Precise Regulation of Juvenile Hormone III R-Stereoisomer Synthesis by Apis mellifera through Specifically Binding Methyl-(2E,6E)-farnesoate and Strictly Controlling Its Titer.

Juvenile hormone III (JH III) is a crucial hormone synthesized exclusively as R-stereoisomer in most insects. Herein, we established a mature Tris-HCl culture system for essential biochemical reactions and applied stable instrumental detection methods to analyze JH III, methyl farnesoate (MF) and juvenile hormone acid (JHA) using UPLC-MS/MS. Our results revealed that the R-JH III terminal synthesis pathway in Apis mellifera follows the "esterify then epoxidize" sequence, with precise methyl-(2E,6E)-farnesoate titer regulation and its spatial cis-trans isomerism, achieving selective R-JH III synthesis. Furthermore, we observed that the preferred generation of S/R-JH III chiral enantiomers varied depending on the spatial cis-trans isomerism of different MFs. Our results suggest that S-JH III could theoretically exist in insects, offering a novel perspective for understanding the synthesis mechanism of diverse complex juvenile hormones in different insect species.

S-dinotefuran affects the social behavior of honeybees (Apis mellifera)and increases their risk in the colony.

The toxic effects of neonicotinoid pesticides on honeybees is a global concern, whereas little is known about the effect of stereoisomeric pesticides among honeybee social behavior. In this study, we investigated the effects of stereoisomeric dinotefuran on honeybee social behavior. We found that honeybees exhibit a preference for consuming food containing S-dinotefuran, actively engage in trophallaxis with S-dinotefuran-consuming peers, and consequently acquire higher levels of S-dinotefuran compared with R-dinotefuran. In comparison to R-dinotefuran, S-dinotefuran stimulates honeybees to elevate their body temperature, thereby attracting more peers for trophallaxis. Transcriptome analysis revealed a significant enrichment of thermogenesis pathways due to S-dinotefuran exposure. Additionally, metabolome data indicated that S-dinotefuran may enhance body temperature by promoting lipid synthesis in the lysine degradation pathway. Consequently, body temperature emerges as a key factor influencing honeybee social behavior. Our study is the first to highlight the propensity of S-dinotefuran to raise honeybee body temperature, which prompts honeybee to preferentially engage in trophallaxis with peers exhibiting higher body temperatures. This preference may lead honeybees to collect more dinotefuran-contaminated food in the wild, significantly accelerating dinotefuran transmission within a population. Proactive trophallaxis further amplifies the risk of neonicotinoid pesticide transmission within a population, making honeybees that have consumed S-dinotefuran particularly favored within their colonies. These findings may contribute to our understanding of the higher risk associated with neonicotinoid use compared with other pesticides.

First report of Black Spot on Eucalyptus grandis × Eucalyptus urophylla caused by Pseudoplagiostoma eucalypti in Guangxi, China.

Eucalyptus grandis × Eucalyptus urophylla hybrid clone is an economically and ecologically important forest variety and is widely planted in Guangxi, China. Black spot, a newly found disease, occurred nearly 5333.3 hectares in an E. grandis × E. urophylla plantation of Qinlian forest farm (N: 21.866°, E: 108.921°) in Guangxi in October, 2019. Infected plants had lesions of black spots with watery margins on petioles and veins of E. grandis × E. urophylla. The size of spots ranged between 3 to 5 mm in diameter. When lesions expanded to girdle the petioles, wilt and death of leaves was observed, which subsequently affected growth of the trees. To isolate the causal agent, symptomatic plant tissues (leaves and petioles) were collected from two different sites, sampled from five plants each site. In the lab, infected tissues were surface sterilized with 75% ethanol for 10 seconds, then 2% sodium hypochlorite for 120 seconds, and rinsed with sterile distilled water three times. Small segments (5×5 mm) were cut from the margins of the lesions, then placed on potato dextrose agar (PDA) plates. The plates were incubated at 26°C in dark for 7 to 10 days. Fungal isolates YJ1 and YM6 with a similar morphology, which were obtained from 14 of 60 petioles and 19 of 60 veins respectively. These two colonies were initially light orange, then turned to olive brown as time progressed. Conidia were hyaline, smooth, aseptate, ellipsoidal, apex obtuse, and base tapering to flat protruding scar, 16.8 to 26.5µm long, and 6.6 to 10.4 µm wide (n=50). Some conidia had one or two guttules. The morphological characteristics were consistent with the description of Pseudoplagiostoma eucalypti Cheew., M. J. Wingf. & Crous (Cheewangkoon et al. 2010). For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 and T1/Bt2b, respectively (White et al. 1990; O'Donnell et al.1998; Glass and Donaldson 1995). Sequences of the two strains were deposited in GenBank (ITS: MT801070 and MT801071; BT2: MT829072 and MT829073). Phylogenetic tree was constructed with a maximum likelihood method, revealing that YJ1 and YM6 were on the same branch with P. eucalypti. Pathogenicity tests of the two strains were performed on three-month-old E. grandis × E. urophylla seedlings, by inoculating 6 wounded (by stabbing on petioles or veins) leaves of seedlings with mycelial PDA plugs (5 ×5 mm) from the edge of a 10-day old colony of strain YJ1 or YM6. Another 6 leaves were treated in the same manner but with PDA plugs as controls. All treatments were incubated in humidity chambers at 27°C and 80% relative humidity, under ambient light. All experiments were conducted three times. Lesions were observed at the points of inoculation, the petioles or veins turned black on inoculated leaves after 7 days, wilting of the leaves were also observed after 30 days, however the controls remained asymptomatic. Re-isolation was made and the fungus had same morphological measurements as the inoculated fungus, thus completing Koch's postulates. P. eucalypti had been reported as a pathogen of leaf spot on E. robusta in Taiwan island (Wang et al. 2016), leaf and shoot blight on E. pulverulenta in Japan (Inuma et al. 2015). To our knowledge, this is the first report of P. eucalypti affecting E. grandis × E. urophylla in mainland China. This report provides basis for the rational prevention and control of this new disease in the cultivation process of E. grandis × E. urophylla.

