Effect of sorghum phenology on the control of Chilo partellus with Nosema marucae (microspora: Nosematidae)

An aqueous suspension of Nosema marucae spores was sprayed on foliage of sorghum plants that had been infested with neonate Chilo partellus larvae at 3, 5, 7 and 9 weeks after (plant) emergence (WAE). These periods corresponded to plants at the tillering, early booting, soft dough and late maturation phenological stages, respectively. The extent of borer infestation and plant damage was monitored until crop harvest, and compared with plants which had been similarly infested but not sprayed with the pathogen, and with plants in insect‐free control plots. When applied early, infestation with neonate borer larvae caused most damage to the plants and the greatest reduction in yield in the untreated plots. Early treatment with N. marucae spores at 3 and 5 WAE resulted in the greatest reduction in damage to plants and the highest improvement in yield of seeds. It is apparent, therefore, that for maximum crop protection the microsporidian needs to be applied when sorghum is in an early phenological stage.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

Onion fly egg distribution in soil: a new sampling method and the effect of two granular insecticides

Tests were carried out in a screenhouse to determine the potential of a microsporidian pathogen,Nosema marucae (Microspora: Nosematidae), for the control of the spotted stalk borer,Chilo partellus (Lepidoptera: Pyralidae), on sorghum. Foliar application of an aqueous suspension ofN. marucae spores on egg batches pasted on the sorghum plants reduced leaf-damage in plants in the protected plots, and increased the proportion of plants with fully- and partially formed heads compared to unsprayed plots. Further, there was less tunnelling and fewer larvae in the sprayed plots. The pathogen also reduced pest infestation and damage to plants when the plots were infested with newly emerged larvae instead of eggs.N. marucae has a potential to controlC. partellus.     

In vitro propagation of Nosema locustae using fat body cell line derived from Mythimna convecta (Lepidoptera: Noctuidae).

Nosema locustae, a microsporidian parasite of locusts and grasshoppers, was successfully propagated in a fat body cell line from Mythimna convecta (BPMNU-MyCo-1). The fat body cells were grown in MGM-448 medium supplemented with 5% fetal bovine serum and 3% Bombyx mori serum at 25 degrees C. Cultures were inoculated with Nosema spores and agitated for 2 min. Infection appeared 3 days post-inoculation and by 7th day, some cells were filled with spores. At the 15th day post-inoculation, 32% of the fat body cells were infected. After isolation, the spore yield ranged from 1.4 x 10(6) spores/ml. Infected cells were subcultured and by the 4th passage spore production decreased. Harvested spores were found infectious to Locusta migratoria.     

Physiological responses to Nilaparvata lugens in susceptible and resistant rice varieties: allocation of assimilates between shoots and roots

Nilaparvata lugens (St?l) is a typical vascular feeder, primarily sucking the phloem sap of host plants. Its feeding on rice, Oryza sativa L., plants changes the pattern of allocation of assimilates between roots and shoots, and the root:shoot (R/S) ratio of assimilates is often measured as an index of physiological responses to N. lugens. The current study investigated changes in the R/S ratio of biomass, sucrose, and soluble sugar contents of rice plants in a susceptible variety (TN1) and a resistant variety (Xieyou 963). The results demonstrated that root and shoot biomasses in the two varieties linearly decreased with the increase of N. lugens infestation density. However, the relationship between changes in the R/ S ratio ofbiomass and N. lugens density differed between rice varieties, with the R/S increasing with infestation density in TN1 and decreasing in Xieyou 963. Sucrose and soluble sugar contents and their R/S values were also significantly different between the two varieties. Compared with the control that was not infested by N. lugens, the R/S values of sucrose and soluble sugar at 3 days after infestation (DAI) increased but decreased at 6 DAI in TN1. The R/S values of sucrose and soluble sugar were higher at 6 DAI than those at 3 DAI in TN1, whereas these values were lower at 6 DAI than at 3 DAI in Xieyou 963. These contrasting results suggest that physiological responses to N. lugens infestation differ between the susceptible and tolerant rice varieties.     

