نشریه مهار زیستی در گیاه پزشکی
سال هفتم شماره 2 (پاییز و زمستان 1398)

Early blight incited by Alternaria spp. is one of the destructive diseases of tomato which causes considerable crop yield losses. Biological control of the disease with epiphytic fungi as phyllosphere competent microorganisms is a suitable alternative to chemical pesticides. In order to isolating the fungi, healthy tomato plants were collected and small pieces of leaves and stems were placed in distilled water on a shaker and the aliquot was cultured. In search for biocontrol strains, isolated fungi were screened against the fungal pathogen (Alternaria alternata SN1–1) using dual culture as well as the volatile and non–volatile metabolites tests in vitro andeffective isolates were selected to study their efficacy on disease control under greenhouse conditions. Conidial suspension of epiphytic fungi was sprayed two times on the aerial parts of the plant in greenhouse. Two weeks after inoculation of the pathogen, the disease severity was evaluated based on a 0–9 scale. Results of screening test showed that epiphytic fungi inhibited the mycelial growth of the pathogen in various degrees. Maximum inhibition in dual culture, volatile and non–volatile metabolites tests were 70%, 53% and 68%, respectively. In greenhouse, the least disease indexes were recorded for the isolates F1–22 and P4––1 (81.4% disease control) together with P1–1 and P2–1 (74.4% disease control). Isolates F1–22, P4–1 and P2–1 identified as Trichoderma harzianum and isolate P1–1 identified as Trichoderma longibrachiatum.

Given the compatibility of Beauveria bassiana and plant extracts, it is feasible to increase efficacy of the fungus in microbial control of date storage pests. The aim of this study was to evaluate the interaction of plant extracts and B. bassiana on sawtoothed beetle populations. The vegetative and reproductive parts of myrtle, mugwort, mentha, yarrow, and neem plants were evaluated. The fungal mycelium growth and spore germination were evaluated to checking the consistency of extracted from plants by extract medium mixing method. Then, the compounds toxicity were evaluated individually and conjunction with B. bassiana by bioassay on the adult and larvae of sawtoothed beetle. Studies have shown that mycelial growth and spore germination of B. bassiana occur only in artemisia and neem. The spore germination rate of B. bassiana in neem was higher than artemisia in all concentrations. A concentration equivalent to 1949.4 microliters/liter of neem was required to reduce 50% of the germination of the fungus. The lowest and highest mycelial growth reduction (3.1% and 20.1%) were observed in 250 microliters/liter of neem and 1000 microliters/liter of artemisia, respectively. So, the concentration of neem equivalent of 36679.4 microliters/liter was required for 50% reduction of mycelial growth. The highest compatibility index (T) was 76.89 for 500 microliters/liter in combination with neem and the lowest level consistent with artemisia in 1000 microliters/liter. Both plant extracts had synergistic effect on the pathogenicity of B. bassiana on adult and larva of sawtoothed beetle. However, synergistic effect of neem extract was higher.

Botrytis gray mold of kiwifruit is one of the most important fungal diseases that causes a significant damage to the kiwifruit during harvest and postharvest stages. The aim of this study was to find some effective isolates of native yeasts that have a good biocontrol effects on the pathogen, Botrytis cinerea. Fifity one plant samples were collected from different part of Iran during 2015–2016. Thirthy four yeast isolates were selected as representative based on the morphological characteristics of the colonies and the size of the cells. The experiment carried out to examine the yeasts ability in pathogen inhibition on Hayward cultivar of kiwifruit based on two assays, in well on fruit and on fruit slices in Petri dishes. Results based on partial ITS region sequence region showed and according to Duncan’s multiple range test at significance level 1% as well as index disease (ID), the yeasts including  Aureobasidium pullulans, Candida membranifaciens, Cryptococcus albidus, Metschnikowia koreensis, Ogataea cortices, Papilioterama flavescens, Rhodotorula mucilaginosa and R. glutinis have the most potential to pathogen growth inhibition.

