A simple and efficient method for isolating small RNAs from different plant species

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.     

Diofenolan: a novel insect growth regulator in common citrus butterfly, <Emphasis Type="Italic">Papilio demoleus</Emphasis>

Penultimate and last instar larvae of the common citrus swallowtail butterfly, Papilio demoleus L. (Papilionidae: Lepidoptera), were treated with different doses of a novel juvenoid, diofenolan. Several deformities were observed as a result of topical administration of diofenolan which include delay in larval–larval and larval–pupal ecdysis, ecdysial failure, mortality, severe reduction in pupation, deformed pupae and complete inhibition of adult emergence. The juvenoid diofenolan severely hampers the normal growth, development and metamorphosis of P. demoleus and can be incorporated into integrated pest management programs for successful control of this important pest of citrus all over the world.     

Note on <Emphasis Type="Italic">Wahlgreniella nervata</Emphasis> (Gillette, 1908) (Hemiptera: Aphididae): a new pest of Damask rose in Turkey

Alate viviparous females of Wahlgreniella nervata (Gillette) were found on a Damask rose (Rosa damascena Mill.) plantation in Isparta Province, Turkey. The aphid is recorded for the first time from Turkey. Besides, Damask rose is a new host for this species. Brief information about this aphid including a key to the alate females of aphid species known to infest Damask rose in Turkey is provided.     

Resistance of <Emphasis Type="BoldItalic">Spodoptera exigua</Emphasis> to ten insecticides in Shandong,China

Spodoptera exigua (Hübner) collected from three regions (two scallion-producing regions and one ginger-producing region) in Shandong, China and a laboratory colony of Wuhan (WHLC) were evaluated for their susceptibilities to ten insecticides (emamectin benzoate, chlorfenapyr, indoxacarb, spinosyn, tebufenozide, methoxyfenozide, chlorfluazuron, beta-cypermethrin, chlorpyrifos and methomyl) in 2008, 2009 and 2010 using a leaf-dip bioassay method. The results indicate that the resistance ratios of S. exigua to newer insecticides such as emamectin benzoate, chlorfenapyr and indoxacarb were all below 20-fold, with no obvious change in all 3 years. S. exigua exhibits moderate resistance to spinosyn, and its resistance ratios increased from 1.98–5.31-fold in 2008 to 14.31–64.20-fold in 2010 from three regions as compared with WHLC. S. exigua showed moderate to high resistance to insect growth regulators such as tebufenozide, methoxyfenozide, chlorfluazuron in 2008, 2009 and 2010, in which resistance to chlorfluazuron was rapidly increased from 31.49–88.19-fold in 2008 to 1184.39–2789.67-fold in 2010. Resistance of S. exigua to beta-cypermethrin and chlorpyrifos varied greatly among the three regions, ranging from 95.31–437.97-fold and 25.05–40.64-fold in 2008, 951.81–1304.40-fold and 44.91–186.33-fold in 2009, to 27.27–1095.31-fold and 19.12–267.98-fold in 2010. In contrast, S. exigua showed low resistance to methomyl, and the resistance ratio was below 5-fold in 3 years. There are several reasons accounting for varying degrees of resistance, including selection pressure, cropping structure and migration, in which the migration of S. exigua may play an important role.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.     

Pathogens of bark beetles (Coleoptera: Curculionidae) in Bulgarian forests

The occurrence and prevalence of bark beetle pathogens in forest stands in Bulgaria were investigated in 944 specimens belonging to 21 bark beetle species. Protozoa, microsporidia, fungi and nematodes occurred in 19 of all investigated species. The infections were found in the gut (nematodes, gregarines, microsporidia), gonads (microsporidia) and hemolymph (nematodes) of the infected insects. Protozoan species (Gregarina typographi, Gregarina spp.) were detected in eight bark beetle species. Morphometric data about G. typographi and Gregarina spp. are presented. The prevalence of the gregarines varied between 1.4% and 64.2%. Microsporidia of the genera Nosema and Chytridiopsis were revealed in three bark beetle species. The prevalence of microsporidia ranged between 1.5% and 11.8%. This is the first report of a microsporidium in Taphrorychus villifrons and of gregarines in T. villifrons, Pityogenes bistridentatus, P. conjunctus, and Orthotomicus erosus. The fungus Beauveria bassiana was found in 3.4% of Hylurgops palliatus specimens. Nematodes (in gut and haemolymph) were revealed in 19 bark beetle species and their prevalence varied between 10% and 98.5%.     

High Performance Thin Layer Chromatography (HPTLC) profile of an insecticidal compound of <Emphasis Type="Italic">Trochus radiatus</Emphasis> and assessment of mucus-associated bacterial extracts

The current study is based on the screening of novel insecticides from new sources that have not being exploited hitherto. The major objectives of this research work were to extract marine molluscs, Lambis lambis, Trochus radiatus and Chicoreus ramosus, from Tuticorin coastal waters using different solvents to test their insecticidal properties and partially purify the active components. The ethyl acetate extracts of L. lambis and T. radiatus showed 100% mortality of Sitophilus oryzae, at a concentration of 200 μg ml⁻¹. The LC₅₀ values for ethyl acetate extracts of L. lambis, T. radiatus and C. ramosus were found to be 67.08, 348.86 and 571.42 μg ml⁻¹, respectively. With regard to bacterial metabolites, at a concentration of 200 μg ml⁻¹ the ethyl acetate extract of A3 was able to elicit an activity of 40% and that of strain A1 – 20%. The LC₅₀ values of the bacterial metabolites were also investigated. Upon chromatographic separation of active ethyl acetate extracts of T. radiatus, the 100% methanol column-purified fraction was found to have an activity of 30% at a concentration of 10 μg against S. oryzae. The purity of the partially pure active compound was observed to be 72.78% on analysis with high pressure thin layer chromatography.     

Combining sub-lethal heating and on-farm wastes: effects on <Emphasis Type="BoldItalic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="BoldItalic">cumini</Emphasis> causing wilt of cumin

In a 2-year field study, the effects of four heating regimes established by varying temperature or duration of heating on efficiency of on-farm wastes as soil amendments in controlling Fusarium oxysporum f.sp. cumini (Foc) causing wilt on cumin (Cuminum cyminum L.), were ascertained. Significant improvement in reduction of Foc propagules was achieved with the increase in duration and amount of heat. In 2000, mild heating under shade (heat level 4), 31.8–65.9% reduction in Foc propagules was estimated in all the amendments at 0–30 cm soil depth, which improved by 75.7–86.5% in the treatment where Foc-infested soil brought from the laboratory was exposed to direct heat (heat level 3) and amendments and irrigation were applied. Foc propagules were reduced by 76.6–88.3% when infested soil was exposed continuously to dry heat for 56 days (heat level 2), improving efficiency of amendments by 0.9–13.5% compared with heat level 3. After 56 days of exposure to dry heat, elevating the temperature by mulching amended soil with a transparent polyethylene film (50 μm thick) for 20 days (heat level 1) augmented reduction by 80.2–95.5%. In the second season, combining 0.04% of onion, Verbisina encelioides or mustard oil-cake with mustard residues (0.18%) improved the reduction in Foc propagules at all heat levels even at the lower soil depth. Maximum reduction (94.9–100%) in Foc propagules at heat levels 1 to 3 was achieved when residues of Verbisina were supplemented with those of mustard. These results suggest a new approach to improve the control of Foc by combining prolonged heating with on-farm wastes such as Verbisina residues and one summer irrigation.