Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to plant volatiles.

Entomopathogenic nematodes (EPNs) use the chemical cues emitted by insects and insect-damaged plants to locate their hosts. Steinernema carpocapsae, a species of EPN, is an established biocontrol agent used against insect pests. Despite its promising potential, the molecular mechanisms underlying its ability to detect plant volatiles remain poorly understood. In this study, we investigated the response of S. carpocapsae infective juveniles (IJs) to 8 different plant volatiles. Among these, carvone was found to be the most attractive volatile compound. To understand the molecular basis of the response of IJs to carvone, we used RNA-Seq technology to identify gene expression changes in response to carvone treatment. Transcriptome analysis revealed 721 differentially expressed genes (DEGs) between carvone-treated and control groups, with 403 genes being significantly upregulated and 318 genes downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the responsive DEGs to carvone attraction were mainly involved in locomotion, localization, behavior, response to stimulus, and olfactory transduction. We also identified four upregulated genes of chemoreceptor and response to stimulus that were involved in the response of IJs to carvone attraction. Our results provide insights into the potential transcriptional mechanisms underlying the response of S. carpocapsae to carvone, which can be utilized to develop environmentally friendly strategies for attracting EPNs.

Deciphering the Chemical Fingerprint of Astragalus membranaceus: Volatile Components Attractive to Bruchophagus huonchili Wasps.

Bruchophagus huonchili is a pest that poses a serious threat to the yield and quality of Astragalus membranaceus seeds. In this study, we employed solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) to identify volatile organic compounds (VOCs) in A. membranaceus pods during the pod-filled period. Additionally, we utilized a Y-tube olfactometer to measure the behavioral response of B. huonchili to different individual VOCs and specific VOC-based formulations. The most effective formulations were further evaluated for their effectiveness in attracting wasps in the field. Our findings revealed that A. membranaceus pods emit 25 VOCs, including green leaf volatiles (GLVs) and terpenoid and aromatic compounds. Among these compounds, five were found to be most attractive to B. huonchili at the following concentrations: 10 µg/µL cis-ß-ocimene, 500 µg/µL hexyl acetate, 100 µg/µL hexanal, 1 µg/µL decanal, and 10 µg/µL ß-caryophyllene, with respective response rates of 67.65%, 67.74%, 65.12%, 67.57%, and 66.67%. In addition, we evaluated 26 mixed VOC formulations, and three of them were effective at attracting B. huonchili. Furthermore, field experiments showed that one of the formulations was significantly more effective than the others, which could be used for monitoring B. huonchili populations.

Parasitoids as taxonomists: how does the parasitoid Chouioia cunea distinguish between a host and a non-host?

BACKGROUND: How parasitoids distinguish hosts from non-hosts remains an unknown question. Chouioia cunea Yang (Eulophidae) is an important fall webworm parasitoid that attacks many forest and agricultural pests. To study the differences in the chemical clues used by C. cunea to distinguish host and non-host plants, we used gas chromatography-mass spectrometry (GC-MS) to identify volatile compounds of two C. cunea hosts (Hyphantria cunea and Helicoverpa armigera) and two non-hosts (Spodoptera exigua and Spodoptera frugiperda). Additionally, we used behavioral assays to compare the attraction of C. cunea to various compounds. RESULTS: The two natural host species were more attractive than the two non-host species, in the following order: Hyphantria cunea > Helicoverpa armigera > S. exigua = S. frugiperda. The pupae of the natural hosts contained 1-dodecene, which was not produced by the two natural non-hosts. When the 'attractants' based on the difference between the species-specific blend emitted by pupae and the optimal blend were sprayed onto the natural non-host pupae, they significantly improved the attraction of C. cunea to the non-host pupae. CONCLUSION: These results revealed that specific host-produced volatile compounds guide C. cunea to distinguish between natural hosts and non-hosts. Overall, this study provides a foundation for developing a behavior-modifying strategy to re-direct C. cunea attacks to control important non-host pests. © 2023 Society of Chemical Industry.

First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 µm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 µm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and ß-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.

Behavioural regulator and molecular reception of a double-edge-sword hunter beetle.