For knowledge localization of Bacillus thuringiensis (Bt) production as biological pest control, optimization of culture medium is necessary. In this study, Bacillus thuringiensis var. tenebrionis–BN1 (Btt–BN1) was used as a biological pesticide to control elm leaf beetles. The aim of optimizing the desire culture medium is to increase the production efficiency of Bt biopesticide using agricultural and food wastes as cheap raw materials. To optimize the parameters of the culture medium, experimental design of response surface methodology (RSM) was applied with a central composite design (CCD) approach. The parameters including the amount of carbon (sugar cane molasses, oats and wheat bran sources) and the amount of nitrogen (corn syrup source) at three levels (0.6 to 1 percent of sugar cane molasses, 0.3 to 0.9 percent of oat, 0.3 to 0.9 percent of wheat bran and 1 to 5 percent of corn syrup) were investigated with colony forming unit (CFU) response. The results obtained under operating conditions of pH = 7, temperature of 30oC after 72 hours. The results showed that the maximum CFU for the minimum concentration of oats (0.3%), the minimum concentration of wheat bran (0.3%), the maximum concentration of sugar cane molasses (1%) and the minimum concentration of corn syrup (1%) were equivalent to 8.36 11 spores.ml–1. In addition, the results of Btt–BN1 bioassay survey on elm leaf beetle showed that two concentrations of 0.5 and 1.0 percent cause 67.7% and 80.0% of mortality, respectively.

In this study the possibility of inducing resistance was tested using five endophytic bacterial strains against wheat take–all disease caused by the Gaeumannomyces graminis var. tritici (Ggt) under greenhouse conditions. The results showed that disease severity in treated plants compared to control samples decreased 56.6% and wheat resistance against the pathogen has been substantially increased. Effect of five endophytic bacterial strains including Pseudomonas fluorescens, Serratia marcescens, Microbacterium phyllosphaerae, Bacillus pumilus and Streptomyces argenteolus were studied on some enzymes related to plant resistance at the 0, 24, 48 and 168 hours after inoculation with Ggt. Results showed the highest levels of the β–1, 4–glucanase and chitinase at 48 and 168 hours after inoculating of pathogen. The level of β–1, 4–glucanase in plant samples inoculated with Serratia marcescens showed 0.0291 U/mg protein compared to c+ (healthy control without endophytic bacteria and pathogen) with 0/0055 and cp (pathogen– infected control and untreated with endophytic bacteria ) with 0/0075. The level of chitinase for plant samples inoculated with Pseudomonas fluorescens and Serratia marcescens strains were 0.0056 and 0.0045 U/mg protein compared to c+ (healthy control without endophytic bacteria and pathogen) with 0/0030 and cp (pathogen– infected control and untreated with endophytic bacteria) with 0/0031, respectively.

It is proved that nutrients have an important role in growth, reproduction, immune and activity digestive enzyme. If insects feed on suitable and valuable food, they will have a strong immune capacity and could be better against foreign agents. In addition, larval feeding lead to produce of insects that they have high fecundity potention. This is important purpose of rearing of intermediate hosts for parasitoides. So, in the present study, some physiological properties of Ephestia kuehniella by feeding with vitamin C and sorbitol and mannitol were determined as a dietary supplement in vitro. Condition of rearing was 25±1 ºC, RH=45% and L: D=14:10 h. Treatments were including first treat: flour (99 g) + ascorbic acid (1 g), second treat: flour (97g) +sorbitol (3 g), third treat: flour (97 g) +mannitol (3 g) and forth treat: flour (100 g) as control. Experiments were done with completely randomized design. Relative growth rate (RGR) showed a significant increase in all treatments compared to control. Third instar larvae reared on nutritional diet containing vitamin C and mannitol had the highest efficiency of conversation of ingested food (ECI) (7.66±0.66%) and highest feeding deterrent index (FDI) (37.2±2.72%). The highest index of efficiency of conversation of digested food to biomass (ECD) (16.38±0.9%) was found in larvae fed on diet containing vitamin C. Chymotrypsin activity significantly increased in larvae fed on vitamin C–enriched food. Whereas, the activity of another protease, trypsin, increased by both sugars and vitamin C treatments compared to those of control. Nutritional diets containing sorbitol and mannitol sugars increased the activity of carbohydrase enzymes, alpha (0.74±0.07) and beta glucosidase (0.44±0.008) µmol/min/mg protein compared to other treatments, and decreased lipase activity in larvae fed on diet containing sorbitol (1.3±0.06) and vitamin C (0.6±0.01) µmol/min/mg protein. Mannitol did not have a positive or negative effect on lipase activity. Regarding immunology, injection of Beauveria bassiana (isolate 47) to third instar larvae resulted in a significant increase in total blood cell and immunocyte counts 4 h after treatment. In fact, the role of vitamin C was clearer in altering blood cell counts (625±48.7) in mm3 hemolymph compared with other treatments. The number of blood cells showed a decreasing trend over time up to 24 hours. In general, among the treatments, vitamin C and mannitol sugar can be considered as good choices in the Mediterranean flour moth diet. It seems that improving the quality of the larval food is effective in their digestion, digestive enzymes, and insect physiological defense potential and the objective of rearing of Ephestia kuehniella as an alternative host to natural enemies will be achieved more strongly.

