Isolation of a novel Carica papaya α-amylase inhibitor with deleterious activity toward Callosobruchus maculatus

Cowpea (Vigna unguiculata) is a subsistence crop for small and poor farmers from Latin America and Africa. This culture is commonly damaged by cowpea weevil (Callosobruchus maculatus), which burrow into stored seeds to fed on. Due to impact of larval predation, several plant defense studies have been developed, indicating that α-amylase inhibitors are able to impede and/or reduce bruchids digestive process. In this report, a novel α-amylase inhibitor from papaya seeds (Carica papaya) with activity against cowpea weevil enzymes was purified and biochemical characterized. Peeled seeds were macerated and extracted with a 0.6 M NaCl and 0.1% HCl solution. Crude extract was precipitated with ammonium sulphate (100%). After dialyses, this rich fraction was applied onto a CM-Cellulose column and retained peak was submitted to an analytic reversed-phase column HPLC (Vydac C-18TP) yielding several peaks. Only one fraction, with molecular mass of 4562 Da, showed significant inhibitory activity against C. maculatus α-amylases. Otherwise, no inhibitory activities against mammalian α-amylases were observed. Bioassays using artificial seeds containing C. papaya α-amylase inhibitor rich fraction (0.5% and 1.0%) were also conduced showing that α-amylase inhibitors were able to increase larval mortality (50%) and also decrease insect fecundity and adult longevity. These results showed the presence of an α-amylase inhibitor from C. papaya seeds with high specificity to insect enzymes, indicating that this inhibitor probably could be used, through genetic engineering, in the construction of transgenic plants with enhanced resistance toward cowpea weevil.     

Inhibition of insect pest α-amylases by little and finger millet inhibitors

The inhibitory effect of three proteinaceous inhibitors isolated from little and finger millet was examined on gut α-amylases for four stored grain and four phytophagous insect-pests. Additionally, using native PAGE, several α-amylases isozymes were observed in all insect-pests studied. Furthermore, thermostabilities and the pH optimum for insect-pests α-amylases, which varied from acidic to alkaline, were also determined. On the other hand, proteinaceous inhibitors from little millet seeds Panicum sumatrense (LMCO3) and from finger millet (FMCO11 and FMCO13) inhibited insect-pests α-amylases with different proportions. The highest inhibition percent was recorded for LMCO3 and FMCO13 against Callosobruchus chinensis α-amylase, where the inhibition percent was approximately 70 and 50%, respectively. Furthermore, millet α-amylase inhibitors also reduced significantly digestive α-amylolytic activities of Acaea janata, C. cephalonica, Sitophilus oryzae, and Tribolium castaneum, indicating that these α-amylase inhibitors could be used toward crop insect-pests.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

Investigation of insecticidal activity of rye α-amylase inhibitor gene expressed in transgenic tobacco (Nicotiana tabacum) toward cotton boll weevil (Anthonomus grandis)

Innumerable proteinaceous α-amylase inhibitors have been isolated and identified from different plant species. Among them, an α-amylase inhibitor gene with bioinsecticidal potential toward Anthonomus grandis (cotton boll weevil) was previously identified in rye seeds (Secale cereale). This cereal inhibitor was expressed in tobacco plants (Nicotiana tabacum) under control of phytohemaglutinin promoter by using Agrobacterium tumefasciens - mediated transformation. Presence of αBIII-rye gene and further protein expression were confirmed by PCR and Western blot analysis, respectively. Immunological assays indicated that the recombinant inhibitor was expressed in concentration range from 0.1% to 0.28% (w:w) of the total protein in tobacco seeds of R₀ plants. From 14 independent transformants, five plants with expression levels between 0.20% and 0.28% in seeds were in vitro assayed against A. grandis amylolytic enzymes causing clear inhibition. Moreover, bioassays using transgenic seed flour mixture for artificial diet produced 74% mortality in A. grandis first larval instar. These data suggest that rye inhibitor could be a promising biotechnological tool for produce transgenic cotton plants with an increased resistance to cotton boll weevil. Moreover, αBIII-rye gene should be considered a potential compound for a pyramiding strategy aiming to delay insect-resistance.     

坝上高原白边地老虎生物学特性及防治研究

白边地老虎Euxoa oberthuri Leech是坝上高原地区作物苗期的重要害虫之一。经1984至1986年研究,1年发生1代,以不脱壳的幼虫在卵内越冬。4月底至5月底三龄前幼虫占85％—89.6％,是幼虫防治的关键时期。当地胡麻播种期多在5月上中旬,正值三龄幼虫发生期。采用0.5％(有效成份)甲胺磷拌种。经室内毒力试验,田间小区及大面积防治示范,结果具有90％的杀虫效果及80.2％—91.7％的保苗效果。省工,成本低,操作简便,适宜大面积推广应用。     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

Mode of transmission and morphological structures of two <Emphasis Type="Italic">Eremothecium</Emphasis> species between <Emphasis Type="Italic">Riptortus pedestris</Emphasis> and soybean

