First Report of Colletotrichum truncatum Causing Anthracnose in Tobacco in China.

In July 2022, large spots were observed on the leaves of tobacco in Guangxi province, China, whose shape was round and elliptical or irregular. The margins of spots were brown or dark brown with a pale yellow centre and several small black fruiting bodies. The pathogen was isolated by tissue isolation. Diseased leaves collected were cut into small pieces, sterilized with 75% ethanol for 30s and 2% sodium hypochlorite (NaCIO) for 60s, and rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 28℃ for 5 to 7 days in the dark (Wang et al. 2022). A total of six isolates were isolated, with differences in colony shape, edge type and colony colour, and aerial mycelium morphology, with the colony shape round or subrounded, and the edge rounded crenate, dentate or sinuate. The color of the colony was initially light yellow, then gradually changed to yellow and dark yellow. After 3-4 days, white aerial mycelia gradually grew up, which was peony-like or covered the whole colony, thus the color of the colony appeared white, and then gradually changed to orange, gray or nearly black, and all six isolates rarely produced conidia, which was consistent with the description of previous reports(Mayonjo and Kapooria 2003, Feng et al. 2021, Xiao et al. 2018). Conidia were hyaline, aseptate, and falcate, with the size of 7.8 to 12.9 × 2.2 to 3.5 µm. For molecular identification, the colony PCR method was used to amplify the internal transcribed spacer(ITS), actin(ACT), chitin synthase(CHS), and beta-tubulin(TUB2) loci of the six isolates using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b, respectively(Cheng et al. 2014). Partial sequences were amplified, sequenced, and uploaded to GenBank (GenBank accession Nos. OP484886，OP518265，OP518266，OP756065，OP756066, and OP756067 for ITS, OP620430 to OP620435 for ACT, OP620436 to OP620441 for CHS, and OP603924 to OP603929 for TUB2). These sequences had 99 to 100% similarity with C. truncatum isolates C-118(ITS), TM19(ACT), OCC69(CHS), and CBS 120709(TUB2) in GenBank. Homology matching was performed using BLAST and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA (7.0) software based on ITS, ACT, CHS, and TUB2 sequences, which showed that all six isolates clustered in the same score as the C. truncatum. A pathogenicity test was performed with healthy tobacco infected with mycelial plugs (about 5 mm in diameter) of six isolates of C. truncatum from a 5-day-old culture, while negative controls on the other leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25℃ to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves had diseased spots, whereas no symptoms appeared on negative controls. The same pathogen, C. truncatum, was identified from the inoculated leaves on the basis of morphological and molecular charchseristics as described above, fulfilling Koch's postulates. In this study, it is the first time to report that the anthracnose on tobacco was caused by C. truncatum. Thus, this work provides a foundation for controlling tobacco anthracnose in the future.

Description and complete mitochondrial genome of Atkinsoniella zizhongi sp. nov. (Hemiptera: Cicadellidae: Cicadellinae) from China and its phylogenetic implications.

A new species, Atkinsoniella zizhongi sp. nov. of the subfamily Cicadellinae, was described and illustrated from China. The new species is similar to A. nigrominiatula (Jacobi, 1944), A. limba Kuoh, 1991, A. dormana Li, 1992, A. peaka Yang, Meng et Li, 2017, and A. divaricata Yang, Meng et Li, 2017. But the characteristics of aedeagus and pygofer process can be used to distinguish them easily. The complete mitochondrial genome of the paratype was sequenced and assembled. The mitogenome of A. zizhongi sp. nov. was 16,483 bp in length, with an A+T content of 75.9%, containing 37 typical genes and a control region (CR). The gene order was consistent with the inferred insect ancestral mitochondrial genome. All of the PCGs were determined to have the typical stop codon TAA or TAG, while COX2 and ND5 ended with incomplete termination codons T and TA, respectively. In addition, phylogenetic trees were reconstructed based on PCGs and rRNAs using both the maximum likelihood (ML) and Bayesian inference (BI) methods. The results showed that the intergeneric and interspecific relationships within the subfamily Cicadellinae were completely consistent in all of the phylogenetic trees, except that the different interspecific relationships within the genus Bothrogonia were detected in the ML analysis based on the amino acid sequences. This study enriches the species diversity of Cicadellinae and further promotes research on its phylogeny.

Comparative Analysis of Mitochondrial Genomes among Twelve Sibling Species of the Genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae) and Phylogenetic Analysis.