BACKGROUND: The black carabid beetle Calosoma maximoviczi is a successful predator that serves as both a beneficial insect and a severe threat to economic herbivores. Its hunting technique relies heavily on olfaction, but the underlying mechanism has not been studied. Here, we report the electrophysiological, ecological and molecular traits of bioactive components identified from a comprehensive panel of natural odorants in the beetle-prey-plant system. The aim of this work was to investigate olfactory perceptions and their influence on the behaviours of C. maximoviczi. RESULTS: Among the 200 identified volatiles, 18 were concentrated in beetle and prey samples, and 14 were concentrated in plants. Insect feeding damage to plants led to a shift in the emission fingerprint. Twelve volatiles were selected using successive electrophysiological tests. Field trials showed that significant sex differences existed when trapping with a single chemical or chemical mixture. Expression profiles indicated that sex-biased catches were related to the expression of 15 annotated CmaxOBPs and 40 CmaxORs across 12 chemosensory organs. In silico evaluations were conducted with 16 CmaxORs using modelling and docking. Better recognition was predicted for the pairs CmaxOR5-(Z)-3-hexenyl acetate, CmaxOR6-ß-caryophyllene, CmaxOR18-(E)-ß-ocimene and CmaxOR18-tetradecane, with higher binding affinity and a suitable binding pocket. Lastly, 168Y in CmaxOR6 and 142Y in CmaxOR18 were predicted as key amino acid residues for binding ß-caryophyllene and tetradecane, respectively. CONCLUSION: This work provides an example pipeline for de novo investigation in C. maximoviczi baits and the underlying olfactory perceptions. The results will benefit the future development of trapping-based integrated pest management strategies and the deorphanization of odorant receptors in ground beetles. © 2022 Society of Chemical Industry.

Molecular basis of (E)-ß-farnesene-mediated aphid location in the predator Eupeodes corollae.

(E)-ß-farnesene (EBF) is an important chemical cue mediating interactions between plants, aphids, and natural enemies. This chemical has two origins, being secreted by aphid as an alarm pheromone and also produced by the attacked plants as a semiochemical attracting natural enemies. Despite the important role of this volatile chemical, little is known on the molecular mechanisms mediating the attraction of natural enemies to EBF. Here, we first verified that the larvae and adults of aphid predator hoverfly Eupeodes corollae detect and are attracted to EBF. Then, we found a neuron housed in type III basiconic sensilla of adult antenna responding to EBF. We further verified that in both adults and larvae odorant receptor EcorOR3 and odorant-binding protein EcorOBP15 mediate detection of EBF and structurally similar volatiles. Finally, we provide evidence that larvae of E. corollae may use aphid-derived EBF for prey location in the short-range, whereas adults could detect plant-derived EBF to find attacked plants from longer distances. Thus, while dissecting the molecular basis for attraction to EBF produced by two different sources, our results may find potential applications in integrated aphid management approaches.

First Report of Paramyrothecium foliicola Causing Stem Canker of Cucumber (Cucumis sativus L.) seedlings in China.

In April 2017, stem canker symptoms were observed on cucumber seedings grown in a greenhouse (0.1 ha) in Wuqing District, Tianjin(39°34' N; 117°07' E), China. Initially, the observed symptoms included small necrotic lesions of a light brown color on the stem base. These lesions subsequently spread and turned a darker brown. The leaves of the affected plants turned yellow and wilted. As the disease progressed the plants eventually died. Years of growing cucumbers and sufficient soil moisture in the greenhouse, might have led to a disease incidence of approximately 7%. Symptomatic tissue pieces were surface disinfested in 2% sodium hypochlorite for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA) incubated at 25°C . At three days of incubation, mycelia appeared, turned into white and floccose isolated colonies around the excised tissue, and developed olivaceous green concentric rings of sporodochia in the following days. A total of 20 isolates with similar morphology were examined. Five single-spore isolates of isolates designated TJWQPF1-TJWQPF5 were obtained and maintained on PDA at 25°C. Hyaline, cylindrical conidiogenous cells measuring 9.53 to 16.51 × 1.51 to 2.49 µm (n=50) developed in whorls of three to six on terminal branches. Conidia were single-celled, hyaline, and rod-shaped with rounded ends. Conidia size averaged 5.07 - 7.15 × 1.13 - 2.32 µm (n=50). These characteristics are similar to the morphology of Paramyrothecium foliicola (Lombard et al. 2016). To further identify the isolate TJWQPF1, genomic DNA was extracted and the internal transcribed spacer (ITS, White et al. 1990), ß-tubulin (tub2, Glass & Donaldson 1995), RNA polymerase II largest subunit (rpb2, O'Donnell et al. 2007) and calmodulin (cmdA, Carbone & Kohn 1999; Groenewald et al. 2013) genes regions were amplified using the primer pairs ITS4/ITS5, Bt2a /Bt2b, RPB2-5F2 /RPB2-7cR, CAL-228F /CAL2Rd , respectively. All sequences were obtained and deposited in GenBank. BLAST searches of the NCBI database revealed that the ITS ( MW092223 ), tub2( MW110635 ) , rpb2 ( MW110637 ) and cmdA ( MW110636 ) sequences of the isolate TJWQPF1 were 100% identical to Paramyrothecium foliicola (GenBank accession numbers MT415351 and MT415352 for ITS sequences; MT415353 for tub2 sequences; MN398028-MN398043 for rpb2 sequences; MN593698- MN593713 for cmdA sequences). We also sequenced the other four single isolates and identified them as P. foliicola. Pathogenicity tests were conducted and repeated three times. Briefly, ten healthy 45-day-old cucumber seedlings (cultivar:Jinlv No.3) were inoculated with 100 µL of conidial suspension of P. foliicola (5×105 conidia per ml). Inoculum was applied to the stem with a syringe. Three healthy cucumber seedlings had 100 µL sterile water injected into the stem to serve as controls. All treated plants were incubated in a climate-controlled growth chamber at 25℃ (90% humidity, 12:12 h light:dark). Symptoms appeared on all inoculated plants after 7 days. In contrast, control seedlings exhibited no symptoms. The fungus was re-isolated from symptomatic tissues and re-identified to be P. foliicola, thereby fulfilling Koch's postulates. To our knowledge, this is the first known instance of P. foliicola inducing stem canker on cucumber plants in China. Stem canker caused by P. foliicola could pose a threat to cucumber production in China. Our results also provide a basis to monitor and manage this potential disease.