Eremothecium ashbyi and E. coryli were present in the maxillary and mandibular stylet pouches of the contaminated heads of the true bug Riptortus pedestris collected during a field survey, 2007. Ascospores of E. ashbyi and E. coryli were taken up in the stylet pouch when R. pedestris fed on soybean seeds infected with the respective fungus. In the case of E. ashbyi, the ascospores had subsequently germinated and were recognized as large masses of mycelia in the stylet pouch. In contrast, masses of E. coryli including buds of irregular size and shape were recognized about 3 days after. These results proved that these yeasts are taken up and become lodged in the stylet pouch when the insect feeds on infected host plants. In stylet sheaths stained with erythrosine, E. ashbyi was found as fragments of mycelia and E. coryli was found as small buds. These observations yield evidence to confirm that E. ashbyi and E. coryli are transmitted as fragments of mycelia and as small buds, respectively, after the insect has fed on infected host plants.     

转cry1Ab基因粳稻对稻纵卷叶螟成虫产卵行为的影响

为评价转cry1Ab基因粳稻(KMD1和KMD2)对稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)成虫产卵行为的影响,采用"Y"型嗅觉仪和笼罩以及田间试验等方法对稻纵卷叶螟成虫对转基因水稻的趋性以及其产卵选择性进行了研究,并利用固相微萃取和GC-MS技术测定了这2种Bt水稻及其对照亲本秀水11挥发物的组成及其相对含量。结果表明,稻纵卷叶螟成虫在"Y"型嗅觉仪和小型笼罩内对Bt水稻和对照亲本的选择性差异不显著,而在大型笼罩和田间条件下均显著趋向于在非转基因水稻上产卵,其中大田中稻纵卷叶螟在KMD1、KMD2和对照中的百叶卵量分别为2.20±1.28、1.00±0.77和5.60±2.16粒。水稻挥发物的组成及其相对含量在Bt水稻及其对照亲本之间差异不显著。表明相对于Bt水稻,在田间条件下稻纵卷叶螟成虫趋向于在非转基因水稻产卵,而水稻挥发物可能不是引起这种行为趋性的直接原因。     

Note: Survey of seedborne fungi of sudanese cultivars of onion,with new records

Through incubation of onion (Allium cepa L.) seed samples on potato dextrose agar and moist filter papers at 28±2°C, 32 species and four varieties belonging to 19 genera of fungi were determined as seedborne in this crop. Among them, 23 species are new reports to the mycoflora of onion seeds.Aspergillus (11 species and three varieties, ∼42.1% of the total colony count of fungi) was the most prevalent genus:A. niger (∼19.7%) was found at the highest rate in the seeds, followed byA. parasiticus (∼17.3%,A. oryzae (∼17.3%) andA. flavus (∼14.5%). The genusAspergillus was followed byPenicillium (∼12.4%),Sclerotium (∼7.1%),Fennellia, Rhizopus, Chaetomium, Drechslera andAlternaria (∼12.4%),Fusarium, Emericella andByssochlamys (∼7.1%), whereas the remaining eight genera displayed a low level of infection (∼10%). Twenty-seven species, three varieties and 12 genera represent Hyphomycetes; four genera, three species and one variety — Ascomycotina; one genus and one species — Zygomycotina; two genera and two species — other Deuteromycotina; and one genus — Oomycetes. Some species, which are known to cause devastating pre- and postharvest diseases to onion crops, were recovered from the seeds of this crop, suggesting the high possibility of their transmission by seed. http://www.phytoparasitica.org posting July 14, 2004.     

Impact of alfalfa mosaic virus subgroup I and II isolates on terpene secondary metabolism of Lavandula vera D.C., Lavandula×alardii and eight cultivars of L. hybrida Rev.

Lavandula vera D.C., Lavandula×alardii and eight different L. hybrida Rev. cultivars cultivated at the Herb Garden of Casola Valsenio (Italy), found naturally infected by Alfalfa mosaic virus (AMV), have been evaluated for the influence of their pathologic status on essential oil biosynthesis and quality. The viral disease, consisting of a “yellow mosaic” on leaves and stems, was identified by means of electron microscopy, RT-PCR-RFLP and PAS-ELISA assays. Molecular characterization of the coat protein gene of four AMV isolates suggested that three of them belonged to subgroup I (from cvs. Ordinario and Grosso, Lavandula×alardii), while the isolate from cv. Sumiens showed molecular features of subgroup II. The infection of Lavandula plants by AMV (of the two subgroups) was found to decrease essential oil secretion. Changes in the relative composition regarded the linalool-linalyl acetate ratio; the overall increase in terpenic esters and in some cases increased sesquiterpenic abundance. Infection caused by AMV subgroup II isolate influenced negatively the quality of the essential oil much more than AMV isolates belonging to subgroup I. The importance of the phytopathological status of essential oil bearing crops is outlined.     