First Report of Leaf Spot Caused by Lasiodiplodia theobromae on Kadsura coccinea in China.

Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 µm (average 26.07×14.51 µm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and ß-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 µL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 µL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.

Diversity of Dictyostelid Cellular Slime Molds, Including Two Species New to Science, in Forest Soils of Changbai Mountain, China.

Dictyostelid cellular slime molds (dictyostelids) are protists that are common inhabitants of most soils, where they feed upon bacteria. Changbai Mountain is the highest mountain in northeast China. Soil samples collected on Changbai Mountain yielded 11 isolates representing six species of dictyostelid samples. Two of these species (Dictyostelium robusticaule and Heterostelium recretum) were found to be new to science, based on morphology, SSU rDNA sequences, and an ATPase subunit 1 gene (atp1) phylogeny. The present study also demonstrated that the increased accuracy and lower costs associated with the use of atp1 sequences make them a complement of SSU rDNA sequences for identifying dictyostelids. Changbai Mountain is characterized by a higher diversity of dictyostelids than indicated by the few previous reports. Moreover, the data for Changbai Mountain, compared with comparable data for Taiwan, suggest that differences in diversity at the family level are possibly related to latitude. Mixed broadleaf-conifer forests produced more isolates and species than broadleaf forests at the same elevation and also had the highest species richness, which indicates an effect of vegetation on dictyostelids. However, the pattern of slightly decreasing diversity with increasing elevation in dictyostelids was also apparent. IMPORTANCE Dictyostelium robusticaule and Heterostelium recretum are two new species of dictyostelids reported in this study. The potential use of atp1 sequences is a complement of SSU rDNA sequences for the identifying dictyostelids. A pattern of slightly decreasing diversity with increasing elevation in dictyostelids was observed, with the conditions that exist at lower elevations apparently more suitable for dictyostelids, whereas differences of diversity observed at the family level are possibly related to latitude.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Illicium difengpi in China.

Illicium difengpi B. N. Chang et al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initial leaf symptoms were round spots with gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomatic leaves were collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affected leaves. Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n = 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n = 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 for ITS, OL539429 for ACT, OL539430 for tef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the corresponding ITS (MT649668), ACT (MN958710), tef1-α (MN958711) and GPDH (KU716077) sequences of P. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACT by the maximum likelihood method showed that isolate GX-2 clustered together with P. capitalensis. The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 µL spore suspension (1 × 106 spores/ml). Another four sterilized leaves were inoculated with 20 µL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy. P. capitalensis was successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensis caused leaf spots on various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report of P. capitalensis causing leaf spot on I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology of this disease should be investigated in further research.

Juvenile Hormone III R Stereoisomer Is Specifically Synthesized by Honeybees (Apis mellifera ligustica) and Shows a Higher Biological Activity in Regulating Their Social Behavior.

The juvenile hormone (JH) plays a key role in the development of honeybee larvae and the alteration of adult behavior. Verification methods of types and stereoisomers of JHs in honeybees were established in this study. The regulatory modes of different stereoisomers of JH III on the social behaviors of honeybees were revealed by the disparity of interaction and RNA-seq. This result represented the first assessment of the effects of R-JH III and S-JH III in honeybee interactions; the former (367 times in total) was significantly higher than the latter (235 times in total); honeybees with high JH titers are always welcome in the colony because the effect of JH III on bees involves the sensing and signaling of hormones, and R-JH III is much more active than S-JH III in this regulation. Efficient R-JH III may be the insurance for bees to establish their social system advantages.

