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.

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.

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.

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.

First Report of Anthracnose on Kadsura coccinea Caused by Colletotrichum siamense in China.

Kadsura coccinea (Lem.) A. C. Smith, an evergreen liana, is widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown numerous biological activities, such as anti-tumor, anti-HIV, and anti-oxidant (Yang et al. 2020). In June 2019, an anthracnose on K. coccinea was observed in a plantation in Longan (23°03´N, 107°54´E), Guangxi province. Disease incidence was up to 30% in a plantation. Its symptoms began as small brown spots that expanded into nearly circular spots (Fig. 1A). To isolate pathogen, diseased leaves were collected. The leaves were sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 90 s, then rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed into potato dextrose agar (PDA) plates. The plates were incubated in an incubator at 25°C in dark for 2-3 days. Fungal colonies with similar morphology of 27 isolates were isolated from the 30 infected tissues. Six representative isolates (YB1 to YB6) were selected to further study their characterization. Fungal colonies were grayish-white, orange-yellow conidial masses could be observed in colonies (Fig. 1C). The mature conidia were colorless and transparent, elliptical, and single-celled, 13.0-21.0 × 4.0-8.0 µm (average 16.92 × 5.92 µm; n =100) (Fig. 1B). The DNA sequences of ribosomal internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1) and ß-tubulin (TUB2) were amplified by PCR using the primer pairs ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b (Wang et al. 2020), respectively. Sequences were submitted to GenBank (Accession nos. MZ040489 to MZ040494 for ITS, MZ069043 to MZ069048 for GAPDH, MZ069049 to MZ069054 for CAL, MZ069055 to MZ069060 for ACT, MZ069061 to MZ069066 for CHS-1, and MZ069067 to MZ069072 for TUB2). These sequences were 98%-100% identical to that of reference isolates JX010278, JX010019, JX009709, GQ856775, GQ856730, and JX010410 of Colletotrichum siamense CBS 125378 ex-type recorded in GenBank. Phylogenetic analysis of combined ITS, GAPDH, CAL, ACT, CHS-1, TUB2 genes with 16 sequences obtained from GenBank using maximum likelihood method showed that the six isolates clustered with two reference isolates of Colletotrichum siamense as a distinct clade (Fig. 2). Based on morphological characteristics and phylogenetic analysis, six isolates were identified as C. siamense. Pathogenicity tests were performed on young, fully expanded leaves of 1-year seedlings. Every leaf was punctured at 6 points on the right half and 6 points on the left half using a sterile needle. 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 2 days and after removing the bags, plants were maintained in a growth chamber at 28°C, 80% relative humidity, and a 12-h photoperiod. Anthracnose spots formed 2 to 3 days after inoculation, whereas the control leaves remained symptomless. Morphological characters matched the descriptions of C. siamense. The pathogen was previously reported to cause anthracnose on Aloe vera (Azad et al. 2020), postharvest anthracnose in mango (Liu et al. 2017), pod rot in cacao (Serrato-Diaz et al. 2020). To our knowledge, this is the first report of anthracnose on K. coccinea caused by C. siamense in China.

First report of Colletotrichum fructicola causing anthracnose on Pouteria campechiana in China.

Pouteria campechiana (Kunth) Baehni (=Lucuma nervosa A. DC.) is a fruit crop planted in southern China (Gao et al. 2019). It is originally from Central America, and also grown there commercially as well as in some American states (Fadzilah et al. 2018). In March 2019, a leaf spot disease was found on P. campechiana in Baoshan, Yunnan, China. Field surveys were done in a 0.06 ha orchard in Yunnan Province. Leaf spots were found on 90% of six-year-old plants in this field and were observed in other planting areas. The symptoms initially appeared as small, round, brown spots. As the disease developed, the center of the lesions was sunken with a dark brown border (Fig. 1). Under severe conditions, some spots were joined into larger irregular spots, and even whole leaves died. The disease severity of different plants varied, and some leaves showed only a few brown spots while others showed many spots. Small fragments of diseased tissues (3×3 mm) were disinfected in 75% ethanol for 10 s, 1% NaClO for 1 min, and rinsed three times in sterilized water. Then, tissues were placed onto potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days. Fungal isolates with similar morphology were consistently recovered from diseased tissues. The 25 colonies were initially cottony, pale white to pale gray on the upper side and greyish-green with black zonation on the underside of plates. Conidia were single-celled and hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig. 1B). The length and width of 200 conidia were measured for two representative isolates, DHG-1 and DHG-2, and these averaged 14.48 × 5.59 µm and 14.92 × 5.57 µm. Appressoria were ovoid, sometimes clavate, brown, averaged 7.47 × 5.86 µm and 7.25 × 5.85 µm (n=30). Brown and globose ascocarp were observed on the leaves of Pouteria campechiana. Asci were unitunicate, thin-walled, 6-8 spored, clavate, averaged 51.53×13.01 µm and 50.21 × 13.32 µm (n=30). Ascospores were hyaline, one-celled, slightly curved to curved with obtuse to slightly rounded ends, averaged 14.64×5.97 µm and 15.19 × 6.23 µm (n=30). These two isolates were selected for molecular identification. DNA was extracted from mycelia with the DNA secure Plant Kit (TIANGEN, Biotech, China). For further molecular identification, the internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), and the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) genes of the strains (DHG-1, DHG-2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, T1/Bt-2b, and AM-F/AM-R (Weir et al. 2012; Silva et al. 2012), respectively.The sequences were obtained and compared with GenBank and they all showed over 99% identity to the type strain of Colletotrichum fructicola ICMP 18581 (Accession nos. JX010165, JX010033, JQ807838, FJ907426, JX010405, JX009866, and FJ917508) (Weir et al. 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS-1, TUB2, GAPDH and ApMat sequences using the Neighbor-joining algorithm revealed that the isolates were C. fructicola (Fig. 2). The sequences were deposited into GenBank with accession MN955541, MN955542, and MN966581 to MN966592. Pathogenicity tests were conducted on eighteen healthy and tender leaves of six 1-year-old P. campechiana plants in a greenhouse. The experiment was repeated twice. The length and width of the inoculated leaves were between 8-13 cm × 2.5-3.6 cm. The epidermis of each tested leaf was lightly scratched in six separate areas with a sterilized needle. Each isolate was inoculated onto at least three wounded leaves by placing 20 µL of a conidial suspension (106 conidia/mL) on the wound sites. Control leaves were also wounded and inoculated with distilled water. All the plants were then sprayed with distilled water and covered with plastic bags. After 10 days, initial symptoms appeared as circular and deep yellow spots. After a few more days, the spots became brown, enlarged to up to 4.0 mm which was similar to symptoms observed in the field, whereas controls remained symptomless. Koch's postulates were fulfilled by re-isolation of C. fructicola from diseased leaves, and identification confirmed by sequencing. Colletotrichum fructicola has been associated with anthracnose on mango, apple, pear and cassava (Oliveira et al. 2018). To our knowledge, this is the first report of C. fructicola associated with anthracnose of P. campechiana worldwide. These results will provide crucial information for future epidemiological studies and for management of this disease.