Pythium oligandrum is known as an important biological agent against plant pathogens. Isolates from different regions of Iran including Bushehr, Bakhtiari, Chaharmahal, Fars, Golestan, Ilam, Kermanshah, Kerman, West Azerbaijan and Yazd were identified based on morphological and molecular characteristics. The highest recovering frequency of isolates was observed in Kermanshah and Fars provinces. The functional diversity of isolates based on their growth temperature and biological control effect under laboratory conditions was evaluated. The antagonistic effects of native P. oligandrum isolates were evaluated against soil–borne fungal pathogens including Rhizoctonia solani, Phytophthora parasitica, Fusarium solani, F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. pisi using nonparametric statistics, and compered with commercial products of various biocontrol agents such as Clonostachys rosea, Fusarium oxysporum, P. oligandrum and Trichoderma spp. The native isolates of P. oligandrum were diverse in their growth temperature and biocontrol activities. The highest mycelial growth inhibition was found in the interaction between P. oligandrum isolates and R. solani. A significant difference between P. oligandrum isolates was found by evaluating their volatile compounds. The isolates of P. oligandrum showed maximum inhibition on P. parasitica and R. solani and minimum inhibition on F. oxysporum f. sp. lycopersici. The non–volatile compounds had the highest significant effect on P. parasitica as well. The result of this study showed that the growth characteristics and the in vitro biocontrol ability of the native isolates of P. oligandrum on various plant pathogens were different. This variety can be employed in biochemical and greenhouse screening of the isolates.

unctional and numerical responses of Aphidius matricariae Haliday were studied on Nasonovia ribisnigri Mosely at 20±1°C, 65±5% RH and a photoperiod of 16:8 (L: D) h. Different densities (2, 4, 8, 16, 32 and 64) of third instar nymphs of N. ribisnigri were provided to mated female wasp (< 24 h old) with ten replicates in 24 hours. The functional response was determined using logistic regression and the parameters were estimated by non–linear regression. The results revealed a type II functional response of A. matricariae to different densities of third instar nymphs of N. ribisnigri. Attack rate and handling time were obtained as 0.0367 h–1 and 0.4114 h, respectively. Also, maximum attack rate (T/Th) was estimated 58.33 nymphs. The data of numerical response of A. matricariae indicated that with increase of host density, the average number of mummified aphids in life span of parasitoid increased from 7.8 (density of 2) to 140.7 mummies (density of 64). According to these results, it seems that A. matricariae is potentially suitable biological control agent in suppressing N. ribisnigri population.

