First Report of Phyllosticta capitalensis Causing Black Spot on Radermachera hainanensis Merr. in China.

Radermachera hainanensis Merr. plants are native in south-central and southeast of China. Plants produce large flowers, and are widely cultivated in China as ornamentals. In April 2020, R. hainanensis Merr. plants grown in Cixi Lvpin Garden (30°26'54″N, 121°25'48″E), Zhejiang Province, were found to have many black circular necrotic lesions. In the early infection stage, the lesions appeared in lower leaves as small black circular spots which developed later into large spots (11 to 38 mm diameter) with grey centers and chlorotic edges. Ultimately, the spots spread and merged. Moreover, infected leaves showed premature leaf fall. Disease intensity reached approximately 20% of plants in the affected field (0.5 ha). After effective chemical control, this disease did not spread to other healthy plants in the same garden. To identify the causative pathogen associated with the disease, ten symptomatic leaves were collected from ten different plants. Leaf tissues were cut from the lesion margins and sterilized as follows: surface sterilized with 75% ethanol for 30 seconds and washed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 3-4 minutes, then washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were cut to small pieces (3×3 mm) and transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under 12 h photoperiod. A total of 15 isolates were obtained from the affected leaves, and all the isolates displayed the same colony characteristics. Then, three single-spore isolates were randomly selected (F2, F5 and F8) for further study. The fungal colonies were dark green with a granular surface, and irregular white edges, later turning black. Conidia were one-celled, oval, and narrow at the end with a single apical end, measuring from 7.8 to 11.1 × 4.6 to 5.9 µm (av. 9.5 × 5.2 µm, n=50). These morphological characteristics were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013; Guarnaccia et al. 2017). The identity of three representative isolates were confirmed by a multilocus approach. The DNA of three isolates were extracted and partial sequences of ribosomal internal transcribed spacer (ITS), actin (ACT), and translation elongation factor 1-alpha (TEF1-α) were amplified and sequenced as previously described (White et al. 1990; O'Donnell et al. 1998; Carbone & Kohn et al. 1999). The three selected isolates shared 100% identical sequence of ITS, ACT and TEF1-α. Then representative isolate F8 was selected for further study. BLAST analysis in GenBank showed that the obtained sequence of ITS (MZ317550) had 99% identity to P. elongata isolate eSX25240811. Other two sequences of ACT (MZ326837) and TEF1-α(MZ326839) showed 99% and 98% identity to P. capitalensis isolate YLWB01, respectively. The phylogenetic trees were constructed by Bootstrap method with 1000 replications using Maximum Likelihood model implemented in the MEGA 7. Results showed that the isolate F8 clustered with P. capitalensis with 100% bootstrap support. Pathogenicity of strain F8 was tested by Koch's postulates. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 28°C. 20 healthy plants were sprayed with a 1×106 conidia ml-1 suspension (three leaves from each individual plants) and another 20 healthy plants were sprayed with sterile distilled water (three leaves from each individual plant) as control. Conidia was obtained from PDA plates after 7 days of incubation in the biochemical incubator at 28°C and concentration was counted in hemacytometer. After 15 days, disease symptoms were observed on all inoculated leaves, whereas the control plants remained asymptomatic. After that, P. capitalensis was re-isolated only from the infected leaves and identified by morphological and sequence analyses. Early identification of P. capitalensis as a causal agent for black spot is crucial to employ effective disease management strategies to control disease in the field. P. capitalensis has been reported on many crops in China (Cheng et al. 2019; Tang et al. 2020; Liao et al. 2020). However, to our knowledge, this is the first report of black spot disease caused by P. capitalensis on Radermachera hainanensis Merr. in China.

Five new 2-(2-phenylethyl)chromone derivatives and three new sesquiterpenoids from the heartwood of Aquilaria sinensis, an aromatic medicine in China.