Observations on the endogenous stages of Eimeria crandallis in domestic lambs (Ovis aries)

Lambs reared coccidia-free were inoculated orally with various numbers of sporulated oocysts of E. crandallis and were killed between 1 and 22 days after inoculation; tissues were examined histologically. Sporozoites were seen 1, 2 and 3 days after inoculation (DAI) in crypt epithelial cells in the mid-jejunum. Infected cells migrated into the lamina propria where the parasite within them developed into a firstgeneration meront containing about 250,000 merozoites at 10 DAI. A second generation of meronts was seen at 10–12 DAI, each containing up to about 10 merozoites, situated mainly at the bases of crypts in the jejunum and ileum but also in the caecum. From 11 DAI pro-gamonts were seen which were enveloped by the host cell nucleus and which divided in synchrony with the host cell for an undetermined number of generations. Mature gamonts began to develop from them by 16 DAI. Oocyst output began at 16 DAI and rose to a peak at about 22 DAI. Up to 10⁸ oocysts were produced per oocyst inoculated. They showed wide variation in size and colour.     

Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia)

In this report, an experimental infection of Apis mellifera by Nosema ceranae, a newly reported microsporidian in this host is described. Nosema free honeybees were inoculated with 125,000 N. ceranae spores, isolated from heavily infected bees. The parasite species was identified by amplification and sequencing the SSUrRNA gene of the administered spores. Three replicate cages of 20 honeybees each were prepared, along with one control cage (n=20) supplied with sugar syrup only. The infection rate was 100% at the dosage administered. The presence of Nosema inside ventricular cells was confirmed in the samples using ultrathin sectioning and transmission electron microscopy. By day 3 p.i. a few cells (4.4%+/-1.2) were observed to be parasitized, whereas by 6 days p.i. more than half of the counted cells (66.4%+/-6) showed different parasite stages, this value increasing on day 7 p.i. (81.5%+/-14.8). Only one control bee died on day 7 p.i. In the infected groups, mortality was not observed until day 6 p.i. (66.7%+/-5.6). Total mortality on day 7 p.i. was 94.1% in the three infected replicates and by day 8 p.i. no infected bee was alive. After the infection, the parasites invaded both the tip of folds and the basal cells of the epithelium and the autoinfective capacity of the spores seemed to spread the infection rapidly between epithelial cells. On day 3 p.i., mature spores could be seen inside host cell tissue implying that the developmental cycle had been completed. The large number of parasitized cells, even the regenerative ones, the presence of autoinfective spores and the high mortality rate demonstrate that N. ceranae is highly pathogenic to Apis mellifera. Possible relation with bee depopulation syndrome is discussed by authors.     

Effects of environmental conditions on the fixation and transfer of nitrogen from alfalfa to associated timothy

Nitrogen fixation (NF) by alfalfa and nitrogen transfer (NT) from alfalfa to associated timothy was studied under different environmental conditions in controlled growth chambers, using the¹⁵N dilution technique. Evidence was obtained of NT from alfalfa to the associated timothy. Conditions that favored NF by alfalfa resulted in an increase in its NT. Of 3 different temperature regimes (25/20, 16/14, and 12/9°C day/night), 16–25/14–20°C was the best range for NF by alfalfa and resulted in the greatest NT. High light intensity (550 uE.m⁻².sec⁻¹) and long days (16–20 h) also caused increased NF by alfalfa and benefitting timothy more than in a regime of low light intensity (by shading 50% or 75%) or short days (12/12 or 16/8 h day/night). When the inoculated (Rhizobium meliloti) root systems of plants were kept free from other microorganisms (axenic condition) to minimize possible decomposition of dead tissues, lower NT from alfalfa was observed, especially at later cuts, compared to non-axenic plants. This suggests that both direct excretion and decomposition of dead alfalfa tissues are sources of N benefit from alfalfa to associated timothy. Contribution no 1065 of the Plant Research Centre.     