The herbivorous leafhopper genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae), a large genus of subfamily Cicadellinae, consists of 98 valid species worldwide and 88 species recorded in China. Some species of the genus are very similar in morphological characteristics, so they are difficult to identify accurately. In this study, 12 mitochondrial genomes of Atkinsoniella species with similar morphological characteristics were first obtained through high-throughput sequencing, which featured a typical circular molecule of 15,034-15,988 bp in length. The arrangement and orientation of 37 genes were identical to those of typical Cicadellidae mitogenomes. The phylogenetic relationship within the subfamily Cicadellinae was reconstructed using maximum-likelihood (ML) and Bayesian inference (BI) methods based on three concatenated datasets. The topological structures of the six obtained phylogenetic trees were highly consistent. The results suggested that Atkinsoniella was recovered as a monophyletic group and emerged as a sister group with the monophyletic clade of Bothrogonia, Paracrocampsa (part), and Draeculacephala (part). The branches of the 12 newly sequenced species were clearly separated, with most nodes receiving strong support in all analyses. In addition, the key to the 12 Atkinsoniella species was provided to identify species according to morphological characteristics. This study further promotes research on the classification, genetics, evolution, and phylogeny of the genus Atkinsoniella and subfamily Cicadellinae.

Host Deprivation Effects on the Functional Response and Parasitism Rate of Habrobracon hebetor (Hymenoptera: Braconidae) on Ephestia elutella (Lepidoptera: Pyralidae) in the Laboratory.

Habrobracon hebetor (Say) is an important biological control agent for lepidopteran pests of stored products. In this study, the age-specific functional response, paralysis rate, and parasitism rate of H. hebetor under different host deprivation treatments (PC: without host deprivation, used as the control, P1d: host deprivation, but the host was removed after 1 d contact, and PW: host deprivation from beginning) were evaluated at different larval densities (5, 10, 20, 40, and 80) of the Ephestia elutella (Hübner) at 28 ± 1°C, 75 ± 5% RH and 16:8 h L:D. Ages of parasitoid females used were 2, 5, 10, and 20 d old. The logistic regression results indicated that the functional response of H. hebetor females under different host deprivation treatments was type II. The longest handling time was observed in 20-d old females, while the shortest handling time and highest maximum attack rate (T/Th) were estimated at the age of 2 d in all treatments. The paralysis and parasitism rates of H. hebetor were the highest at 2, 5, and 10-d old in all treatments. The results of this study suggest that H. hebetor females up to 10-d old can be used as an efficient biological control agent against E. elutella. The data of this study can also be used to predict the efficacy of different aged H. hebetor females in controlling E. elutella populations.

Characterization of Two Complete Mitochondrial Genomes of Atkinsoniella (Hemiptera: Cicadellidae: Cicadellinae) and the Phylogenetic Implications.

The complete mitochondrial genomes of Atkinsoniella grahami and Atkinsoniella xanthonota were sequenced. The results showed that the mitogenomes of these two species are 15,621 and 15,895 bp in length, with A+T contents of 78.6% and 78.4%, respectively. Both mitogenomes contain 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (CR). For all PCGs, a standard start ATN codon (ATT, ATG, or ATA) was found at the initiation site, except for ATP8, for which translation is initiated with a TTG codon. All PCGs terminate with a complete TAA or TAG stop codon, except for COX2, which terminates with an incomplete stop codon T. All tRNAs have the typical cloverleaf secondary structure, except for trnS, which has a reduced dihydrouridine arm. Furthermore, these phylogenetic analyses were reconstructed based on 13 PCGs and two rRNA genes of 73 mitochondrial genome sequences, with both the maximum likelihood (ML) and Bayesian inference (BI) methods. The obtained mitogenome sequences in this study will promote research into the classification, population genetics, and evolution of Cicadellinae insects in the future.

Host deprivation effects on population performance and paralysis rates of Habrobracon hebetor (hymenoptera: Braconidae).

BACKGROUND: Habrobracon hebetor (Hymenoptera: Braconidae) is a gregarious ectoparasitoid that attacks the larvae of several species of pyralid and noctuid moths. The reproduction and population dynamics of parasitoids in general are affected by host deprivation. However, how host deprivation affects H. hebetor is unknown. The effect of host deprivation on the parental generation, life table parameters, and the paralysis rate of the F1 generation of H. hebetor were evaluated using the age-stage, two-sex life table under laboratory conditions. RESULTS: The results indicated that the greatest longevity and the least lifetime fecundity of the F0 generation occurred after 19 days of host deprivation (PW-20 treatment). The life table parameters (intrinsic rate of increase, r; finite rate of increase, λ; and net reproductive rate, R0 ) and the paralysis rate parameters (net paralysis rate, C0 ; transformation rate, Qp ; stable paralysis rate, ψ; and finite paralysis rate, ω) of F1 individuals after PW-20 treatment were significantly higher than those of individuals subjected to the control treatment (no host deprivation). However, no difference was detected between the two host deprivation treatments: host deprivation after 1 day of host contact and immediate host deprivation (PW treatment). CONCLUSION: Our results demonstrated that the effectiveness of H. hebetor did not decrease even during host deprivation for 19 days. Meanwhile, it was observed that mass rearing of the parasitoid could be improved by providing 10 individuals of 5th instar larvae of Ephestia elutella (Lepidoptera: Pyralidae) with a 20% honey-water solution. © 2020 Society of Chemical Industry.

