Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.     

Isolation and screening of fungi associated with eggs and females of root-knot nematodes and their biocontrol potential against Meloidogyne incognita in Bangladesh

A total of 297 fungal isolates belonging to 20 genera and 33 species were isolated and identified from eggs and females of Meloidogyne spp. in Bangladesh. The predominant genera were Fusarium, Aspergillus and Penicillium; and the significant ones were Purpureocillium, Trichoderma and Pochonia. The 24 well tissue culture plate screening technique was applied for pathogenicity tests against Meloidogyne incognita in vitro. The average percentages of egg parasitism, egg hatch inhibition and juvenile mortality varied significantly and were ranged from 8.2 to 64.9% (p = 0.05), 24.8 to 72.4% (p = 0.05), and 2.3 to 33.1% (p = 0.05), respectively. Two isolates of Purpureocillium lilacinum (PLSAU 1 and PLSAU 2) and one isolate of Pochonia chlamydosporia (PCSAU 1) reduced more than 60% average root galls of tomato, eggplant and cucumber in greenhouse experiments. This is the first investigation of fungi associated with nematodes in the country and their biological control potential against M. incognita.     

Actinomycetes and fungi isolated from plant-parasitic nematode infested soils: screening of the effective biocontrol potential,indole-3-acetic acid and siderophore production

Root-knot nematodes are serious pathogens that severe damage to major crops. They damage plant root system that caused significant yield losses. Moreover, the predisposition of nematode-infected plants is secondary infection from fungal plant pathogen that additional adverse effects on plant growth. Our target is to find the antagonist for control nematode, and secondary infection agents and stimulate plant growth. Twenty-three plant-parasitic nematode infested soils were taken from some provinces in the northern and center of Thailand and actinomycetes and fungi were isolated. Eighty-three isolates belong to actinomycete and 67 isolates were fungi. The predominant actinomycete taxa was Streptomyces (97.6%). The predominant fungal taxa were Penicillium (37.3%) and Fusarium (32.8%). All actinomycete and fungal isolates were subjected for primary screening in vitro for their effects on egg hatching and juvenile mortality of Meloidogyne incognita. Secondary screening was evaluated for antagonist effect on plant pathogenic fungi collected from nematode-infected plant, plant growth hormone (indole-3-acetic acid; IAA) and siderophore production. From primary screening, 7 actinomycete and 10 fungal isolates reduced egg hatching and kill juveniles of M. incognita after 7 days incubation. In secondary screening, 10 nematophagous microbes produced IAA and 9 isolates produced hydroxamate siderophore. Streptomyces sp. CMU-MH021 was selected as a potential biocontrol agent. It reduced egg hatching rate to 33.1% and increased juvenile mortality rate to 82% as contrasted to the control of 79.6 and 3.6%, respectively. This strain had high activity to against tested fungi and high ability on IAA (28.5 μg ml⁻¹) and siderophore (26.0 μg ml⁻¹) production.     

根结线虫放线菌及其生物防治活性研究

从感染植物根部的根结线虫卵和雌虫中,分离得到放线菌20株。形态、细胞壁氨基酸组分和16S rRNA基因序列分析表明:分离菌株分属于链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和假诺卡氏菌属(Pseudonocardia),其中链霉菌占80.0%。分离菌株对根结线虫Meloidogynespp.卵的平均寄生率、卵的孵化率、幼虫死亡率分别为54.1%、40.4%和26.2%。根据体外测试的结果,选择具有高致病力的3株链霉菌(1-17,2-6,9-47)和1株诺卡氏菌(5-1)进行温室番茄防效测试,其生防效率分别为31.4%、37.7%、56.4%和42.4%。     

Fungi associated with eggs and first-instar larvae of the European corn borer

Fungi isolated from field-collected egg masses of the European corn borer, Ostrinia nubilalis, were identified as Alternaria spp., A. porri, Fusarium spp., Fusarium oxysporum, Beauveria bassiana, Mucor spp., and an unidentified yeast. Most fungi were associated with predator injury to the egg mass. Bioassay of fungi on egg masses, however, showed that Alternaria spp. and A. porri reduced the hatch of both injured and uninjured egg masses, and Mucor sp. reduced the hatch only when the egg mass was injured.     

