Insect Bioassays Research Articles

Functional characterization of BbEaf6 in Beauveria bassiana: Implications for fungal virulence and stress response

ABSTRACT The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana. We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the ΔBbEaf6 mutant. Furthermore, the ΔBbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the ΔBbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana, providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

CRISPR–Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases (CDPKs) act as key signal transduction enzymes in plants, especially in response to diverse stresses, including herbivory. In this study, a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses. We developed a strategy for screening insect-resistance genes from a CRISPR–Cas9 mutant library of GhCPKs. The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants. The average target-gene coverage was 86.18%, the genome editing rate was 89.49%, and the editing heritability was 82%. An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects. The mutant that showed the clearest insect resistance, cpk33/74 (in which the homologous genes GhCPK33 and GhCPK74 were knocked out), was selected for further study. Oral secretions from Spodoptera litura induced a rapid influx of Ca2+ in cpk33/74 leaves, resulting in a significant increase in jasmonic acid content. S-adenosylmethionine synthase is an important protein involved in plant stress response, and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2. In addition, virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S. litura. This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Role of chitinase expression in the virulence of Lecanicillium lecanii against citrus black aphid (Toxoptera aurantii)

Chitinase plays a vital role in the virulence of entomopathogenic fungi (EPF) when it infects host insects. We used gene recombination technology to express chitinase of three strains of Lecanicillium lecanii: Vl6063, V3450, and Vp28. The ORF of ChitVl6063, ChitV3450 and ChitVp28 were inserted into the fungal expression vector pBARGPE-1, which contained strong promoter and terminator, respectively, to construct a chitinase overpressing plasmid, then transformed the wild-type strain with blastospore transformation method. The virulence of the three recombinant strains against Toxoptera aurantii was improved by overproduction of ChitVl6063, ChitV3450, and ChitVp28, as demonstrated by significantly lower 3.43 %, 1.72 %, and 1.23 % fatal doses, respectively, according to an insect bioassay. Similarly, lethal times of recombinants (ChitVl6063, ChitV3450 and ChitVp28) were also decreased up to 29.51 %, 30.46 % and 33.90 %, respectively, compared to the wild-type strains. Improving the expression of chitinase is considered as an effective method for the enhancement of the EPF value. The efficacy could be enhanced using recombinant technology, which provides a prospecting view for future insecticidal applications.

Chemical Synthesis and Insecticidal Activity Research Based on α-Conotoxins.

The escalating resistance of agricultural pests to chemical insecticides necessitates the development of novel, efficient, and safe biological insecticides. Conus quercinus, a vermivorous cone snail, yields a crude venom rich in peptides for marine worm predation. This study screened six α-conotoxins with insecticidal potential from a previously constructed transcriptome database of C. quercinus, characterized by two disulfide bonds. These conotoxins were derived via solid-phase peptide synthesis (SPPS) and folded using two-step iodine oxidation for further insecticidal activity validation, such as CCK-8 assay and insect bioassay. The final results confirmed the insecticidal activities of the six α-conotoxins, with Qc1.15 and Qc1.18 exhibiting high insecticidal activity. In addition, structural analysis via homology modeling and functional insights from molecular docking offer a preliminary look into their potential insecticidal mechanisms. In summary, this study provides essential references and foundations for developing novel insecticides.

Two ferrous iron transporter-like proteins independently participate in asexual development under iron limitation and virulence in Beauveria bassiana

Reductive assimilation pathway involves ferric reductase and ferrous iron transporter, which is integral for fungal iron acquisition. A family of ferric reductase-like proteins has been functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. In this investigation, two ferrous iron transporter-like proteins (Ftr) were functionally annotated in B. bassiana. BbFtr1 and BbFtr2 displayed high similarity in structure and were associated with the plasma and nuclear membrane. Their losses had no negatively influence on fungal growth on various nutrients and development under the iron-replete condition. Single mutants of BbFTR1 and BbFTR2 displayed the iron-availability dependent developmental defects, and double mutant exhibited the significantly impaired developmental potential under the iron-limited conditions. In insect bioassay, the double mutant also showed the weaker virulence than either of two single disruption mutants. These results suggested that two ferrous iron transporter-like proteins function independently in fungal physiologies under the iron-deficient condition. Intriguingly, a bZIP transcription factor BbHapX was required for expression of BbFTR1 and BbFTR2 under iron-depleted conditions. This study enhances our understanding of the iron uptake system in the filamentous entomopathogenic fungi.

