Molecular Identification of Fungal Endophytes of Medicinal Plant Citrullus colocynthis (L.) Schrad as a Medicinal Plant: Role of Tissue Type and Sampling Location on the Diversity.

Endophytic fungi are an important group of organisms in association with plants which are able to colonize all plant internal tissues and improve their fitness. The present research aims to isolate and identify endophytic fungi of Citrullus colocynthis plant and then investigate the effects of sampling location and tissue type on the fungal endophyte diversity of this plant. To do so, a sampling program was done in 11 geographically isolated C. colocynthis growing areas of Hormozgan province, Iran. For molecular identification of endophytic fungi of C. colocynthis, the internal transcribed spacer region (ITS1-5.8S-ITS4), as a universal DNA barcode marker for fungi, was amplified using primer sets. Totally, 12 taxa (Alternaria solani, Cladosporium halotolerans, Setosphaeria rostrata, Aspergillus niger, A. allahabadii, A. terreus, A. occultus, A. cristatus, Penicillium chrysogenum, Talaromyces purpureogenus, Fusarium sp., and Pseudozyma flocculosa) were isolated. Our findings also showed that the diversity of fungal endophytes isolated from C. colocynthis was affected by the tissue type and sampling site. Accordingly, the leaves and seeds were found to have the highest and lowest rates of endophyte colonization and richness in all sampling seasons, respectively. Simpson's diversity index of 0.8165 in root tissue indicated the high diversity of endophytes in this organ. In addition, Shannon's diversity index in the root (1.846) was higher than that in the other organs. The highest Shannon's and Simpson's indices were observed in Khoon Sorkh and Minab regions. Generally, at least two factors (region and type of tissue) played the most important roles in determining the composition of fungal endophytes in C. colocynthis.

Monitoring of Mutual Interference Behavior of Trichogramma brassicae (Hymenoptera: Trichogrammatidae) over 45 Generations of Rearing on Angoumois Grain Moth.

Trichogramma brassicae (Bezdenko) is one of the most common species of natural enemies used in augmentative biological control programs in many countries. Understanding of the foraging behavior of a parasitoid can help us to improve its performance under field conditions. This study is the first assessment of trends in mutual interference behavior of T. brassicae under long-term mass rearing (over 45 generations) on a common factitious host, Sitotroga cerealella (Olivier). Our results revealed that the total parasitism rate of T. brassicae reared on S. cerealella eggs was significantly affected by parasitoid densities and number of generations under continuous rearing. Also, parasitoid density and number of generations in rearing had significant effects on the per capita parasitism rate. Meanwhile, per capita searching efficiencies were different in sequential generations and at different densities. The number of hosts parasitized per parasitoid decreased on day 1 of the experiment with increasing parasitoid density, showing the effect of mutual interference. The linear regression between the natural logarithm of per capita searching efficiency and the natural logarithm of parasitoid density showed an inverse relationship. While the m (interference coefficient) values increased, the Q (quest constant) values had a decreasing trend over 45 generations. The highest (- 0.167) and lowest (- 0.242) values of m were observed in G45 and G5, respectively. Accordingly, G5 and G45 had the highest (0.053) and lowest (0.023) Q values, respectively. Thus, it seems the negative effects of mutual interference decreased over generations.

Generation-Dependent Functional and Numerical Responses of a Naturally Fungus-Infected Colony of Habrobracon hebetor (Hymenoptera: Braconidae) Reared on Ephestia kuehniella (Lepidoptera: Pyralidae) in Iran.