Aquatic Actinomycete-Fungal Interactions and Their Effects on Organic Matter Decomposition: A Microcosm Study

The role of fungi in the decomposition of organic matter in streams has been well examined, although the role of bacterial antagonists in such processes has gained little attention. To examine bacterial-fungal interactions, experiments involving pairwise combinations of four actinomycete isolates (A1+ and A2+ could remove chitin from chitin-containing media, and A1? and A2? could not) and two fungal isolates (F+ a true fungus, F? an oomycote) were conducted. For each bacterial-fungal combination, 250-ml microcosms were sampled at 8 day intervals for 32 days. Microbial biomass and organic matter, as well as the activities of five extracellular enzymes, were measured. Each experiment consisted of a control group and four treatment groups. Controls comprised sterilized stream water and macrophytes. The first treatment was inoculated with only actinomycetes (~103 cells ml-1), the second treatment was inoculated with only fungi (~102 cells ml-1), the third group was inoculated simultaneously with actinomycetes and fungi, and the fourth group was inoculated with actinomycetes 2 days after fungal establishment. For all combinations, the lowest rates of organic matter decomposition were expected in the controls, as a result of only physical degradation. In contrast, the greatest rates of organic matter decomposition were predicted in treatments inoculated with F+ 2 days prior to A1? or A2?. Greater than 50% of the organic matter was decomposed in each of the fungal treatments. Fungal-actinomycete interactions resulted in reduced fungal biomass relative to the fungal-only treatments. However, when inoculated 2 days apart, combinations of F? and actinomycetes resulted in enhanced rates of organic matter decomposition, as well as greater levels of extracellular enzyme activities. These results demonstrate that actinomycete-fungal interactions and their colonization dynamics affect the accumulation of biomass, extracellular enzyme activities, and rates of organic matter decomposition.     

Pathogenicity of Fungi to Eggs of Heterodera glycines

Twenty-one isolates of 18 fungal species were tested on water agar for their pathogenicity to eggs of Heterodera glycines. An egg-parasitic index (EPI) for each of these fungi was recorded on a scale from 0 to 10, and hatch of nematode eggs was determined after exposure to the fungi on water agar for 3 weeks at 24 C. The EPI for Verticillium chlamydosporium was 7.6, and the fungus reduced hatch 74%. Pyrenochaeta terrestris and two sterile fungi also showed a high EPI and reduced hatch 42-73%. Arthrobotrys dactyloides, Fusarium oxysporum, Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, Fusarium solani, and Exophiala pisciphila were moderately pathogenic to eggs (EPI was 2.0-4.5, and hatch was reduced 21-56%). Beauveria bassiana, Hirsutella rhossiliensis, Hirsutella thompsonii, Dictyochaeta heteroderae, Dictyochaeta coffeae, Gliocladium catenulatum, and Cladosporium sp. showed little parasitism of nematode eggs but reduced hatch. A negative correlation was observed between hatch and fungal parasitism of eggs. Fusarium oxysporum, H. rhossiliensis, P. lilacinus, S. heteroderae, V. chlamydosporium, and sterile fungus 1 also were tested in soil in a greenhouse test. After 3 months, the nematode densities were lower in soil treated with H. rhossiliensis and V. chlamydosporium than in untreated soil. The nematode population densities were correlated negatively with the EPI, but not with the percentage of cysts colonized by the fungi. Plant weights and heights generally increased in the soil treated with the fungi.     

In-vitro Assays of Meloidogyne incognita and Heterodera glycines for Detection of Nematode-antagonistic Fungal Compounds

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.     

Field Assessment of Adhesion and Hatch ofChrysoperlaEggs Mechanically Applied in Liquid Carriers

A mechanical technique was evaluated for releasing green lacewing eggs in liquid suspensions. Deposited eggs were enclosed within a circle of nonpoisonous adhesive to protect them from predation and to prevent escape of hatched larvae. Released eggs were monitored daily for 5 days after release by measuring three response variables: adhesion rate of eggs to foliage, hatch rate of eggs, and “yield” of larvae from discharged eggs; “yield” was the product of egg adhesion and egg hatch. Factors tested were: egg conditioning prior to release (incubated or refrigerated), carrier (distilled water or commercial carrier solution), application technique (mechanical or hand application), and row facing (North or South). Release technique did not significantly effect egg hatch on any day. Conditioning eggs prior to release had the greatest effect on hatch of eggs and resulting yield of larvae during the 5-day monitoring period. Carrier had a significant effect on adhesion of eggs to leaves and hatch of eggs. Commercial carrier solution increased egg adhesion but decreased egg hatch compared to water. Overall mean yield of larvae from incubated eggs distributed mechanically was not significantly different for eggs suspended in water (36.4% on day 5 post-release) and for eggs suspended in commercial carrier solution (36.1% on day 5 post-release). Hand-applied eggs had a higher hatch and subsequent yield of larvae than mechanically released eggs; however, the hand technique was labor intensive.     