Powdered neem seed and cake for management of the banana weevil,cosmopolites sordidus, and parasitic nematodes

Field trials were conducted in Kenya with ‘Nakyetengu’, an East African highland banana cultivar (AAA-EA), highly susceptible to banana pests. Regardless of soil fertility levels, incorporation around the plant base of powdered neem(Azadirachta indica A. Juss.) seed or cake at 60-100 g/mat at 4-month intervals, gave better control of the banana weevil,Cosmopolites sordidus (Germar), and of parasitic nematodes, than that achieved with soil application of Furadan 5G (carbofuran) at 60 g/mat at 6-month intervals. Compared with untreated control, fruit yield in most of the neem treatments was significantly higher, particularly during the second cycle of crop production. Neem application conferred a net economic gain, whereas Furadan application proved uneconomical. Application of powdered neem seed or cake at higher rates (200–400 g/mat) at 6-month intervals caused phytotoxicity, resulting in drying up of banana plants before fruiting, or in ‘chokethroat’,i.e., inflorescence emergence failure.     

7种林木植物对华北大黑鳃金龟取食和繁殖的影响

为明确华北大黑鳃金龟Holotrichia oblita(Faldermann)的嗜食植物,优化其室内人工饲养技术及为诱虫植物提供候选材料,以花生叶片为对照,研究了7种林木植物叶片对华北大黑鳃金龟取食和繁殖的影响。结果表明,华北大黑鳃金龟对8种植物日均取食量由高到低依次为金叶女贞、刺槐、核桃、榆树、花生、柳树、香椿、毛白杨,其中金叶女贞、香椿和毛白杨与花生间有极显著差异,金叶女贞与刺槐、核桃、榆树间无显著差异;单雌产卵量由高到低依次为金叶女贞、柳树、核桃、香椿、花生、榆树、毛白杨、刺槐,其中金叶女贞、柳树、核桃、刺槐与花生间有极显著差异,而金叶女贞与柳树间无显著差异。研究表明,饲养华北大黑鳃金龟成虫适宜饲料植物为金叶女贞,日均取食量最高为4.70 g,产卵期最长为56.00 d,单雌产卵量最多为58.88个,其次为核桃和柳树。     

Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

Passalora blight of anise (Pimpinella anisum) and its control in Turkey

Passalora blight of anise, caused byPassalora malkoffii (Bubák) U. Braun, is an important disease of anise in Turkey. The disease affects all the aboveground parts of plants including flower clusters. Infected seeds have dark, linear stromata. Detection of the pathogen on seeds was studied by the blotter method, agar method, washing test and sowing infected seeds in disease-free soils. The pathogen was recovered only by the washing test and to a limited extent by water agar + seed decoction agar. Sixteen of 24 seed samples from diseased regions were found to be infected. The pathogen was not detected by any other methods. However, several indigenous fungi,e.g. Alternaria alternata, were isolated, which may have prevented the growth of the pathogen. Seed washings of infected seed samples had typical spores of the pathogen up to 10⁶ conidia per gram of seed. Transmission of the pathogen was shown by sowing infected seeds in disease-free field soils in two locations where anise had not been grown previously. Azoxystrobin, chlorothalonil + carbendazim and flutriafol seed treatments at 0.04 g a.i. kg⁻¹ seed, 1.0 g + 4.5 g a.i. kg⁻¹ seed and 0.015 g a.i. kg⁻¹ seed reduced the disease by 92.5%, 89.6% and 36.2% in 2002 and by 78.9%, 75.8% and 41.2% in 2003, respectively. Three foliar applications of axosystrobin, chlorothalonil + carbendazim and flutriafol at the rates of 187.5 g a.i. ha⁻¹, 1500 g + 6750 g a.i. ha⁻¹ and 31.3 g a.i ha⁻¹ reduced disease incidence by 92.5%, 86.0% and 96.8% in 2002 and by 97.5%, 90.8% and 97.0% in 2003, respectively. http://www.phytoparasitica.org posting May 17, 2005.     

Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.     

Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato

We have previously shown that flagellin of Pseudomonas syringae pv. tabaci is an elicitor that induces a hypersensitive reaction (HR) in nonhost tomato cells. Flagellin is the major HR elicitor produced by this pathogen, as shown by the inability of a flagellin-defective mutant, ΔfliC, to induce HR. Also, a ΔfliD mutant that secretes large amounts of monomer flagellins induces a strong HR in tomato. In this study, the possible involvement of an Hrp type III secretion system (TTSS) in flagellin-induced HR was investigated using flagella-defective mutants or Hrp TTSS-defective mutants. The hrcC gene encodes HrcC protein, which is required for Hrp pilus formation in the outer membrane. An hrcC mutation, introduced into the wild-type, ΔfliC, and ΔfliD mutants of P. syringae pv. tabaci did not affect swimming motility or flagellin secretion, whereas all ΔhrcC, ΔfliC, and ΔfliD mutants lost the ability to cause disease on host tobacco leaves. However, the ΔhrcC mutant and the ΔfliD/ΔhrcC double mutant were still able to induce HR cell death, expression of one of the defense-related genes hsr203J, and the generation of hydrogen peroxide in nonhost tomato cells. Thus, flagellin is required for both pathogenicity in host tobacco and HR in nonhost tomato. On the other hand, hrp TTSS is necessary for pathogenicity on host tobacco but is not indispensable to induce HR in nonhost tomato. These results clearly show that flagellin-induced HR is hrp-independent in tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB049570