Five new 2-(2-phenylethyl)chromone derivatives, (5S,6R,7R,8S,7'R)-7'-hydroxyagarotetrol (1), (5S,6R,7R,8S,7'S)-7'-hydroxyagarotetrol (2), (6S,7S,8R)-2­[2­(4-methoxyphenyl)ethyl]­6,7,8­trihydroxy­5,6,7,8­tetrahydrochromone (3), (6S,7S,8R)-2­(2-phenylethyl)­6,7,8­trihydroxy­5,6,7,8­tetrahydrochromone (4), (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)-7-methoxychromonyl-6-oxy]chromone (5), three new sesquiterpenoids, (4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (6), (4S,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (7), and (4R,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (8), along with 14 known compounds were isolated from the resinous heartwood of Aquilaria sinensis (Thymelaeaceae). The chemical structures of these new compounds were elucidated by 1D and 2D NMR and MS data, single-crystal X-ray diffraction analysis, and electronic circular dichroism (ECD) calculations. The neuroprotective activities of these isolates were evaluated using an in vitro model of rat adrenal pheochromocytoma (PC12) cell injury induced by corticosterone. At concentrations from 5 to 40 µM, compounds 4 and 6, agarotetrol (9), and 6-hydroxy-2-(2-phenylethyl)chromone (17) showed significant protective activities against corticosterone-induced PC12 cell injury (P < 0.001).

First Report of Colletotrichum siamense Causing Anthracnose on Zinnia elegans Jacq. in China.

Zinnia elegans (syn. Zinnia violacea), known as common zinnia, is one of the most spectacular ornamental plants in the family Asteraceae. Zinnia plants are widely cultivated in China for their impressive range in flower colours and profuse bloom over a long period. In April 2019, Zinnia plants grown in Ningbo Botanical Garden (29°56'57″N, 121°36'20″E) were found to have many circular necrotic lesions. In the early infection stage, the lesions appeared as small circular specks which developed later into large spots (15 to 32 mm diameter). Typical symptoms appeared to be grayish white centers with a chlorotic edges and disease incidence reached approximately 80% of plants in the affected field. Moreover, the growth of Zinnia plants was seriously affected by the disease. To identify the causative pathogen associated with the disease, 10 symptomatic leaves were collected from ten different Zinnia plants. Leaf tissues were cut from the lesion margins, surface sterilized with 75% ethanol for 30 seconds and rinsed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 2-3 minutes, washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under the 12 h photoperiod. A total of ten pure fungal isolates were obtained and all the isolates displayed the same colony structure. Afterwards, three pure strains were randomly selected (F1, F3 and F5) for further study. The fungal colonies showed gray to brownish aerial mycelia with pink-colored masses of conidia. Conidia were one-celled, hyaline, cylindrical to subcylindrical, spindle-shaped with obtuse ends, measuring from 15.6 to 17.3 × 4.6 to 5.1 µm with both ends rounded. These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides complex (Weir et al. 2012). The identity of a representative isolate, F3, was confirmed by a multilocus approach. Genomic DAN of isolate F3 was extracted and partial sequences of actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal internal transcribed spacer (ITS), manganese-superoxide dismutase (SOD2) , glutamine synthatase (GS), beta-tubulin (TUB2) and calmodulin (CAL) were amplified and sequenced as previously described (Weir et al. 2012). These nucleotide sequences were deposited in GenBank (accession MN972436 to MN972440, and MT266559 to MT266561; all sequences in FASTA format are shown (Supplementary S1). BLAST analysis of ITS, ACT, CHS, GAPDH and GS sequences from the F3 isolate revealed similarity to C. gloeosporioides voucher strain ZH01 with 100%, 100%，99%, 99% and 99% identity, respectively. SOD, TUB2 and CAL sequences showed similarity to C. siamense with 100%, 100% and 100% identity, respectively. The phylogenetic trees were constructed by Maximum Likelihood method (ML) using JTT model implemented in the MEGA 7. Results inferred from the concatenated sequences (ACT, CHS, GAPDH, ITS, SOD, GS, TUB2 and CAL) placed the isolate F3 within the C. siamense cluster (Supplementary S2). To confirm pathogenicity of the fungus, Koch's postulates were conducted by spraying 20 Zinnia plants (60-day-old) with a 1 × 106 conidia/ml suspension. Plants were maintained in the growth chamber at 25°C and 85% relative humidity. After 10 to 15 days, symptoms were observed on all inoculated leaves and resembled those observed in the field, whereas the control plants remained asymptomatic. Here, C. siamense was isolated only from the infected Zinnia leaves and identified by morphological and gene sequencing analyses. C. siamense has been reported in many crops in China (Yang et al. 2019; Chen et al. 2019; Wang et al. 2019). However, to our knowledge, this is the first report of anthracnose caused by C. siamense on Zinnia elegans in China. References Chen, X., Wang, T., Guo, H., Zhu, P. K., and Xu, L. 2019. First report of anthracnose of Camellia sasanqua caused by Colletotrichum siamense in China. Plant Dis. 103:1423-1423. Wang, Y., Qin, H. Y., Liu, Y. X., Fan, S. T., Sun, D., Yang, Y. M., Li, C. Y., and Ai, J. 2019. First report of anthracnose caused by Colletotrichum siamense on Actinidia arguta in China. Plant Dis. 103:372-373. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud. Mycol. 73: 115-180. Yang, S., Wang, H. X., Yi, Y. J., and Tan, L. L. 2019. First report that Colletotrichum siamense causes leaf spots on Camellia japonica in China. Plant Dis. 103:2127-2127.

Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against Tetranychus urticae.

Bioassay-guided fractionation of the supernatant of the biocontrol strain Bacillus amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the active fractions by column chromatography separation and HPLC purification. The chemical structures of these compounds were identified as cyclo-(Gly-l-Phe), 2, cyclo-(l-Phe- trans-4-OH-l-Pro), 3, cyclo-(Gly-l-Tyr), 4, cyclo-(l-Ala-l-Pro), 5, cyclo-(l-Pro- trans-4-OH-l-Pro), 6, cyclo-(Gly-l-Pro), 7, cyclo-(l-Pro-l-Pro), 8, and cyclo-(l-Tyr- trans-4-OH-l-Pro), 9. Those cyclodipeptides displayed significant acaricidal activities with LC50 values of 13.85-98.24 µM. Cyclo-(l-Tyr- trans-4-OH-l-Pro) (LC50 13.85 µM) was five times more effective than the positive control abamectin (LC50 72.06 µM). The results indicated that the hydroxyl group is an important component. This is the first report on the acaricidal capabilities of cyclodipeptides against Tetranychus urticae. The results revealed that the acaricidal activity of the biocontrol strain B. amyloliquefaciens W1 was dependent on its constituent cyclodipeptides, which have the potential to be safe and environmentally friendly acaricides.

Age influences the olfactory profiles of the migratory oriental armyworm mythimna separate at the molecular level.

BACKGROUND: The oriental armyworm Mythimna separata (Walk) is a serious migratory pest; however, studies on its olfactory response and its underlying molecular mechanism are limited. To gain insights to the olfactory mechanism of migration, olfactory genes were identified using antennal transcriptome analysis. The olfactory response and the expression of olfactory genes for 1-day and 5-day-old moths were respectively investigated by EAG and RT-qPCR analyses. RESULTS: Putative 126 olfactory genes were identified in M. separata, which included 43 ORs, 13 GRs, 16 IRs, 37 OBPs, 14 CSPs, and 3 SNMPs. RPKM values of IR75d and 10 ORs were larger than co-receptors IR25a and ORco, and the RPKM value of PR2 was larger than that of other ORs. Expression of GR1 (sweet receptor) was higher than that of other GRs. Several sex pheromones activated evident EAG responses where the responses of 5-day-old male moths to the sex pheromones were significantly greater than those of female and 1-day old male moths. In accordance with the EAG response, 11 pheromone genes, including 6 PRs and 5 PBPs were identified in M. separate, and the expression levels of 7 pheromone genes in 5-day-old moths were significantly higher than those of females and 1-day-old moths. PR2 and PBP2 might be used in identifying Z11-16: Ald, which is the main sex pheromone component of M. separata. EAG responses to 16 plant volatiles and the expression levels of 43 olfactory genes in 1-day-old moths were significantly greater than that observed in the 5-day-old moths. Heptanal, Z6-nonenal, and benzaldehyde might be very important floral volatiles for host searching and recognized by several olfactory genes with high expression. Some plant volatiles might be important to male moths because the EAG response to 16 plant volatiles and the expression of 43 olfactory genes were significantly larger in males than in females. CONCLUSIONS: The findings of the present study show the effect of adult age on olfactory responses and expression profile of olfactory genes in the migratory pest M. separate.