Nematophagous fungi of Toxocara canis eggs in a public park of La Plata, Argentina

Fungi have showed a great potential for the biological control of nematodes. However, they have not been evaluated for the control of animal and/or human parasites transmitted by egg contaminated soils. Environmental contamination with Toxocara spp. eggs is a public health problem. Accidental swallowing of Toxocara canis eggs (a nematode of dogs) usually results on a zoonotic infection (toxocarosis). The objectives of this research were: 1) To test the presence of antagonistic fungi against T. canis in the soil in public places of La Plata city, Argentina, infected with eggs of this parasite, 2) To determine the possible association between biotic and abiotic factors of the soil with the presence of fungal parasites of egg nematodes. Soil samples were tested for: textural type, organic matter (%), pH, presence of egg-parasite fungi, of larvae and of nematode eggs, in particular of Toxocara spp. The studied area showed the following characteristics: pH: 6.6-8.0, organic matter: 1.2-70%, with a predominantly loam texture. The following antagonistic fungal genera were identified: Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Mortierella, Paecilomyces and Penicillium. A prevalence of 70% was detected for nematode eggs, of 33% for Toxocara spp. eggs and of 90% for larvae. No association between the presence of egg-parasite fungi and the considered factors was found. More studies are necessary to know the natural antagonism factors to T. canis eggs for its in situ biological control.     

Detection of mycotoxins in foodstuffs by use of chick embryos

A method of extracting fungal cultures with aqueous methanol and hexane is described. The extract from 100 grams of foodstuff, or pure fungal cultures on cereals (maize, wheat and rye), was inoculated into groups of twenty fertile hens eggs, both into the air sac and into the yolk, which were then incubated through to hatch at twenty-two days. The results show good sensitivity to aflatoxin and sporidesmin. The fungal cultures are recorded as toxic (below 50% hatch) non toxic (above 68% hatch) and doubtful. Five isolates ofAspergillus repens isolated from one batch of mouldy hay gave a high (43%) incidence of deformed embryos. This method is suggested as a general biological test for toxicity in foodstuffs.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

Heterodera glycines Cyst Components and Surface Disinfestants Affect H. glycines Hatching

We investigated the effects of Heterodera glycines cyst components and surface disinfestants on hatching of H. glycines eggs in vitro. Eggs were incubated in either H. glycines cyst wall fragments, cyst wall and egg rinsate, egg homogenate, or control solutions of soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch in cyst wall and egg rinsate, and egg homogenate, was greater (α = 0.05) than hatch in sterile distilled water; however, it was not different from hatch in zinc sulfate according to Dunnett''s test. Hatch in cyst wall fragments was similar to hatch in sterile distilled water. To determine whether surface disinfestants affected hatch, eggs were treated first with chlorhexidine diacetate, mercuric chloride, sodium hypochlorite, or streptomycin sulfate and then incubated in H. glycines egg homogenate, soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch of eggs treated with chlorhexidine diacetate, mercuric chloride, and streptomycin sulfate was reduced (α = 0.05), and hatch of eggs treated with sodium hypochlorite was increased (α = 0.05) relative to hatch of nontreated eggs in all incubation solutions except zinc sulfate according to Dunnett''s Test. Hatch in zinc sulfate was similar among all surface disinfestants except mercuric chloride, where hatch was reduced relative to hatch of nontreated and other surface disinfestant-treated eggs.     

Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

大豆胞囊线虫的休眠

以前研究发现,辽宁地区大豆生长期间及收获期土壤中胞囊孵出的二龄幼虫量很少,推测线虫卵的休眠与大豆生长时期或季节相关。为明确该地区大豆胞囊线虫的休眠特点,2002-2003年采用田间随机多点取样、室内分离及模拟自然条件孵化等方法对大豆胞囊线虫的休眠进行深入研究。结果表明:在生长季节,感病品种辽豆10根围土壤中的白色雌虫、卵囊及褐色的胞囊均可孵出二龄幼虫,且孵化持续时间较长,第21d仍有幼虫孵出,白色雌虫及卵囊内的卵孵化率高于褐色胞囊;不同作物对其根围土壤中胞囊内卵的孵化影响不大,寄主作物大豆、非寄主作物玉米根围及休闲地土壤中的胞囊在条件适宜均可孵出二龄幼虫;季节对胞囊内卵的孵化有较大的影响,出苗期孵化率最高,收获期最低,2周时平均1个胞囊孵出幼虫分别为83.8和9.7条;胞囊皮对线虫卵的孵化有显著的影响。表明沈阳地区大豆胞囊线虫在正常和逆境条件下均有部分卵表现休眠。     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.     

