Possible antagonism of (Z)‐2‐hexenyl (E)‐3‐hexenoate against the attractiveness of (E)‐2‐hexenyl (Z)‐3‐hexenoate to Ooencyrtus nezarae (Hymenoptera: Encyrtidae)

Ooencyrtus nezarae (Hymenoptera: Encyrtidae) is an egg parasitoid of bean bug Riptortus pedestris (Hemiptera: Alydidae) which is a major pest of beans. Females of O. nezarae are attracted to (E)‐2‐hexenyl (Z)‐3‐hexenoate (EZ), one of the components of aggregation pheromone of R. pedestris. Effects of three isomers (ZE, EE and ZZ) of EZ on the attractiveness of O. nezarae were tested using electroantennography (EAG) and field bioassays. EAG analyses revealed that the response of O. nezarae to ZE was significantly higher than those to air, hexane and two other isomers, even though the response was lower than that to EZ. ZE affected the attractiveness of EZ dose‐dependently in the field. Addition of ZE (100 mg) to EZ (10 mg) caused a significant reduction in the catches of O. nezarae females. Single or binary addition of two other isomers (EE and ZZ) to EZ could not decrease or increase significantly the number of O. nezarae catches of EZ. Even though addition of ZZ (10, 50 or 100 mg) to EZ (10 mg) caused dose‐dependent reduction in the number of O. nezarae female catches, the reductions were not significantly different from that of EZ. EZ and its three isomers were not attractive to O. nezarae males at all.     

Engineering soya bean seeds as a scalable platform to produce cyanovirin‐N,a non‐ARV microbicide against HIV

There is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC₅₀ of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.     

Characterization of the Proteome of Theobroma cacao Beans by Nano‐UHPLC‐ESI MS/MS

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano‐UHPLC‐ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species‐specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non‐specific databases confirmed that using a species‐specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.     

Antiureolytic Activity of Substituted 2,5‐Diaminobenzoquinones

A series of 2,5‐bis(alkyl/arylamino)‐1,4‐benzoquinones ( 1 – 12 ) were investigated in vitro for their potential to inhibit the activity of jack bean urease. Compounds 1–6 , 8 , 9 , 11 and 12 effectively inhibited the jack bean urease activity by 90.8 % when tested at 5 μm , whereas 7 and 10 had relatively little effect. The IC₅₀ for most compounds was in the nanomolar range (31.4 nm and 36.0 nm for 2 and 8 , respectively). The mechanism of enzyme inhibition shown by 2 and 8 is typical of mixed‐type inhibitors, whose affinity for the active site is over 6‐ and 2‐fold higher (K_i=30.0 and 22.8 nm , for 2 and 8 , respectively) than that of an allosteric site. Molecular docking studies revealed that both 2 and 8 establish hydrogen bonds with the amino acids residues Asp494, Met588, His593 and Ala636 in the active site of jack bean urease. These results indicate that such aminoquinones are useful leads for the development of more efficient urease inhibitors of wider utility.     

Contest‐type competition between age classes in scramble‐type Callosobruchus maculatus (Coleoptera: Bruchidae)

We examined the effect of age differences on competition type in individuals of a scramble‐type strain of Callosobruchus maculatus (F.). When oviposition of two individuals on a bean was manipulated to introduce time intervals using two lines with different adult body colors, the frequency of two‐adult emergence decreased with the introduction of sequential oviposition. This result indicates that an age difference between two individuals induces contest competition. The frequency of adult emergence in older individuals decreased, whereas in younger individuals it increased with the introduction of sequential oviposition. Using a dissecting microscope, we observed that bodies of older individuals that died in the bean during the 4‐day oviposition interval were crushed at the pupal stage under the pupal chambers of younger individuals. These results show that an age difference between two larvae in a bean causes contest competition due to one‐sided interference by a younger individual during pupation of an older individual. Based on these experimental results, we discuss the ecological cause of contest competition and the population‐level consequences of identified interactions in scramble‐type C. maculatus.     

Multiplex CRISPR/Cas9‐mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus

Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant–environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone‐3‐hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9‐mediated multiplex gene‐editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII‐1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T₀ transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T₀ triple‐mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T₃ generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one‐third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T₂ triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.     

Generation of a multiplex mutagenesis population via pooled CRISPR‐Cas9 in soya bean

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.     