Silencing an ATP-Dependent Caseinolytic Protease Proteolytic Subunit Gene Enhances the Resistance of Rice to Nilaparvata lugens.

The ATP-dependent caseinolytic protease (Clp) system has been reported to play an important role in plant growth, development, and defense against pathogens. However, whether the Clp system is involved in plant defense against herbivores remains largely unclear. We explore the role of the Clp system in rice defenses against brown planthopper (BPH) Nilaparvata lugens by combining chemical analysis, transcriptome, and molecular analyses, as well as insect bioassays. We found the expression of a rice Clp proteolytic subunit gene, OsClpP6, was suppressed by infestation of BPH gravid females and mechanical wounding. Silencing OsClpP6 enhanced the level of BPH-induced jasmonic acid (JA), JA-isoleucine (JA-Ile), and ABA, which in turn promoted the production of BPH-elicited rice volatiles and increased the resistance of rice to BPH. Field trials showed that silencing OsClpP6 decreased the population densities of BPH and WBPH. We also observed that silencing OsClpP6 decreased chlorophyll content in rice leaves at early developmental stages and impaired rice root growth and seed setting rate. These findings demonstrate that an OsClpP6-mediated Clp system in rice was involved in plant growth-defense trade-offs by affecting the biosynthesis of defense-related signaling molecules in chloroplasts. Moreover, rice plants, after recognizing BPH infestation, can enhance rice resistance to BPH by decreasing the Clp system activity. The work might provide a new way to breed rice varieties that are resistant to herbivores.

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Abstract Background Chitinase is an enzyme that hydrolyzes chitin, a major component of the exoskeleton of insects, including plant pests like whiteflies. The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton (Gossypium hirsutum) through Agrobacterium-mediated transformation. Fifty-five putative transgenic cotton plants were obtained, out of which fifteen plants successfully survived and were shifted to the field. Using gene-specific primers, amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation. These five plants were further evaluated for their mRNA expression levels. The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field, were selected to raise their subsequent progenies. Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10 (2) for the ch1 gene and 3.7-fold in P2 (1) for the chi2 gene. Fluorescent in situ hybridization (FISH) confirmed a single copy number of ch1 and chi2 (hemizygous) on chromosome no. 6. Furthermore, the efficacy of transgenes on whitefly was evaluated through an insect bioassay, where after 96 h of infestation, mortality rates of whitefly were calculated to be 78%–80% in transgenic cotton plants. The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant. Conclusion Based on these findings, it can be concluded that the chemically synthesized barley chitinase genes (ch1 and chi2) have the potential to be effective against insects with chitin exoskeletons, including whiteflies. The transgenic cotton plants expressing these genes showed increased resistance to whiteflies, resulting in reduced egg numbers and higher mortality rates.

Defence and nutrition synergistically contribute to the distinct tolerance of rice subspecies to the stem borer, Chilo suppressalis.

Damage caused by the rice striped stem borer (SSB), Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is much more severe on indica/xian rice than on japonica/geng rice (Oryza sativa) which matches pest outbreak data in cropping regions of China. The mechanistic basis of this difference among rice subspecies remains unclear. Using transcriptomic, metabolomic and genetic analyses in combination with insect bioassay experiments, we showed that japonica and indica rice utilise different defence responses to repel SSB, and that SSB exploited plant nutrition deficiencies in different ways in the subspecies. The more resistant japonica rice induced patterns of accumulation of methyl jasmonate (MeJA-partof a defensive pathway) and vitamin B1 (VB1-a nutrition pathway) distinct from indica cultivars. Using gene-edited rice plants and SSB bioassays, we found that MeJA and VB1 jointly affected the performance of SSB by disrupting juvenile hormonelevels. In addition, genetic variants of key biosynthesis genes in the MeJA and VB1 pathways (OsJMT and OsTH1, respectively) differed between japonica and indica rice and contributed to performance differences; in indica rice, SSB avoided the MeJA defence pathway and hijacked the VB1 nutrition-related pathway to promote development. The findings highlight important genetic and mechanistic differences between rice subspecies affecting SSB damage which could be exploited in plant breeding for resistance.