The functional and numerical responses of Habrobracon hebetor (Say) were assessed over 30 sequential generations (G) on different densities (2, 4, 8, 16, 32, 64, and 128 fifth-instar larvae) of the Mediterranean flour moth, Ephestia kuehniella (Zeller). Seven tested generations (G2inf, G4inf, G6inf, G8inf, G10inf, G15inf, and G30inf) had already been naturally infected by a fungus species, whereas only the second generation (G2) had the colonies of both infected (G2inf) and uninfected (G2uninf) individuals. All infected generations, as well as the uninfected generation, showed a Type III functional response. A high variation was found in the handling times (Th) of the parasitoid through 30 sequential generations, and G10inf had the longest handling time. The shortest handling time and the maximum estimated attack rate (T/Th) were observed in G2inf. No significant difference in terms of the number of parasitized hosts was found among all infected generations, as well as between G2inf and G2uninf. Although the handling times in G2inf and G2uninf were close to each other, the attack coefficient of G2uninf was more than that of G2inf. The attack coefficient and handling time of infected generations increased from G2inf to G10inf and then decreased in the higher generations. The most plausible parameters of functional response of infected generations were observed in Ginf. A negative relationship between eggs laid and host densities was observed in the infected generations (G2inf, G4inf, G6inf, G8inf, and G10inf). G2uninf and, among infected generations, Ginf were the best generations for practical utilization in inundative release programs.

Quality control of the parasitoid wasp Trichogramma brassicae (Hymenoptera: Trichogrammatidae) over 45 generations of rearing on Sitotroga cerealella.

Trichogramma brassicae (Bezdenko) is an important biological control agent that has been used widely against many lepidopteran pests. Commonly, colonies of Trichogramma are mass reared on factitious hosts such as Sitotroga cerealella (Olivier) over many generations. In this study, we evaluated the quality and performance of a colony of T. brassicae that had been reared for over 45 generations (G) using two-sex life table parameters and parasitism capacity. We found that female adult longevity was significantly different among sequential generations, ranging from 5.58 ± 2.5 d (at G5) to 3.75 ± 1.42 d (at G45). However, no significant difference was found in male adult longevity among different generations. Although female wasps survived longer until the 15th generation, they allocated more days for egg laying at G5 and G10. The highest values of gross reproductive rate (GRR), net reproductive rate (R0 ), intrinsic rate of increase (r), finite rate of increase (λ) and mean generation time (T) were found in G5 and G10, which also showed significantly higher c0 . No significant difference in the finite parasitism rate (ω) was found among generations up to G15. These results suggest that T. brassicae wasps held under continuous laboratory rearing can be used successfully in biological control programs until the 15th generation without any loss of quality or performance. However, laboratory mass rearing colonies declined in quality after 15 generations and we suggest that they should be rejuvenated regularly by adding field-collected parasitoids periodically.

Age stage, two-sex life table of Habrobracon hebetor (Braconidae) on Spodoptera exigua (Noctuidae) reared on different sugar beet genotypes.

The present study was carried out to unveil interactive relevance among consecutive and alternate members of a tritrophic system comprised of sugar beet genotypes, beet armyworm, Spodoptera exigua (Hübner), and its parasitoid, Habrobracon hebetor (Say) using demographic parameters. To do so, H. hebetor was reared on S. exigua fed on 10 sugar beet genotypes, including SB26; SB27; SB29; SB33; SB34; (7112*SB36)*Sh-1-HSF-5; FC220; FC301; SBSI006; and HM 1339RZ in a growth chamber at 25 ± 1 °C, 60 ± 5% RH, and 16:8 (L: D) h photoperiod. The data was analyzed based on the age-stage, two-sex life table theory. Our results revealed high variation in duration of different life stages of H. hebetor on S. exigua reared on different sugar beet genotypes examined. The shortest (10.605 days) and longest (13.721 days) pre-adult period of H. hebetor was on S. exigua reared on SB26 and SB34, respectively. The longest (17.2 days) and shortest adult longevity (7.5 days) was on S. exigua reared on SB26 and SB27, respectively. The highest values of the intrinsic rate of increase (r) (0.209 day-1) and finite rate of increase (λ) (1.233 day-1) were observed on S. exigua reared on SB34 and their lowest values (0.159 and 1.172 day-1, respectively) were recorded on SB27. Resistant and susceptible genotypes to S. exigua, FC301 and (7112*SB36)*Sh-1-HSF-5, respectively, were only genotypes on which H. hebetor had greater and approximately equal r compared with S. exigua. This finding indicates high capability of H. hebetor to be successfully employed against S. exigua on sugar beet genotypes which are extremely different in resistance to this pest.