Mitochondrial genome of Bolanusoides shaanxiensis (Cicadellidae: Typhlocybinae: Typhlocybini), with its phylogenetic analysis.

The mitochondrial genome of one leafhopper species Bolanusoides shaanxiensis was sequenced and annotated. The mitogenome is 15,724 bp in length, containing 37 typical genes and a control region. The A + T content of the whole mitogenome is 78.9%. Most of PCGs started with ATN and stopped with TAA, except for ATP8 started with TTG, COX2, COX3 and ND5 used incomplete T as stop codon. The phylogeny tree is monophyletic among 31 related species. The relationships of B. shaanxiensis and Typhlocyba sp. were closer than others. This study further enriched mitogenome database of the tribe Typhlocybini.

Differences in fungicide resistance profiles and multiple resistance to a quinone-outside inhibitor (QoI), two succinate dehydrogenase inhibitors (SDHI), and a demethylation inhibitor (DMI) for two Stagonosporopsis species causing gummy stem blight of cucurbits.

BACKGROUND: Gummy stem blight (GSB) is a devastating disease of cucurbits that has been effectively managed with fungicide applications. However, the Stagonosporopsis spp. that cause GSB have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, resistance profiles of unique and clonal genotypes of 113 Stagonosporopsis citrulli and 19 S. caricae isolates to four different fungicides were determined based on in vitro mycelial growth assays and molecular markers based on genes encoding fungicide targets. RESULTS: All 19 S. caricae isolates screened were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All 113 S. citrulli isolates were sensitive to tebuconazole and sensitive to fluopyram, with one exception that was fluopyram-resistant. All isolates of S. citrulli except two were resistant to azoxystrobin. Phenotypic differences in response to boscalid were detected among S. citrulli isolates, but the phenotypes were not associated with multilocus genotypes (MLG) determined by 16 microsatellite loci. Additionally, isolates sharing the same MLG varied by SdhB genotype. A unique mutation of I229V in SdhB, a target of succinate dehydrogenase inhibitor fungicides, was detected for the fluopyram-resistant isolate of S. citrulli. CONCLUSION: Both the lack of association of fungicide resistance profiles with genetic similarity of isolates based on microsatellite loci and the finding that widely distributed MLG varied in fungicide resistance profiles suggest that independent evolutionary events for resistance to boscalid have likely occurred. Frequent genetic recombination within populations may be responsible for resistance to multiple fungicides. This study provides useful information for effectively managing both species of GSB fungi present in the southeastern USA and understanding the evolution of fungicide resistance within populations of plant-pathogenic fungi. © 2019 Society of Chemical Industry.

The complete mitochondrial genome sequences of two insect-tea producers in Pyralidae (Lepidoptera) from South China: Pyralis farinalis and Orthopygia glaucinalis.

Complete mitochondrial genomes of Pyralis farinalis and Orthopygia glaucinalis were sequenced, respectively. Both contains 13 protein-coding genes (PCGs), 22 tRNA, two rRNA genes, and one AT-rich region. Pyralis farinlis mitogenome was 15,204 bp, with 11,234 bp coding 3732 aa. The rRNA had 1004 bp LSU and 802 bp SSU. Mitogenome of O. glaucinalis was 15,032 bp, with 11,038 bp coding 3668 aa. The rRNA contained 1406 bp LSU and 814 bp SSU. All PCGs used TAN as stop codon, except for both ND4 and ND5 of O. glaucinalis. Phylogenetic relationship of both species was also shown with 13 references.

Organization and evolution of mating-type genes in three Stagonosporopsis species causing gummy stem blight of cucurbits and leaf spot and dry rot of papaya.

Population divergence and speciation of closely related lineages can result from reproductive differences leading to genetic isolation. An increasing number of fungal diseases of plants and animals have been determined to be caused by morphologically indistinguishable species that are genetically distinct, thereby representing cryptic species. We were interested in identifying if mating systems among three Stagonosporopsis species (S. citrulli, S. cucurbitacearum, and S. caricae) causing gummy stem blight (GSB) of cucurbits or leaf spot and dry rot of papaya differed, possibly underlying species divergence. Additionally, we were interested in identifying evolutionary pressures acting on the genes controlling mating in these fungi. The mating-type loci (MAT1) of three isolates from each of the three species were identified in draft genome sequences. For the three species, MAT1 was structurally identical and contained both mating-type genes necessary for sexual reproduction, which suggests that all three species are homothallic. However, both MAT1-1-1 and MAT1-2-1 were divergent among species showing rapid evolution with a much greater number of amino acid-changing substitutions detected for the reproductive genes compared with genes flanking MAT1. Positive selection was detected in MAT1-2-1, especially in the highly conserved high mobility group (MATA_HMG-box) domain. Thus, the mating-type genes are rapidly evolving in GSB fungi, but a difference in mating systems among the three species does not underlie their divergence.