The Bbotf1 Zn(Ⅱ)2Cys6 transcription factor contributes to antioxidant response, fatty acid assimilation, peroxisome proliferation and infection cycles in insect pathogenic fungus Beauveria bassiana

The abilities to withstand oxidation and assimilate fatty acids are critical for successful infection by many pathogenic fungi. Here, we characterized a Zn(II)2Cys6 transcription factor Bbotf1 in the insect pathogenic fungus Beauveria bassiana, which links oxidative response and fatty acid assimilation via regulating peroxisome proliferation. The null mutant ΔBbotf1 showed impaired resistance to oxidants, accompanied by decreased activities of antioxidant enzymes including CATs, PODs and SODs, and down-regulated expression of many antioxidation-associated genes under oxidative stress condition. Meanwhile, Bbotf1 acts as an activator to regulate fatty acid assimilation, lipid and iron homeostasis as well as peroxisome proliferation and localization, and the expressions of some critical genes related to glyoxylate cycle and peroxins were down-regulated in ΔBbotf1 in presence of oleic acid. In addition, ΔBbotf1 was more sensitive to osmotic stressors, CFW, SDS and LDS. Insect bioassays revealed that insignificant changes in virulence were seen between the null mutant and parent strain when conidia produced on CZP plates were used for topical application. However, propagules recovered from cadavers killed by ΔBbotf1 exhibited impaired virulence as compared with counterparts of the parent strain. These data offer a novel insight into fine-tuned aspects of Bbotf1 concerning multi-stress responses, lipid catabolism and infection cycles.

The indirect effect of nectar-inhabiting yeasts on olfactory responses and longevity of two stink bug egg parasitoids

Adult parasitoids are well known to feed on sugar-rich resources such as floral nectar. Recently, an increasing body of evidence has shown that nectar is ubiquitously colonized by microorganisms and, as a consequence, microbial metabolic activity can affect several traits of floral nectar. Yet, how the fermentation of nectar by yeasts impacts the olfactory responses and performance of parasitoids is largely understudied, especially in the case of egg parasitoids. In this study, we investigated whether fermentation by the nectar yeasts Metschnikowia gruessii and M. reukaufii affects the olfactory responses of Trissolcus basalis and Ooencyrtus telenomicida, two egg parasitoid species associated with the southern green stink bug Nezara viridula. We also investigated how yeast fermentation affects the longevity and survival of the egg parasitoids. Results of static four-chamber olfactometer tests showed that nectar fermented by M. gruessii (but not by M. reukaufii) was attractive to both egg parasitoid species, whereas no significant yeast-mediated effects were found in terms of wasp longevity. Gas chromatography coupled with mass spectrometry (GC-MS) showed a clear separation of the volatile profiles among M. gruessii, M. reukaufii and non-fermented control nectar supporting the results of the insect bioassays. The results of our study highlight the need to consider the role of microbes when studying interactions between flower nectar and egg parasitoids and could have implications from a conservation biological control perspective.

The clip cage conundrum: Assessing the interplay of confinement method and aphid genotype in fitness studies.

Behavior and fitness are important ecological traits frequently measured in insect bioassays. A common method to measure them in soft-bodied herbivorous insects involves confining individuals to plant leaves using clip cages. Although studies have previously highlighted the negative effects of clip cages on leaf physiology, little is known about the impact that using this confinement method has on insect fitness. The responses of different aphid genotypes/clones to different containment methods have not previously been investigated. Here we measured key fitness traits (intrinsic rate of natural increase, mean relative growth rate, time to reach reproductive adulthood and population doubling time) in the potato aphid, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), when confined to plants using two methods: (1) clip cages to confine aphids to individual strawberry leaves and (2) a mesh bag to confine aphids to whole strawberry plants. Our study identified a strong negative impact on all the measured aphid fitness traits when using clip cages instead of mesh bags. We also identified genotype-specific differences in response to confinement method, where clip cage confinement differentially affected the fitness of a given aphid genotype compared to the same genotype on whole plants. These results suggest that clip cage use should be carefully considered when experiments seek to quantify insect fitness and that whole plants should be used wherever possible. Given the prevalence of clip cage use in insect bioassays, our results highlight the need for caution when interpreting the existing literature as confinement method significantly impacts aphid fitness depending on their genotype.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Characterization of bacterial pathogens in Muga silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae)