One of the parameters used in natural enemies evaluation is functional response studies to predict the efficacy of the predator in biological control programs. The vine mealybug, Planococcusficus (Signoret) is an important vineyard pests in all parts of the world. Cryptolaemusmontrouzieri Mulsant, is a well–known natural enemy of mealybugs. The experiment was conducted under laboratory condition with 26±1°C with 70±5 % RH and 12:12 D:L photoperiod with four densities of adult females of vine mealybug, P. ficus, as host insect. Adult female and male and 3rd instar larval stage of C. montrouzieri and four densities (3, 5, 7 and 9, each with 10 replicates). The number of host killed after 24 hours were recorded. Determination type of functional response model and estimation of related parameters were carried out based on two–stage analysis method by using SAS software. A type 2 functional response was concluded under this experimental condition for all stages of predator using logistic regression with parameters estimated through nonlinear regression. The results for searching efficiency and handling time for 3rd instar larval, adult male and adult female of the predator were (0.6922 and 0.9549 hours), (0.4901 and 1.3542 hours) and (0.5977 and 1.0996 hours), respectively. Results showed that 3rd instar larvae of predator showed a better searching efficiency and lowest handling time rather than adults which is a fine character for a predator.

The success of pest control using insect pathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae depends on practical and economical methods for their mass production. In this study, the effects of light periods (including 12-12, 18-6 and 18-6 light and dark), incubation times (48, 72- and 96-hours) and incubator rotations (100, 150 and 200 rpm) were investigated based on blastospore concentrations of the two fungi in 4 replications. The results showed that different treatments including light periods, incubation times and incubator rotations had significant difference on B. bassiana and M. anisopliae fungi in terms of spores concentrations, germination percentage, wet and dry weights production in complete liquid phase for blastospore at 1% probability level. The highest concentration of blastospore, germination percentage, wet weight and dry weight of B. bassiana and M. anisopliae were 18 hours of light and 6 hours of darkness, rotation rate of incubator of 150 rpm and incubation period of 75 hours. Also, the results of this study showed that B. bassiana and M. anisopliae can be replicated with good performance of sugarcane byproducts and maintain the germination capacity of blastospore. The highest concentration of blastospore, germination percentage, fresh weight and dry weight of B. bassiana and M. anisopliae were incubated at 150 rpm. According to the results of this study, two species of pathogenic fungi can be produced by using a combination of environmental conditions and incubator device settings with good production performance and appropriate economic efficiency.

Mulberry whitefly Aleuroclava jasmini sensu lato Takahashi 1932 as a sap feeder insect is one of the most serious pest in mulberry trees in Tehran province, Iran since 2016. The population of the species has increased due to lack of natural enemies in the new area. In this paper, virulence of four Iranian isolates of B. bassiana on second nymphal stage of A. jasmini sensu lato in five concentrations through spraying under laboratory conditions were investigated. The number of dead nymphs was recorded daily to 10 days. Mean mortality percentages were analyzed by AVONA under 95% of confidence level in SPSS. LC50 and LT50 were calculated by SAS. The final results from Probit test showed that the lowest LC50 produced by to two isolates, PTCC5197 and IRAN1359 as 1.3 × 105 conidia ml–1 indicated higher pathogenicity against A. jasmini sensu lato. The highest LC50 belonged to IRAN1228 with the value of 9.9 × 105 conidia ml–1 . LT50 results showed that by increasing the isolates concentration, mean lethal time decreases. It can be concluded that the studied isolates had high level of pathogenicity on A. jasmini sensu lato nymphs and it can be applied through IPM programs, indeed their environmentally safety would be so valuable.

Biosurfactants are surface active biomolecules that are produced by microorganisms and have diffetent applications. In recent years, these biological molecules have received much attention due to their special properties such as specificity, low toxicity and easy preparation. The effect of biosurfactant produced by Staphylococcus hominis on red flour beetle larvae (Tribolium castaneum) was investigated. This experiment was done with three replications by different concentrations, and the results were analysed by SAS software. The results of the mean mortality percentage showed that the mentioned bacterial biosurfactant at a concentration of 10000 µg/g on the ninth day of treatment with 66.67% had the highest effect on larval mortality. LD50 was evaluated as 6395758/49, 1131823.60 and 15359.4 µg/g on the fifth, seventh and ninth days, respectively. The results showed that the biosurfactant produced by S. hominis had an acceptable ability to control T. castaneum.