Adult size and timing of reproduction in five species of Asobara parasitoid wasps.

The majority of adult parasitoid wasps are unable to synthesize lipids and therefore face a trade-off between the investment of lipids in eggs or in the maintenance of soma. It has been shown that resource allocation should depend on body size in parasitoids. Given that smaller females have shorter expected life times, they should concentrate their reproductive effort into early life. To test this prediction, we investigated the relationship between body size and the timing of egg production in parasitoids. We measured body size, lipid reserves, and reproductive investment (number of eggs, ovigeny index equivalent [OIE] and egg size) at eclosion in five species of Asobara (Hymenoptera: Braconidae) originating from different geographic and climatic environments. Our results show significant interspecific variation in all these traits. A diagnostic test for phylogenetic independence revealed that closely related species did not resemble each other more closely than expected by chance for all traits measured. Lipid reserves scaled positively with body size both between and within species. In agreement with theory, OI correlated negatively with body size both between and within species. Total egg area at eclosion correlated negatively with lipid reserves both between and within species. This indicates the existence of a trade-off between allocation of lipids to current reproduction and survival/future reproduction. With the exception of the most extreme pro-ovigenic species, A. persimilis, we found that pro-ovigeny was compensated for by small egg size. Our results indicate the role of habitats in shaping interspecific variation in resource allocation strategies.

Effect of Arsenophonus Endosymbiont Elimination on Fitness of the Date Palm Hopper, Ommatissus lybicus (Hemiptera: Tropiduchidae).

The date palm hopper, Ommatissus lybicus de Bergevin, is one of the most important pests of the date palm in the Middle East and North Africa. This insect uses its needle-like sucking mouthparts to feed on phloem, which is devoid of most essential amino acids and many vitamins. The absence of essential nutrient in its diet is suggested to be ameliorated by endosymbionts in O. lybicus. Arsenophonus is one of the main bacterial endosymbionts widely prevalent in O. lybicus. In this study, we used antibiotics to eliminate Arsenophonus from O. lybicus originating from three populations (Fin, Qale'e Qazi, and Roodan) and studied the effects on the fitness of the pest. Our results revealed that the removal of Arsenophonus increased the developmental time of the immature stages and reduced the values of different life-history parameters including nymphal survival rate and adult longevity in the host. Furthermore, elimination of Arsenophonus completely obliterated offspring production in all O. lybicus populations investigated. These results confirm the dependency of O. lybicus on Arsenophonus for fitness and give a new insight regarding the possibility of symbiotic control of O. lybicus.

Diversity and Spatiotemporal Distribution of Fungal Endophytes Associated with Citrus reticulata cv. Siyahoo.