Egg chamber production, egg protection and clutch size among fungivorus beetles of the genus Eumicrota (Coleoptera: Staphylinidae) and their evolutionary implications

Observations of members of the staphylinid genus Eumicrota show that they breed on a variety of relatively persistent gilled mushrooms (Pleurotus, Hohenbuehelia) , relatively fleshy polypores (Fauolus, Phaeolus) and similar fungi (Climacodon, Hydnellum, Phanerochaete) , and a few more woody polypores (Daedaleopszs). Behaviour features of members of Eumicrota associated with these mushrooms include: construction of an egg chamber by the female; female guarding and care of eggs; and fidelity of the female to the egg chamber before, during, and after hatching. Such energetic investment by the female results in increased survival and hatching of eggs by preventing egg predation by conspecific larvae and adults, and by preventing fungal hyphae from growing over the eggs. This behaviour differs significantly from that of related genera (Phanerota, Gyrophaena) which live and breed on more ephemeral gilled mushrooms. This is especially noticable in comparison with members of the genus Gyrophaena which are exclusively limited to those mushrooms. Members of Gyrophaena do not form an egg chamber or guard their eggs (though some cover their eggs with a layer of fungal frass), adults leave the mushroom soon after larvae hatch, and females lay significantly fewer eggs per clutch (Gyrophaena mean = 4.94, N= 18; Eumicrota mean = 11.78, N = 23; P < 0.05). It is hypothesized that these differences in behaviour of closely related genera reflect life cycle strategies evolved in response to host fungi which differ in persistence and predictability. Comparison of known behavioural features among lineages of gyrophaenine staphylinid beetles did not resolve the phylogenetic patterns of evolution of these life history features, primarily because of lack of comparative data for some groups.     

Fungal ovicidal activity on Toxocara canis eggs

BackgroundVisceral toxocariasis is a parasitic zoonosis caused by Toxocara canis. The prevalence of this parasite in dogs, soil contamination and the resistance of eggs increase human exposure to the disease. Moreover, the difficulties of the control measures justify the need for alternative ones.AimsThe objective of this study was to evaluate the in vitro ovicidal activity of fungi isolated from soils from public places in the city of Pelotas, Rio Grande do Sul, Brazil, on Toxocara canis.MethodsSamples of soil from ten localities were inoculated onto Petri dishes with 2% water–agar (WA) that contained antibiotics, and incubated at 25 °C/21 days. Isolated fungi were tested in vitro for ovicidal activity, with five replicates. One mL of an embryonated Toxocara canis egg suspension (10³ eggs) was poured over the fungal cultures after 10 days of growth. At intervals of 7, 14 and 21 days, 100 eggs were removed from each plaque and evaluated by optical microscopy.ResultsAcremonium, Aspergillus, Bipolaris, Fusarium, Gliocladium, Mucor and Trichoderma were isolated from the soil. A significant ovicidal type 3 effect was observed in Trichoderma, Fusarium solani complex and Acremonium. Those isolates from the genus Trichoderma showed their ovicidal effect on the 14th day of fungus–egg interaction. The other fungal genera tested showed a type 2 effect.ConclusionsThese results suggest that the use of Trichoderma and Fusarium solani complex in biological control of T. canis is promising; however, further studies should be performed.     

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined.     

Influence of temperature, photoperiod and humidity on oviposition and egg hatch of the root-feeding flea beetle Longitarsus bethae (Chrysomelidae: Alticinae), a natural enemy of the weed Lantana camara (Verbenaceae)

The root-feeding flea beetle Longitarsus bethae Savini & Escalona, was introduced into South Africa as a candidate biological control agent for the noxious and invasive weed, Lantana camara L. As part of the study to predict the beetles' survival in its new range, the influence of climatic conditions on its egg development and reproductive performance were investigated in the laboratory. The threshold temperature (T degrees) and degree-days (DD) required for egg hatch were determined after exposing the eggs to various constant temperatures (12, 17, 22, 27 and 32 degrees C) in separate growth chambers. The DD required for egg hatch was 178.6, and the temperature threshold required for egg hatch was 11.3 degrees C. Survival of eggs varied from 27 to 56% at 32 and 17 degrees C, respectively, and was optimum between 17 and 25 degrees C. Oviposition was examined under high and low relative humidity (RH) regimes while egg hatch was determined at six RH levels, each maintained in a separate controlled growth chamber set at a constant temperature (25 degrees C). Whilst RH had no influence on oviposition, eggs were highly susceptible to aridity, and continuous exposure to relative humidity below 63% for more than three days was wholly lethal at 25 degrees C. Optimum egg hatch occurred at RH between 85 and 95% for up to 12 days. The effect of day length on oviposition and subsequent egg hatch was investigated under two photoperiod regimes. Neither oviposition nor subsequent egg hatch was influenced by photoperiod. The knowledge obtained will be useful for mass rearing as well as field release programmes for L. bethae.