Plant growth promotion and suppression of charcoal‐rot fungus (Macrophomina phaseolina) in velvet bean (Mucuna pruriens L.) by root nodule bacteria

Root‐nodulating bacteria are intimate associates of legumes. From a pool of rhizobia isolated from root nodules of Mucuna pruriens (Velvet bean/Kaunch), RMP66 and BMP17 were found to be capable of promoting siderophore and IAA production and phosphate solubilization (insoluble tri‐calcium). Both symbionts were studied further to determine their abilities to promote plant growth and to control root‐rot in Mucuna pruriens caused by the pathogenic plant fungus Macrophomina phaseolina. RMP66 and BMP17 were selected based on their excellent inhibitory activities against M. phaseolina (by 78% and 71%, respectively) in dual culture and in agar‐well assays using cell‐free culture filtrate (CFCF) (by 76% and 62%, respectively). Both strains inhibited fungal growth to a greater extent in iron‐deficient medium (51% and 69%) than in iron‐supplemented medium (37% and 0%), respectively. CFCFs of RMP66 and BMP17 obtained from Pikovskaya's broth and tryptophan‐amended YEM broth inhibited fungal growth by 80%‐55% and 70%‐43%, respectively, and were identified as Sinorhizobium meliloti RMP66 and Bradyrhizobium diazoefficiens BMP17 by 16S rDNA sequencing. Centrifuged and pelleted cells harvested from exponentially grown cultures of S. meliloti RMP66 and B. diazoefficiens BMP17 were used to bacterize seeds of M. pruriens, which then showed enhanced seed germination (by up to 17% and 12%, respectively), and subsequent increases in other plant growth parameters in field trials. Considerable increases in seedling vigour indices (62%: 53% and 110%: 130%) and biomass (8%: 13% and 25%: 28%) were also observed for bacterial treatments. Tn5‐mediated antibiotic‐resistant marker strains showed enhanced nodule occupancy by up to 72% and 68%, respectively. This study describes a multifunctional legume nodule rhizobia that could be utilized in multicropping systems under different agroclimatic conditions as a bioinoculant and alternative to fertilizers.     

CO2‐mediated changes of plant traits and their effects on herbivores are determined by leaf age

1. Concentration of atmospheric CO₂ is predicted to double during the 21st century. However, quantitative effects of increased CO₂ levels on natural herbivore–plant interactions are still little understood. 2. In this study, we assess whether increased CO₂ quantitatively affects multiple defensive and nutritive traits in different leaf stages of cyanogenic wildtype lima bean plants (Phaseolus lunatus), and whether plant responses influence performance and choice behaviour of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis). 3. We cultivated lima bean plants in climate chambers at ambient, 500, 700, and 1000 ppm CO₂ and analysed cyanogenic precursor concentration (nitrogen‐based defence), total phenolics (carbon‐based defence), leaf mass per area (LMA; physical defence), and soluble proteins (nutritive parameter) of three defined leaf age groups. 4. In young leaves, cyanide concentration was the only parameter that quantitatively decreased in response to CO₂ treatments. In intermediate and mature leaves, cyanide and protein concentrations decreased while total phenolics and LMA increased. 5. Depending on leaf stage, CO₂‐mediated changes in leaf traits significantly affected larval performance and choice behaviour of adult beetles. We observed a complete shift from highest herbivore damage in mature leaves under natural CO₂ to highest damage of young leaves under elevated CO_2. Our study shows that leaf stage is an essential factor when considering CO₂‐mediated changes of plant defences against herbivores. Since in the long run preferred consumption of young leaves can strongly affect plant fitness, variable effects of elevated CO₂ on different leaf stages should receive highlighted attention in future research.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> are predominant in root nodules of <Emphasis Type="Italic">Vigna angularis</Emphasis>, a native legume crop in the subtropical region of China

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Mating status regulates post‐mating refractory period in Striacosta albicosta females

In polyandrous insect species, males may transfer substances to reduce sperm competition by affecting female sexual receptivity. In this study, we determined the incidence of polyandry in females of Western bean cutworm (WBC), Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), and investigated the influence of both previous female and male mating history on the duration of mating, the female refractory period, and subsequent calling behavior of females under controlled laboratory conditions. The mating status of WBC males influenced mating duration, with copulations involving previously mated males taking longer, possibly related to the time required to produce an ejaculate. The duration of the female refractory period and the onset time of recalling during the scotophase were both affected by female mating history, but not by that of the males. Females had a shorter refractory period and resumed calling activity earlier after their second and third matings than after their first mating. The earlier onset of calling by previously mated females could reduce competition with virgin females and their shorter refractory period could explain the high incidence of polyandry observed in nature.     