Aim: To identify and characterize the bacterial pathogens associated with bacteriosis in muga silkworm, A. assamensis. Methodology: The mid gut of diseased silkworm, A. assamensis was dissected and bacterial pathogens were cultured. Bacterial pathogens were identified by 16S rRNA gene sequencing. Insect bioassay studies were conducted to understand the pathogenicity of bacterial isolates. Results: Bacterial pathogens were identified as Pseudomonas aeruginosa and Bacillus mycoids. Under laboratory conditions, approximately 70% mortality of A. assamensis larvae was due to P. aeruginosa, while 50% larval mortality was due to B. mycoids. Interpretation: P. aeruginosa was relatively more pathogenic to A. assamensis than B. mycoids. Further, a graph on disease progression was developed to understand the trajectory of bacteriosis in A. assamensis. Key words: Bacteriosis, Disease progression, Muga silkworm, Pseudomonas sp., 16S rRNA sequencing

Isolation of a highly virulent Metarhizium strain targeting the tea pest, Ectropis obliqua.

Tea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with "green" and organic farming practices. To identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control. Eleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae. Isolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

Farnesol dehydrogenase from Helicoverpa armigera (Hübner) as a promising target for pest management: molecular docking, in vitro and insect bioassay studies using geranylgeraniol as potential inhibitor.

Juvenile hormone (JH) plays pivotal roles in several critical developmental processes in insects, including metamorphosis and reproduction. JH-biosynthetic pathway enzymes are considered highly promising targets for discovering novel insecticides. The oxidation of farnesol to farnesal, catalysed by farnesol dehydrogenase (FDL), represents a rate-limiting step in JH biosynthesis. Here, we report farnesol dehydrogenase (HaFDL) from H. armigera as a promising insecticidal target. The inhibitory potential of natural substrate analogue geranylgeraniol (GGol) was tested in vitro, wherein it showed a high binding affinity (kd 595µM) for HaFDL in isothermal titration calorimetry (ITC) and subsequently exhibited dose-dependent enzyme inhibition in GC-MS coupled qualitative enzyme inhibition assay. Moreover, the experimentally determined inhibitory activity of GGol was augmented by the in silico molecular docking simulation which showed that GGol formed a stable complex with HaFDL, occupied the active site pocket and interacted with key active site residues (Ser147 and Tyr162) as well as other residues that are crucial in determining the active site architecture. Further, the diet-incorporated oral feeding of GGol caused detrimental effects on larval growth and development, exhibiting a significantly reduced rate of larval weight gain (P < 0.01), aberrant pupal and adult morphogenesis, and a cumulative mortality of ~ 63%. To the best of our knowledge, the study presents the first report on evaluating GGol as a potential inhibitor for HaFDL. Overall, the findings revealed the suitability of HaFDL as a potential insecticidal target for the management H. armigera.

Two Secretory T2 RNases Act as Cytotoxic Factors Contributing to the Virulence of an Insect Fungal Pathogen.

RNase T2 members are secreted by several pathogens or parasites during infection, playing various roles in pathogen-host interaction. However, functions of those members in biocontrol microbes targeting their hosts are still unknown. Here, we report that an insect fungal pathogen, Beauveria bassiana, produces two secretory RNase T2 members that act as cytotoxic factors, which were examined by insect bioassays using the targeted gene(s) disruption and overexpression strains. Overexpression strains displayed dramatically increased virulence, which was concurrent with few fungal cells and hemocytes in hemocoel, suggesting a cytotoxicity of the overexpressed gene products. In vitro assays using yeast-expressed proteins verified the cytotoxicity of the two members against insect cells, to which the cytotoxic effect was dependent on their RNases enzyme activities and glycosylation modification. Moreover, the excessive humoral immune responses triggered by the two ribonucleases were examined. These results suggested prospects of these two T2 ribonucleases for improvement of biocontrol agents.

UV-induced mutagenesis of Beauveria bassiana (Hypocreales: Clavicipitaceae) yields two hypervirulent isolates with different transcriptomic profiles.