Enhancement of Biocontrol Activities and Cyclic Lipopeptides Production by Chemical Mutagenesis of Bacillus subtilis XF-1, a Biocontrol Agent of Plasmodiophora brassicae and Fusarium solani.

Bacillus subtilis XF-1 has been used as a biocontrol agent of clubroot disease of crucifers infected by Plasmodiophora brassicae, an obligate pathogen. In order to maximize the growth inhibition of the pathogen, random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine was applied to strain XF-1. The efficacy of 226 selected mutants was assessed against the growth of an indicator fungal pathogen: Fusarium solani using agar plate assay and the disruptive effects on the resting spores of P. brassicae. Four mutants exhibited inhibition activity significantly higher than the wild type. The cell extracts of these mutants and the XF-1 were subjected to matrix-assisted laser desorption ionization-time of flight mass spectra analysis, and three families of cyclic lipopeptides (CLPs) fengycin, surfactin and iturin were identified from the parental strain and the screened mutants. However, the relative contents and compound diversity changed after mutagenesis, and there was slight variation in the surfactin and fengycin. Notably, only 5 iturin components were discovered from the wild strain XF-1, but 13 were obtained from the mutant strains, and the relative CLPs contents of all mutant strains increased substantially. The results suggested that CLPs might be one of main biocontrol mechanisms of the clubroot disease by XF-1. The 4 mutants are far more effective than the parental strain, and they would be promising biocontrol candidates not only against P. brassicae but probably other plant diseases caused by fungi.

BZcon1, a SANT/Myb-type gene involved in the conidiation of Cochliobolus carbonum.

The fungal pathogen Cochliobolus carbonum (anamorph, Bipolaris zeicola) causes Northern Leaf Spot, leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China. Asexual spores (conidia) play a major role in both epidemics and pathogenesis of Northern Leaf Spot, but the molecular mechanism of conidiation in C. carbonum has remained elusive. Here, using a map-based cloning strategy, we cloned a single dominant gene, designated as BZcon1 (for Bipolaris zeicola conidiation), which encodes a predicted unknown protein containing 402 amino acids, with two common conserved SANT/Myb domains in N-terminal. The BZcon1 knockout mutant completely lost the capability to produce conidiophores and conidia but displayed no effect on hyphal growth and sexual reproduction. The introduced BZcon1 gene fully complemented the BZcon1 null mutation, restoring the capability for sporulation. These data suggested that the BZcon1 gene is essential for the conidiation of C. carbonum.

Diversity and active mechanism of fengycin-type cyclopeptides from Bacillus subtilis XF-1 against Plasmodiophora brassicae.

Bacillus subtilis XF-1, a strain with demonstrated ability to control clubroot disease caused by Plasmodiophora brassicae, was studied to elucidate its mechanism of antifungal activity against P. brassicae. Fengycin-type cyclopeptides (FTCPs), a well-known class of compounds with strong fungitoxic activity, were purified by acid precipitation, methanol extraction, and chromatographic separation. Eight homologs of fengycin, seven homologs of dehydroxyfengycin, and six unknown FTCPs were characterized with LC/ESI-MS, LC/ESI-MS/MS, and NMR. FTCPs (250 microg/ml) were used to treat the resting spores of P. brassicae (10(7)/ml) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A(260)) and at 280 nm (A(280)) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be cleaved by the FTCPs of B. subtilis XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol.