Proteomic Study of Differentially Expressed Proteins in Seeds between Parents and Offspring of Castor Bean (<i>Ricinus communis</i> L.)

Castor bean (Ricinus communis L.), is one of the top 10 oilseed crops in the world and, therefore, of high economic value. Hybridization is one of the most effective ways to breed new varieties with high yield, high oil content, and better stress resistance. Therefore, prediction of desired traits in castor hybrid offspring is particularly important. In this study, proteomic analysis was performed to identify differentially expressed proteins (DEPs) in seeds between castor hybrid offspring and their female (Lm female line aLmAB₂) and male parents (CSR·181). Among the DEPs upregulated in the seeds of hybrid offspring, the majority were related to seed yield and stress tolerance, while some were related to oil synthesis and fatty acid synthesis and metabolism in seeds. In other words, the hybrid offspring showed heterosis for seed yield, stress tolerance, oil synthesis, and fatty acid synthesis and metabolism when compared with their parents. Further, real-time quantitative polymerase chain reaction assays were performed on 12 genes encoding DEPs involved in oil synthesis, pollen abortion, yield, and stress tolerance of seeds. The results showed that the expression levels of the 12 genes were consistent with those of the DEPs.     

Immunological detection of bean common mosaic virus in French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) leaves

Bean common mosaic potyvirus (BCMV) is an important seed borne pathogen of French bean. Differential inoculation with bean common mosaic virus at cotylodonary trifoliate leaf stage and pre-flowering stage of crop growth revealed that cotyledonary leaf infection favored maximum disease expression. Under immunosorbent electron microscopy (ISEM) the virus particles of filamentous structure having a diameter of 750 nm (l) and 15 nm (w) were observed. These particles gave positive precipitin tests with polyclonal antiserum of Potato virus Y.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Effects of diapause on post‐diapause reproductive investment in the moth Ostrinia scapulalis

Diapause is a strategy used by many insect species to survive adverse environmental conditions. However, diapause incurs costs that may have adverse effects on post‐diapause development and reproduction. We herein investigated the effects of diapause on the post‐diapause reproductive investment of males and females in a multivoltine moth, the adzuki bean borer, Ostrinia scapulalis (Walker) (Lepidoptera: Crambidae). We found that (1) post‐diapause males and females were smaller and had lower mating success than non‐diapause individuals, (2) post‐diapause females had lower fecundity and shorter longevity than non‐diapause females, (3) post‐diapause males transferred similar numbers of eupyrene and apyrene sperm as non‐diapause males, (4) the fecundity and longevity of non‐diapause females mated with post‐diapause males and those mated with non‐diapause males were not significantly different, and (5) no significant relationship was found between diapause duration (short and long) and post‐diapause reproductive investments in both males and females. These results suggest that post‐diapause males did not reduce reproductive investment in spite of the cost of diapause, and the significant decline in reproductive output in post‐diapause females was due to their reduced body weight and longevity, which appeared to be direct consequences of the cost of diapause.     

Experimental evolution of competing bean beetle species reveals long‐term reversals of short‐term evolution,but no consistent character displacement

Interspecific competition for shared resources should select for evolutionary divergence in resource use between competing species, termed character displacement. Many purported examples of character displacement exist, but few completely rule out alternative explanations. We reared genetically diverse populations of two species of bean beetles, Callosobruchus maculatus and Callosobruchus chinensis, in allopatry and sympatry on a mixture of adzuki beans and lentils, and assayed oviposition preference and other phenotypic traits after four, eight, and twelve generations of (co)evolution. C. maculatus specializes on adzuki beans; the generalist C. chinensis uses both beans. C. chinensis growing in allopatry emerged equally from both bean species. In sympatry, the two species competing strongly and coexisted via strong realized resource partitioning, with C. chinensis emerging almost exclusively from lentils and C. maculatus emerging almost exclusively from adzuki beans. However, oviposition preferences, larval survival traits, and larval development rates in both beetle species did not vary consistently between allopatric versus sympatric treatments. Rather, traits evolved in treatment‐independent fashion, with several traits exhibiting reversals in their evolutionary trajectories. For example, C. chinensis initially evolved a slower egg‐to‐adult development rate on adzuki beans in both allopatry and sympatry, then subsequently evolved back toward the faster ancestral development rate. Lack of character displacement is consistent with a previous similar experiment in bean beetles and may reflect lack of evolutionary trade‐offs in resource use. However, evolutionary reversals were unexpected and remain unexplained. Together with other empirical and theoretical work, our results illustrate the stringency of the conditions for character displacement.