Factors influencing biocontrol of jimsonweed (Datura stramonium L.) with the leaf-spotting fungus Alternaria crassa

In greenhouse tests, Alternaria crassa (Sacc.) Rands killed > 80% of inoculated jimsonweed (Datura stramonium L.) seedlings within 14 days following a 9-h dew period at 25°C with 1 × 10⁵ spores/ml, and after 8 h of dew at 1 × 10⁶ spores/ml. At least 10 h of dew with 1 × 10⁵ spores/ml and 9 h of dew with 1 × 10⁶ spores/ml were required to obtain 100% mortality of fungus-inoculated plants. Growth stage and inoculum concentration studies revealed that higher concentrations of inoculum were required to obtain 100% mortality of larger plants. Weed control was significantly reduced by day/night air temperatures of 35°C and 24°C, respectively, at all inoculum concentrations as compared to the controls at lower air and dew temperature regimes. The results of these studies indicate that A. crassa has potential as a biological herbicide for the control of jimsonweed.     

Effect of black bean aphid, Aphis fabae, on transpiration, stomatal conductance and crude protein content of faba bean

This study was initiated to investigate effects of damage by 0, 5 and 10 aphids/plant on the physiology of faba bean plants throughout different feeding periods and at two plant development stages. Immediately following removal of Aphis fabae, measurements showed 84–229% increase in transpiration rate. These changes were proportional to the number of aphids and infestation duration. Injury by A. fabae caused the stomatal conductance to be much higher in the leaves of infested plants. Leaf stomatal conductance of the infested plants increased significantly by 51–224% depending on initial aphid densities and feeding intervals. This increase was proportional to the infestation level for each date. Length of infestation period and plant growth stage seemed to have no clear effect on stomatal apertures. Aphid feeding caused a damage of about 7–33% of crude protein levels in the leaf tissue. This reduction increased with increasing infestation levels and time, except for 28‐day‐old plants on 28 days. The physiological effects of aphid feeding on water vapour and chemical composition of damaged leaves are particularly serious when the population is high.     

Management of Bittergourd yellow mosaic virus (BGYMV) by using virus inhibiting chemical,biocontrol agents,antiviral principles (AVP) and insecticide

Abstract

Bitter gourd Yellow Mosaic Virus (BGYMV) is a Whitefly transmitted geminivirus. BGYMV causes yellow mosaic disease in bitter gourd. This disease attains significance because the virus causing this disease is capable of attacking the crop at all stages. There was a severe yield loss in bitter gourd plants due to the infection of BGYMV. Bitter gourd plants treated with Bougainvillea spectabilis challenge inoculated with BGYMV reduced the disease incidence and increased the plant growth. In the above treatment the disease incidence was 33.33% at 75 Days After Sowing (DAS). But in the inoculated untreated control the disease incidence was 100% at 75 DAS. The mean maximum plant height was 92.24 cm in plants inoculated at 65 DAS. Bougainvillea spectabilis treated plants challenge inoculated with BGYMV showed an increased activity of peroxidase, polyphenoloxidase and phenol content from 4 Days After Inoculation (DAI) to 12 DAI. The activity of all the enzymes was reduced from 16 DAI in all the treatments.     

Impact of a novel species of Nosema on the southwestern corn borer (Lepidoptera: Crambidae)