ESI LC-MS and MS/MS characterization of antifungal cyclic lipopeptides produced by Bacillus subtilis XF-1.

Bacillus subtilis XF-1 (CGMCC No. 2357), a patent strain with good effects for controlling the clubroot of crucifer and many pathogenic fungi, was predicted to produce cyclic lipopeptide (CLP) antibiotics based on its genomic analysis. In this study, the CLPs were purified and determined with the following protocol: the supernatant of XF-1 cultivating mixture was firstly precipitated, then the precipitants were extracted with methanol and further separated by Sephadex LH-20 chromatography to test its antifungal activities. Fungi-inhibiting fractions were further characterized with LC/ESI-MS and LC/ESI-MS/MS. The results show that four molecular ion peaks [M+H]⁺ (m/z 1,464, 1,478, 1,492 and 1,506) from fungi suppression fraction were identified as fengycin A with fatty acid of C16-C19, fengycin B (C14-C17), fengycin C (C15-C18), fengycin D (C15-C18) and fengycin S (C15-C18). Fengycin C (C15 and C18), fengycin D (C15, C16 and C18) and fengycin S (C15, C16 and C18) were reported for the first time. The diversity of the fengycins that exist in this strain will help the elucidation of their biocontrol mechanisms.

[Cerebral pathological evaluation following neural stem cells intraventricular transplantation in neonatal rats with periventricular leukomalacia].

OBJECTIVE: To evaluate the brain pathological changes following exdogenous neural stem cells (NSCs) intraventricular transplantation in neonatal rats with periventricular leukomalacia (PVL), and to explore the feasibility of NSCs transplantation for the treatment of PVL in premature infants. METHODS: NSCs were prepared from E14 embryonic rat brain. Two-day-old neonatal rats were randomly divided into six groups: PVL, PVL+culture medium, PVL+NSCs, sham operation, sham operation+culture medium, and sham operation+NSCs (18-21 rats each group). Intraventricular transplantation of exdogenous NSCs was performed 72 hrs after PVL induction or sham operation. The cerebral pathological evaluation was undertaken by light microscopy 7, 14 and 21 days after transplantation. RESULTS: The pathological changes in the cerebral white matter were gradually improved with the prolonged time after transplantation. After 21 days of transplantation, 50% of the cerebral white matter showed mild pathological changes and 50% of that showed severe pathological changes, with neuronal pathological scores of 1.28+/-0.86, in the untreated PVL group. In the PVL+NSCs group, 30% of normal white matter, 40% of mild and 30% of severe pathological changes in the white matter were observed, with neuronal pathological scores of 0.32+/-0.16, 21 days after transplantation. There were very significant differences in both of pathological changes in the cerebral white matter and neuronal pathological scores between the PVL and PVL+NSCs groups (x2=10.7, P<0.01; F=29.664, P<0.01). CONCLUSIONS: Intraventricular transplantation of exdogenous NSCs can apparently improve cerebral white matter damage. It is suggested that intraventricular transplantation of NSCs is of a great potential feasibility for the treatment of PVL in premature infants.

[Establishment of a neonatal rat model of periventricular leukomalacia and its concomitant cataract].