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) can infest over 300 plant species and cause huge economic losses. Beauveria bassiana (Hypocreales: Clavicipitaceae) is one of the most widely used entomopathogenic fungi (EPF). Unfortunately, the efficacy of B. bassiana against S. frugiperda is quite low. Hypervirulent EPF isolates can be obtained by ultraviolet (UV)-irradiation. Here we report on the UV-induced mutagenesis and transcriptomic analysis of B. bassiana. The wild-type (WT) B. bassiana (ARSEF2860) was exposed to UV light to induce mutagenesis. Two mutants (named 6M and 8M) showed higher growth rates, conidial yields, and germination rates compared to the WT strain. The mutants showed higher levels of tolerance to osmotic, oxidative, and UV stresses. The mutants showed higher protease, chitinase, cellulose, and chitinase activities than WT. Both WT and mutants were compatible with the insecticides matrine, spinetoram, and chlorantraniliprole, but incompatible with emamectin benzoate. Insect bioassays showed that both mutants were more virulent against S. frugiperda and the greater wax moth Galleria mellonella. Transcriptomic profiles of the WT and mutants were determined by RNA-sequencing. The differentially expressed genes (DEGs) were identified. The gene set enrichment analysis (GSEA), protein-protein interaction (PPI) network, and hub gene analysis revealed virulence-related genes. Our data demonstrate that UV-irradiation is a very efficient and economical technique to improve the virulence and stress resistance of B. bassiana. Comparative transcriptomic profiles of the mutants provide insights into virulence genes. These results provide new ideas for improving the genetic engineering and field efficacy of EPF. © 2023 Society of Chemical Industry.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

Engineering of cry genes “Cry11 and Cry1h” in cotton (Gossypium hirsutum L.) for protection against insect pest attack

Cotton is white gold and contributes significantly to the economy of the countries. The process of evolution of resistance in various cotton insects directly affects the life of people. Among the cotton insect pests, pink bollworm (Pectinophora gossypiella) and army bollworm (Spodoptera litura) have turned out to be destructive. The development of seed using gene stacking approach has tremendously reduced the applications of agrochemicals making the technology greener for society. A gene cassette containing Cry11 and Cry1H genes was constructed. The cassette was cloned into the pCAMBIA2300 plant expression vector using the CAMV3-35S promoter. The construct was transformed into cotton line FBS-222 using the shoot apex-cut Agrobacterium-mediated transformation method. The expression of the Cry genes was 3-fold higher in transgenic than the non-transgenic cotton plants using qRT-PCR. Finally, the insect bioassay with transgenic cotton showed 90% mortality against pink bollworms and 80% against cotton army bollworms. The results demonstrate the development of seeds using the gene pyramiding approach is an effective strategy to control insect pest attacks.

Nano-selenium promotes the product quality and plant defense of Salvia miltiorrhiza by inducing tanshinones and salvianolic acids accumulation

Selenium (Se) is a non-essential element for plants, but it has dosage effects on plant’s life in some environments. Although many studies have revealed the beneficial effects of Se on the plant growth and defense of grains, fruits and vegetables, the underlying mechanism is largely unknown. Here, the effects of nanoparticle Se (Nano-Se) on Salvia miltiorrhiza, a valuable traditional Chinese medicine, were described. Ten mg/L Nano-Se was the optimum concentration for foliar application to stimulate plant growth, antioxidant capacity, and the accumulation of tanshinones and salvianolic acids. With the increase in the duration of Nano-Se exposure, plant biomass, nutrients and tanshinones contents gradually increased, while the salvianolic acids contents first increased significantly and then decreased. Transcriptomic analysis showed that the promotion effects of Nano-Se were probably due to the activation of photosynthesis, plant hormone signal transduction and antioxidant response in S. miltiorrhiza. Nano-Se might increase the contents of tanshinones and salvianolic acids by activating the salicylic acid (SA) and jasmonic acid (JA) signaling pathways and the downstream APETALA2/ethylene-responsive element binding factors (AP2/ERF) and WRKY transcription factors. Insect bioassays revealed that Nano-Se could reduce the survival and fecundity of aphids on S. miltiorrhiza by inducing the contents of salvianolic acid B (SalB) and rosmarinic acid (RA). Thus, these results indicate that Nano-Se could promote the crop yield, product quality and pest resistance of S. miltiorrhiza by inducing plant biomass and secondary metabolites. Our study suggests potential applications of Nano-Se in the production of industry crops.