoria, when found, occurred 6 h after the first signs of germination. The percentage of germlings with appressoria increased with increasing temperature to a maximum of 43% at 15°C, with no appressoria formed at 25°C after 48 h of incubation. Increasing wetness duration caused increasing numbers of conidia to germinate at all temperatures tested (5–25°C). The minimum leaf wetness periods required for germination at 5, 10, 15, 20 and 25°C were 24, 12, 9, 9 and 12 h, respectively. At 20°C, a shorter wetness period (6 h) was sufficient if germinating conidia were then placed in 100% RH, but not at 80 or 60%. However, no conidia germinated without free water even after 48 h of incubation at 20°C and 100% RH. The models developed in this study should be validated under field conditions. They could be developed into a forecasting component of an integrated system for the control of olive leaf spot.     

The effects of different temperatures and relative humidity on the development,mortality and nymphal predation of <Emphasis Type="BoldItalic">Anthocoris minki</Emphasis>

Mass rearing techniques are a necessity in developing a suitable and economic biological control method. Anthocoris minki Dohrn (Heteroptera: Anthocoridae), which is successfully reared for use in biological control, is a promising indigenous Anthocoris species for biological control of Agonoscena pistaciae Burckhardt and Lauterer (Homoptera: Psyllidae) in pistachio orchards. Development time of nymphal instars, mortality percentage and prey consumption of A. minki fed on Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs were studied at three constant temperatures (20, 25 and 30 ± 1°C) and two relative humidity levels (40 and 65 ± 5%) under laboratory conditions. Temperature and r.h. significantly affected development time and nymphal prey consumption of A. minki. Duration of incubation and total nymphal development time decreased significantly with each increase in temperature. Total nymphal development time of A. minki at 40% and 65% r.h. was 18.6 and 18.6 days at 20°C; 13.7 and 14.6 days at 25°C; and 10.8 and 11.8 days at 30°C, respectively. The shortest nymphal development time and highest nymphal mortality percentage were recorded at 30°C and 40% r.h. The lowest nymphal mortality was 30.1% and 32.1% at 40% and 65% r.h., respectively, at 25°C; the highest nymphal mortality was 51.5% and 52.1% at 20° and 30°C, respectively, and 40% r.h. The highest average number of E. kuehniella eggs consumed was 123.1 at 20°C and 65% r.h., and the lowest was 86.4 at 30°C and 40% r.h. during nymphal development time of A. minki.     

Survival of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in infected tissues of <Emphasis Type="Italic">Capsicum annuum</Emphasis> and in soils of the state of Pernambuco,Brazil

The survival of Ralstonia solanacearum A1-9^Rif race 1 phylotype I was studied in ten different soil types in the absence of the host plant as well as in infected tissues of the stem and root of bell peppers buried in the soil at 0, 5, and 15 cm. The survival time of R. solanacearum A1-9^Rif in the ten soil types ranged from 42 up to 77 days. Among the chemical and physical characteristics of the soil, clay content, residual moisture, and available water were positively correlated, and pH was negatively correlated, with survival time, population size at 42 days, and area under the population curve. The pathogen survival differed significantly in relation to the plant tissues, but not with respect to the incorporation depth of the infected tissues. The root tissue of bell pepper supported a larger bacterial population at 7 and 21 days (5 × 10⁴ and 3.1 × 10⁴ CFU g⁻¹ tissue, respectively) compared with the stem tissue (0.35 × 10⁴ and 0.48 × 10⁴ CFU g⁻¹ tissue, respectively) and also had a larger area under the population curve. On the other hand, the stem tissues presented a greater decomposition rate and pH compared with the roots. In conclusion, the different types of studied soils as well as the infected bell pepper tissues were considered potential primary sources of R. solanacearum inocula, but only for a short period.     

Thermal requirements and development of<Emphasis Type="Italic">Bracon vulgaris</Emphasis>, a parasitoid of the cotton boll weevil

The development ofBracon vulgaris Ashmead (Hymenoptera: Braconidae), parasitoid of the cotton boll weevilAnthonomus grandis Boheman (Coleoptera: Curculionidae), was studied at constant temperatures of 18, 20, 23, 25, 28, 30, 33, 35 and 38°C, 70±10% r.h. and 14:10 L:D period. The period from egg to adult ofB. vulgaris varied from 8.6 (35°C) to 32.9 days (18°C). The eggs of this parasitoid became desiccated and did not hatch at 38°C. The lower development threshold (LDT) and thermal constants for development varied withB. vulgaris instar, but they were similar for males and females. The duration of the 1st, 2nd, 3rd and 4thB. vulgaris instars required, respectively, 9.36 degree-days above one LDT=12.27°C, 13.48 degree-days above one LDT=6.83°C, 11.65 degree-days above one LDT=9.41°C, and 12.82 degree-days above one LDT=10.78°C.B. vulgaris has physiological adaptations to different threshold temperatures and it shows high potential to be used againstA. grandis in cotton crops.     