Botanical Volatiles Selection in Mediating Electrophysiological Responses and Reproductive Behaviors for the Fall Webworm Moth Hyphantria cunea.

Host-plant volatiles play vital roles for insects to locate foraging, mating, and oviposition sites in the environment. As one of the devastating invasive forestry pests, Hyphantria cunea causes a great annual loss in China, and understanding its chemical ecology is an important task. The current research was done in terms of chemical analysis, electrophysiology, and behavioral assays on H. cunea to assess its olfactory reception toward host-plant volatiles. A screen of possible common host volatiles was done, targeting on five favored hosts of H. cunea, harvesting six potential bioactive compounds from a total of 78 odorant components. Six types of antennal sensilla were investigated on their distributions on the antennae, and sexual dimorphism was described. H. cunea showed responses to all selected host-related volatiles in electroantennogram tests, and linalyl butyrate elicited the strongest responses. Furthermore, mating rates in adult pairs that are exposed to dibutyl phthalate and phytol have been significantly increased, while oviposition rates and female fecundity were not influenced. The results of the current study provide initial evidence showing that universal host-derived volatile cues are essential for H. cunea moth in terms of mating, which can also provide insights into the development of botanical attractants.

Isolation and identification of attractants from the pupae of three lepidopteran species for the parasitoid Chouioia cunea Yang.

BACKGROUND: Chouioia cunea Yang (Hymenoptera: Eulophidae) is a parasitic wasp and natural enemy of several lepidopteran pests during their pupal stage. The volatiles from pupae of three hosts, Hyphantria cunea (Arctiidae), Antheraea pernyi (Saturniidae) and Lymantria dispar (Erebidae), were analyzed and compared to elucidate the chemical cues used by C. cunea to locate its hosts. RESULTS: The attraction of C. cunea to H. cunea pupae has no obvious association with the types of plant leaves consumed by H. cunea before pupation. C. cunea exhibited the strongest attraction to the pupae of H. cunea, followed by those of A. pernyi and L. dispar based on behavioral experiments. Gas chromatography-mass spectrometry and GC-electroantennography (GC-EAD) analyses showed that these three host pupae consisted of essentially the same active volatile components but at different relative amounts. Active components derived from these pupae by GC-EAD were alkanes from C12 to C27, and C. cunea showed different levels of attraction to different single compounds. CONCLUSION: Host location by C. cunea primarily depends on common compounds emanating from the pupae of several host species. The relative amount of each component varies across host species, guiding host preferences by C. cunea. Optimal blends of several components were identified. Understanding the chemical cues used by C. cunea to locate its host could increase the possibility of developing attractants for parasitic wasps and subsequently increasing the parasitism rate of C. cunea on various hosts. © 2019 Society of Chemical Industry.