Complete genome sequence of biocontrol strain Paenibacillus peoriae HJ-2 and further analysis of its biocontrol mechanism.

BACKGROUND: Paris polyphylla is a herb widely used in traditional Chinese medicine to treat various diseases. Stem rot diseases seriously affected the yield of P. polyphylla in subtropical areas of China. Therefore, cost-effective, chemical-free, eco-friendly strategies to control stem rot on P. polyphylla are valuable and urgently needed. RESULTS: In this paper, we reported the biocontrol efficiency of Paenibacillus peoriae HJ-2 and its complete genome sequence. Strain HJ-2 could serve as a potential biocontrol agent against stem rot on P. polyphylla in the greenhouse and field. The genome of HJ-2 consists of a single 6,001,192 bp chromosome with an average GC content of 45% and 5,237 predicted protein coding genes, 39 rRNAs and 108 tRNAs. The phylogenetic tree indicated that HJ-2 is most closely related to P. peoriae IBSD35. Functional analysis of genome revealed numerous genes/gene clusters involved in plant colonization, biofilm formation, plant growth promotion, antibiotic and resistance inducers synthesis. Moreover, metabolic pathways that potentially contribute to biocontrol mechanisms were identified. CONCLUSIONS: This study revealed that P. peoriae HJ-2 could serve as a potential BCA against stem rot on P. polyphylla. Based on genome analysis, the genome of HJ-2 contains more than 70 genes and 12 putative gene clusters related to secondary metabolites, which have previously been described as being involved in chemotaxis motility, biofilm formation, growth promotion, antifungal activity and resistance inducers biosynthesis. Compared with other strains, variation in the genes/gene clusters may lead to different antimicrobial spectra and biocontrol efficacies.

Dictyostelium purpureum var. pseudosessile, a new variant of dictyostelid from tropical China.

BACKGROUND: Dictyostelid cellular slime molds (dictyostelids) are microscopic throughout their entire life cycle. The vegetative phase consists of single-celled amoeboid forms which live in the soil/leaf litter microhabitat of fields and forests along with animal dung, where they feed upon bacteria and other microbes, grow, and multiply until the available food supply is exhausted. When this happens, the amoeboid forms aggregate together in large numbers to form multi-celled pseudoplasmodia, which then give rise to fruiting bodies (sorocarps) that consist of supportive stalks and unwalled sori containing propagative spores. RESULTS: Dictyostelium purpureum var. pseudosessile, a new variant of dictyostelid, is described herein, based on morphological features and molecular data. This new variant was isolated from soil samples collected in two tropical areas of China. The complete spore-to-spore life cycle of this species, which required 50 h, including spore germination, myxamoebae, cell aggregation, pseudoplasmodium, and sorocarp formation, was documented. Descriptions and illustrations are provided for this species based on our collections. Data from ontogeny, morphology and phylogeny analyses (SSU) of D. purpureum var. pseudosessile confirm that it is a Group 4 species according to the newly proposed classification of dictyostelids. CONCLUSIONS: Our results suggest that the violet sori, widens at the midpoint of sorophore and simple recurved sorophore bases represent the prominent features for the new variant D. purpureum var. pseudosessile. The latter is a Group 4 species now known from two tropical areas of China where dictyostelids remains understudied.

Developmental features and associated symbiont bacterial diversity in essential life cycle stages of Heterostelium colligatum.

Dictyostelium discoideum is a specialized amoebozoan protist that can feed on, carry and disperse bacteria. However, the symbiont bacterial diversity in other species of dictyostelids and the diversity associated with essential life cycle stages are still unknown until now. Here, another species of dictyostelids, Heterostelium colligatum, a new record for tropical China, was isolated from the soil collected in Xishuangbanna Tropical Botanical Garden, Yunnan Province, China. We describe the complete life cycle of this species and illustrate details of spore-to-spore development. The symbiont bacterial diversity and relative abundance associated with life cycle stages of H. colligatum, including the aggregation, pseudoplasmodium, and sorocarp stages, were investigated by high throughput metagenomic techniques. H. colligatum appears to be capable of carrying different types of bacteria during its life history in addition to those used as a food resource. The dominant groups of those three stages in its life cycle were the Proteobacteria, Actinobacteria and Firmicutes. The relative abundance of the dominant phyla and shared OTUs were different for the aggregation, pseudoplasmodium, and sorocarp stages. A comparison of the symbiont bacterial assemblages associated with D. discoideum and H. colligatum indicated that different dictyostelid species carried different species of symbiont associated bacteria.