A study was undertaken to elucidate the impact of an undescribed Nosema sp. on the southwestern corn borer (SWCB; Diatraea grandiosella Dyar). The Nosema sp. (isolate 506) included in the study was isolated from an overwintering SWCB larva in Mississippi. It was highly infectious per os, with a median infective dose of 2.0 x 10(3) spores per larva. Even at the highest dosage tested (10(7) spores per larva), minimal mortality (< or = 3%) was observed in infected larvae, pupae, and adults reared in the laboratory on an artificial diet. However, infected pupae (0- and 7-d-old) were smaller, and the time to adult eclosion from pupation was slightly increased. Furthermore, the number of eggs produced by infected SWCB female moths substantially decreased (32%), and this effect was most pronounced on day 2, when the greatest number of eggs were oviposited by infected and noninfected moths. For eggs produced by infected females mated with infected males, hatch was slightly decreased by 16 and 15% for eggs laid on days 2 and 3, respectively. In addition, egg hatch was reduced in eggs oviposited by noninfected females mated with infected males on day 3. A low prevalence of infection (< 6%) was observed in the F1 generation originating from infected females mating with noninfected males, from noninfected females mating with infected males, and from infected females mating with infected males. Nosema 506 spores were observed in the proximity of reproductive tissues of infected female and male moths. Spores also were detected on the chorion surface and within eggs laid by infected females. Furthermore, 1-11% of larvae hatching from surface-sterilized eggs were infected by Nosema 506 indicating a transovarial mechanism of transmission.     

Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation

Spores of the microsporidian Nosema algerae exposed to 1, 2, and 4 hr of sunlight and 121 μW/cm² of germicidal ultraviolet light were fed to first-instar Anopheles albimanus. Twenty days after feeding, the incidence and intensity of infection (spores/mosquito) were recorded from adult mosquitoes. While sunlight-treated spores showed no significant decline in incidence of infection after 1-, 2-, and 4-hr exposures, intensity of infection decreased significantly after the 2- and 4-hr exposures. Incidence of infection of mosquitoes fed bactericidal ultraviolet light-treated spores declined 48.2, 76.2, and 99.9% after 1-, 2-, and 8-min exposures, respectively. Measured by intensity of infection, activity of bactericidal light-treated spores decreased 87.2% after 1 min, 91.7% after 2 min, and 99.9% after 8 min. Levels of radiation required to inactivate spores of N. algerae fell within the range reported for other Microsporidia.     

Cytology of infection,development and expression of resistance to Plasmodiophora brassicae in canola

The timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi‐nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of non‐inoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.     

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

Comparative virulence of Nosema plodiae and Nosema heterosporum in the Indian meal moth, Plodia interpunctella

When larvae of the Indian meal moth, Plodia interpunctella, were fed diets containing spores of Nosema plodiae, the number that survived to the adult stage decreased and the rate of adult emergence was retarded as the concentration of spores was increased; all surviving adults were infected. Also, when larvae were reared on diets containing spores of Nosema heterosporum, the number that survived to the adult stage decreased as the concentration of spores was increased; however, no relationship was apparent between concentration of spores and the rate of adult emergence. The LC₅₀'s of N. plodiae and N. heterosporum were 8.09 × 10⁶ and 4.52 × 10³ spores/g diet, respectively, which confirmed preliminary observations regarding the relative virulence of the two species of Nosema to Indian meal moth larvae.     

Release of lipoxygenase products and monoterpenes by tomato plants as an indicator of Botrytis cinerea-induced stress

Changes in emission of volatile organic compounds (VOCs) from tomato induced by the fungus Botrytis cinerea were studied in plants inoculated by spraying with suspensions containing B. cinerea spores. VOC emissions were analysed using on-line gas chromatography–mass spectrometry, with a time resolution of about 1 h, for up to 2 days after spraying. Four phases were delimited according to the starting point and the applied day/night rhythm of the experiments. These phases were used to demonstrate changes in VOC flux caused by B. cinerea infestation. Tomato plants inoculated with B. cinerea emitted a different number and amount of VOCs after inoculation compared to control plants that had been sprayed with a suspension without B. cinerea spores. The changes in emissions were dependent on time after inoculation as well as on the severity of infection. The predominant VOCs emitted after inoculation were volatile products from the lipoxygenase pathway (LOX products). The increased emission of LOX products proved to be a strong indicator of a stress response, indicating that VOC emissions can be used to detect plant stress at an early stage. Besides emission of LOX products, there were also increases in monoterpene emissions. However, neither increased emission of LOX products nor of monoterpenes is specific for B. cinerea attack. The emission of LOX products is also induced by other stresses, and increased emission of monoterpenes seems to be the result of mechanical damage induced by secondary stress impacts on leaves.