OBJECTIVE: To establish a reliable neonatal rat model of periventricular leukomalacia (PVL) which is expected to be similar to PVL of human preterm infants pathologically, and to explore the concomitant eye lesions in the PVL model. METHODS: Two-old-day neonatal rats were randomly divided into a PVL group and a sham-operated group (n=19 each). The PVL model was established by the ligation of bilateral common carotid arteries, followed by a 30-min exposure to 8% oxygen. The cerebral infarction area was assessed with TTC staining 1 day after operation. Cerebral pathology was examined under a light micsrocope 2 and 21 days after operation. The examinations of eyes under a slip lamp and the pathology of eyeballs under a light microscope were performed 21 days after operation. RESULTS: The TTC staining cerebral slices showed there were extensive white areas of infarction in the brain of the PVL group, with an infarction area of 53.45 +/- 33.90 mm3 and a percentage of infarction of (24.98 +/- 15.44)% . Significant cystic necrosis and apoptosis around the periventricular and subcortical white matter and mild damage in cortical neurons were observed in the PVL group 2 days after operation. The more obvious cystic necrosis around the periventricular area was found in the PVL group 21 days after operation. There were no pathological changes in the brain of the sham-operated group. All of rats in the PVL group had bilateral cataracts, however, no pathological changes were observed in their postbulbar tissues. The sham-operated group did not show eye abnormal. CONCLUSIONS: The PVL animal model that was similar to PVL of human preterm infants pathologically was successfully established by the ligation of bilateral common carotid arteries, followed by 30-min hypoxia exposure, with a positive effect and a good repeatability. Cataract can also be induced by the method.

[Analysis of signal peptides of the secreted proteins in Agrobacterium tumefaciens C58].

The 4554 ORFs of Agrobacterium tumefaciens C58 Cereon were used for the prediction of signal peptides by the network tools, such as SignalP3.0, LipoP1.0, TMHMM2.0 and TargetP1.01. Total 203 signal peptides with conserved amino residues are found, among them, 158 are secretary types, 9 are RR-motif types, 28 are SignalPase II types and 8 are bacteriocin-pheromone types. However, only two signal peptides from the secreted proteins, AGR-C-1878p and AGR-C-1880p have the same amino sequences, showing the signal peptides of the strain are highly variable.

Genetic analysis of rice varietal diversity for rice blast control.

Two Indica hybrid rice of Shanyou63 (A) and Shanyou22 (B), two glutinous landraces of Huanghenuo (C) and Zinuo (D) and three improved Japonica rice of Hexi41 (E), Chujing12 (F) and 8126 (G) were selected and their genetic resistance relationship was estimated using resistance gene analogue (RGA). The results showed that there were similar genetic relationships between hybrid varieties at the genetic similarity (GS) of 0.86,and among improved Japonica varieties at the GS of 0.84, while highly genetic diversifications between traditional varieties, Indica and Japonica varieties, traditional and modern variety ( GS:0.45). The results also showed that clustering analysis based on RGA data were generally corresponded to known pedigrees and blast field resistances of the varieties. Based on varietal differences in RGA data and agronomic traits, plot experiments of five mixed-planting combinations of A/C, A/D, B/C, B/D and A/B and two combinations of E/C and E/F/G were conducted in Jianshui and Shiping counties ( Indica rice growing region) and Luxi County (warm Japonica region) in Yunnan Province in past two years, respectively. The results demonstrated that rice blast management was more effective in five mixed-planting combinations of varieties with different genetic backgrounds (GS: 0.45-0.77) than in two combinations with similar genetic relationships (GS: 0.84-0.90), compared with their monocultures. It is evident for the highly susceptible landraces in mixed-planting to achieve disease control, with significant decreases both in incidence and severity. The blast control efficiencies of landraces in different mixture combinations reached to 54.47%-92.18%. The control efficiencies of improved varieties varied from 15.12% to 25.54% in mixture combinations with closed genetic relationship. In addition,the total yield of 5 varietal combinations with distant genetic relationship increased 539.0-904.0 kg/ha in the mixed-planting plots, at increase rates of 5.6%-10.2%. Mixed rice varieties with similar genetic background did not achieve significant yield increase. Otherwise, the yield of E/F/G decreased 2.7%-4.0% compared with pure stand. The results can provide scientific basis of varietal combinations in diversification experiments for blast control.