The predation consequence of continuous breeding vs starting a new colony of a polyphagous insect predator

The predation rates of a recently collected population of the mirid Macrolophus pygmaeus versus one continuously bred in the laboratory, reared under the same conditions for three generations, were compared in 24-h choice experiments using first to fourth instar nymphs of the aphid Myzus persicae on aubergine and sweet pepper. On aubergine, the mean wet weights for first, second, third and fourth instar nymphs of M. persicae were 46%, 36%, 8% and 14% greater, respectively, compared with aphids on pepper. Adult females from a recently collected field population of M. pygmaeus (MpK) consumed more second and third instars compared with first and fourth instars on aubergine and pepper (79% and 88% of biomass consumed, respectively). M. pygmaeus females (MpV) from a population maintained in the laboratory for more than 15 years consumed more first and second instar nymphs on aubergine. On pepper, consumption by MpV females was relatively constant between instars and unlike other combinations, where third instars contributed the greatest amount of biomass consumed (43–60%), fourth instars contributed the most biomass consumed (51%). The total aphid biomass consumed by the MpK population was twofold greater on pepper and 4.7-fold greater on aubergine when compared with the MpV population. The MpK population consumed more aphid biomass on aubergine compared with pepper while the reverse was the case for the MpV population. Such intraspecific differences may have a significant influence on their efficacy as predators in biological control systems.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

Genetic diversity and differentiation of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> nuclear polyhedrosis virus isolates from India

India has a rich biodiversity of microbes. Soil is the major source for isolation of entomopathogens, after the infected insects themselves. Four isolates of Helicoverpa armigera nuclear polyhedrosis virus (HearNPV) were obtained from the samples collected one each from Anand, Surat and Junagadh of Gujarat and Patancheru of Andhra Pradesh. All the HearNPV isolates appeared as clear, irregular six-sided objects with rounded edges, phase-bright under phase contrast. Junagadh, Surat, Patancheru and Anand isolates gave 27.47–42.80%, 36.83–51.32%, 26.05–43.76% and 42.99–54.85% mortality, respectively, when the percent mortality was pooled over period. The least number of HearNPV polyhedral inclusion bodies (PIBs) (5.1 × 10⁷) of the Anand isolate were required to kill 50% of the H. armigera population within 120 h. The Anand isolate was fastest (90.30 h), followed by Surat (120.26 h), Junagadh (139.53 h) and Patancheru (143.10 h) in killing 50% of the H. armigera population at a dose of 10⁹ PIBs ml⁻¹. RAPD analysis of all 15 arbitrary oligonucleotide primers generated 353 scorable bands with 201 loci. A total of 181 polymorphic bands were obtained, ranging in size from 141 to 1,873 base pairs. The percentage of polymorphic loci was 90.19%. The mean polymorphism information content (PIC) value for 15 primers was found to be 0.99. The similarity coefficient values based on 15 RAPD markers ranged from 0.235 to 0.407. The four isolates were grouped into two clusters: one cluster consisted of Junagadh and Anand and the second cluster consisted of Surat and Patancheru.     

Sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to propamidine: in vitro determination of baseline sensitivity and the risk of resistance

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC₅₀ values of mycelial growth ranging from 0.182 to 1.460 μg ml⁻¹, with a mean of 0.79 ± 0.27 μg ml⁻¹. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10⁻⁹ and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC₅₀ values of 5.0–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹ respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC₅₀ = 0.3–3 μg ml⁻¹; MIC = 10–30 μg ml⁻¹) and conidial germination (EC₅₀ = 0.03–0.1 μg ml⁻¹; MIC = 1–3 μg ml⁻¹) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC₅₀ ∼ 0.3 μg ml⁻¹, ranging from 0.03 to 1 μg ml⁻¹; MIC > 100 μg ml⁻¹) and high (EC₅₀ > 100 μg ml⁻¹) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.