Endophytic fungi are characterized as microorganisms found within internal tissues of living plants without any immediate, overtly negative effects. The present study was carried out to isolate, taxonomically characterize and determine the spatiotemporal distribution of endophytic fungi associated with leaf, stem, trunk, and root of mandarin (Citrus reticulata cv. Siyahoo). To do so, the sampling program was done seasonally in four geographically isolated mandarin growing areas of Hormozgan province of Iran, including Siyahoo, Ahmadi, Sikhoran, and Roudan. In total, 702 fungal isolates were obtained from leaf, stem, trunk, and root of healthy mandarin trees divided into 26 distinct morphotypes based on morphological characteristics. The morphotypes were taxonomically characterized through phylogenetic analysis of the ITS1-5.8S-ITS4 rDNA region sequences. Accordingly, 10 different fungal orders from 5 fungal classes were identified, i.e., Saccharomycetes (Saccharomycetales), Eurotiomycetes (Eurotiales), Dothideomycetes (Capnodiales, Pleosporales, Dothideales), and Sordariomycetes (Diaporthales, Hypocreales, Microascales, Togniniales), all from Ascomycota, which represented 97.2% and Ustilaginomycetes (Ustilaginales) from Basidiomycota which represented 2.8% of the isolates. The Aureobasidium pullulans, Penicillium citrinum, and Dothideomycetes sp. were the most frequent isolates. The trunk and leaf showed the highest and lowest total colonization frequency and species richness of endophytic fungi, respectively, in all sampling periods. The results showed that the colonization frequency of endophytes in Hormozgan province was higher in autumn than that in spring, winter, and summer. The trunk showed the maximum diversity of endophytes over all seasons. The Shannon-Wiener (H') and Simpson indices had significant correlation with sampling cites and tissue type and the maximum value of Shannon and Simpson indices (H' = 3.05 and 1 - D = 0.94) was found in the specimens collected from Siyahoo. In conclusion, the three factors (season, location, and tissue type) all in together could determine fungal endophyte composition of C. reticulata.

Distribution and Variation of Bacterial Endosymbiont and "Candidatus Liberibacter asiaticus" Titer in the Huanglongbing Insect Vector, Diaphorina citri Kuwayama.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.

Mating and Carbohydrate Feeding Impacts on Life-History Traits of Habrobracon hebetor (Hymenoptera: Braconidae).

Rapid population growth and wide host range make Habrobracon hebetor Say (Hymenoptera: Braconidae) highly suitable for biological control programs. To improve performance of the reared wasps in biological control programs we conducted a series of experiments on the effect of mating and carbohydrate feeding on life-history traits of this parasitoid. Mating caused a significant cost to fecundity, host paralysis and parasitism ability of H. hebetor. Mated wasps produced 30.98 and 23.2% less progeny than virgin wasps in unfed and fed treatments, respectively. Carbohydrate feeding increased survival only in the absence of host larvae. Virgin wasps produced only male offspring while the sex ratio produced by mated females was close to 50/50 for both fed and unfed wasps. Virgin wasps demonstrated an aggressive parasitism behavior and significantly higher paralysis and parasitism rates compared to mated wasps. Virgin wasps fed with carbohydrate found significantly more hosts than unfed wasps. To conclude, mating disruption in H. hebetor resulted in an aggressive parasitism behavior in the laboratory, which may also occur in the field. Therefore mating disruption is expected to improve the efficacy of these parasitoids in biocontrol. Carbohydrate feeding only improved searching ability and host allocation and could be applied in situations where hosts are highly dispersed.

Temperature and Exposure Time in Cold Storage Reshape Parasitic Performance of Habrobracon hebetor (Hymenoptera: Braconidae).

Cold storage can extend shelf life of parasitoids for use in biocontrol. However, cold storage may have negative impacts on life history traits of the parasitoids and, therefore, on their performance as biocontrol agents. Here, we examine the effect of cold storage on life history traits of Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of several economic lepidopteran pests. Newly emerged wasps were stored at three constant temperatures (3°C, 5°C, 7°C) for up to 4 wk. Both temperature and exposure time significantly affected longevity, parasitism, fecundity, and sex ratio. Significant reduction in longevity was observed at 3°C and 7°C, whereas longevity of wasps stored at 5°C remained stable up to the second week and then gradually decreased in Weeks 3 and 4. Parasitism rate also significantly decreased after cold storage at 3°C, 5°C, and 7°C (ranked from high to low). Fecundity decreased at T 3°C and T 5°C but this trait was not affected by storage at T 7°C. A significant shift in male production was observed at T 5°C in Week 3, but in Week 4, the only treatment with male biased reproduction was T 3°C. These results show that the effect of temperature and exposure time in cold storage is trait dependent. Overall, storage at 5°C for a period of 3 wk least impacted most life-history traits of H. hebetor wasps.