Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

Study on infection symptoms of root-knot nematode, Meloidogyneja vanica, on the stem of the tomato seedlings in greenhouse

On the first of seeds germination, Meloidogyne javanica induced knots on the stem eventually by to penetrate into the coleoptiles. In this experiment, used soil was loamy-sandy type, pH=7.2, including 1.5% composts. Tomato seedlings of cultivar Rutgers were used as host. This experiment was conducted in Completely Randomized Design with 3 treatments and 10 replicates as fallow: (1) control, (2) 10 J2s/cm3 of soil, (3) 20 J2s/cm3 of soil. In each pot (1000 cm3), 50 seeds were planted. Inoculation was done, a day after to plant seeds, in 2 cm depth. Pre-emergence and post-emergence damping-off, number of stem-knots, height of knots on the stem in 3 treatments was more than 2 treatments. In some of the pots of 2 treatments were not observed stem-knots. As long as four weeks after planting, there was not significant difference in fresh weight and height of the seedlings in treatments. Numbers of adult females in 3 treatments were more than 2. In the stem-knots, collenchymas tissue, endodermis and stem cambium were found deformed J2s and J3s, and adult females.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Effect of various cauliflower cultivars on population density fluctuations of the cabbage aphid,Brevicoryne brassicae (L.) (Hom.: Aphididae) and its parasitoid Diaeretiella rapae (McIntosh) (Hymenoptera: Braconidae)

In this study, population density changes of the cabbage aphid, Brevicoryne brassica (L.), were tested in the field during 2011 under natural infestation at research field of the Shahed University (South of Tehran). This investigation was conducted on the eight cauliflower cultivars including Smilla, Snow mystique, White cloud, Buris, Galiblanka, Snow crown, SG and Tokita in randomised complete block design with five replications. Sampling was done after starting of aphid activity in the field every week and the total number of adult aphids and nymphs were counted in three leaves of the upper, middle and lower parts of the plant. In addition, the percentage of parasitism of the cabbage aphid by Diaeretiella rapae was evaluated. Results of the sampling showed that peak of the aphid population were at the first half of November. The parasitism percent of the cabbage aphid by D. rapae was significantly different among studied cultivars (p?<?0.05). Therefore, it can be concluded that using of Smilla, Buris and SG cultivars probably increases the efficiency of D. rapae in the control of the B. brassica (L.).     

Efficacy of Elettaria cardamomum L. (Zingiberaceae) essential oil on the two spotted spider mite,Tetranychus urticae Koch (Acari: Tetranychidae)

Two-spotted spider mite, Tetranychus urticae Koch is the major pest of various plants worldwide. Now the control is dependent on the use of chemical pesticides. Plant compounds are recently known as biopesticides. Essential oil of Elettaria cardamomum was researched on repellent and oviposition inhibition of T. urticae. The LC₅₀ values of fumigant toxicity of this oil on adults and eggs of the two spotted spider mite were 7.26 and 8.82?μL/L air, respectively. Also LT₅₀ value of essential oil at 45?μL/L air was 23.86?h and LT₅₀ value of essential oil at 60?μL/L air was 9.01?h. In addition, different concentrations of the essential oil of E. cardamomum significantly affected oviposition deterrence and repellency of adults. The results of this study indicated that essential oil of E. cardamomum may be considered as a biopesticide to control two spotted spider mites.     

Effect of eight cauliflower cultivars on biological parameters of the cabbage aphid,Brevicoryne brassicae (L.) (Hem: Aphididae) in laboratory conditions

The cabbage aphid, Brevicoryne brassicae is one of the most important pests with broad host range that does damage to cruciferae plants. In this research, biology parameters of the cabbage aphid was studied on the eight cauliflower cultivars: Smilla, Snow mystique, White cloud, Buris, Galiblanka, Snow crown, SG and Tokita. This study was conducted under controlled conditions (25?±?2?°C, 65?±?5% RH and L-D: 16–8). Statistical analysis showed that there is significant difference between different stages of growth and mean number of laid nymphs period (p?<?0.05), so that the maximum and minimum growth period were observed on the cultivars Galyblanka and Buris, respectively. The longest mean longevity of total nymphal instars was obtained on White cloud, and the shortest on Smilla. The highest pre nymph period, nymph production period, mean number of laid nymphs, Adult longevity and total lifespan on the Galiblanka cultivar was calculated as 8.60, 13.50, 58.60, 17.30 and 25.90, respectively. The results showed that using cultivars that affected on the adult reproductive parameters and provides conditions for reduction of reproduction could be a good solution for the cabbage aphid control management.