Effects of pyriproxyfen on development and hormone of the aphis, Aphis craccivora (Hemiptera: Aphididae).

Pyriproxyfen (PPF) has been shown to affect the pupal stage and ecdysone levels in holometabolous insects, such as silkworms and mealworms. It remains unknown whether it affects hemimetabolous insects with their hormone levels in insects lacking a pupal stage. In this laboratory study, bioassays were conducted to investigate the effects of varying doses of PPF on Aphis craccivora Koch (Hemiptera: Aphididae). Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the types and titers of juvenile hormone (JH) and 20-hydroxyecdysone (20E). Additionally, the effects of PPF on A. craccivora reproduction and molting, as well as its influence on relevant gene expression, were examined. The results revealed LC50 and LC90 values of 3.84 and 7.49 mg/l for PPF, respectively, after 48 h of exposure. The results demonstrated a significant reduction in the titer of JH III and a significant increase in the titer of 20E following treatment with PPF. However, there was no significant decrease observed in the titer of JH III skipped bisepoxide (JH SB3). A sublethal concentration of PPF was found to inhibit Krüppel homolog 1 (kr-h1) gene expression and reduce aphid reproduction, but it did not significantly impact ecdysone receptor expression and aphid molting. The results of this study demonstrate that PPF exhibits a lethal effect on aphids, thereby providing an effective means of control. Additionally, sublethal concentrations of PPF have been found to inhibit the JH in aphids, resulting in a decline in their reproductive ability and achieving the desired control objectives.

Biocontrol potential of lipopeptides produced by Paenibacillus polymyxa AF01 against Neoscytalidium dimidiatum in pitaya.

Pitaya canker, caused by Neoscytalidium dimidiatum, is one of the most important fungal diseases that cause significant losses in production. To replace chemical pesticides, the use of biocontrol strains to manage plant diseases has been the focus of research. In this study, the bacterial strain AF01, identified as Paenibacillus polymyxa, exhibited significant antifungal effects against N. dimidiatum and four other pitaya fungal pathogens. The strain P. polymyxa AF01 produces 13 fusaricidins, which directly inhibit mycelial growth, spore germination and germ tube elongation by causing the membrane integrity and cell ultrastructure to incur irreversible damage. Pot experiment and yield test confirmed that AF01 provided preservative effects by reducing the disease index. In comparison to the untreated control groups, RNA-seq data showed that P. polymyxa AF01 selectively blocked some transcription and translation processes and inhibited RNA and DNA structural dynamics, energy production and conversion, and signal transduction, particularly cell wall biosynthesis, changes in membrane permeability, and impairment of protein biosynthesis. Thus, P. polymyxa AF01 could be potentially useful as a suitable biocontrol agent for pitaya canker.

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.

Cultural studies coupled with DNA based sequence analyses and its implication on pigmentation as a phylogenetic marker in Pestalotiopsis taxonomy.

Previous phylogenetic studies based on DNA sequence data have partially resolved taxonomic relationships among Pestalotiopsis species. There are still some morphological characters whose phylogenetic significance have not been assessed properly due to limited taxon sampling, in particular the degree of pigmentation of median cells. In this study, the stability of pigmentation of median cells of conidia in Pestalotiopsis species was evaluated in subculture, and a molecular phylogenetic analysis was conducted on 45 strains belonging to 26 species in order to reappraise the pigmentation of median cells for its significance in the taxonomy of Pestalotiopsis. Phylogenetic relationships were inferred from nucleotide sequences in ITS regions (ITS1, 5.8S and ITS2) and ß-tubulin 2 gene (tub2). The results showed that pigmentation of median cells was stable and it could be a key character in the taxonomy of Pestalotiopsis species. Instead of "concolorous" and "versicolor" proposed by Steyeart (1949), "brown to olivaceous" and "umber to fuliginous" are described and proposed in this paper.

Weed seed bank dynamics responses to long-term chemical control in a rice-wheat cropping system.

BACKGROUND: It remains an open question if the long-term application of single chemical herbicides would inevitably lead to increased weed populations and result in out-of-control weeds. The annual dynamics of weed seed bank responses to different weeding measures (chemical herbicide, hand weeding and no weeding) in rice-wheat cropping systems were compared to observe the succession of weed communities under different weed selection pressures for 17 years. RESULTS: In unweeded rice-wheat cropping plots, the initially dominant broadleaf weeds were overtaken by grasses and eventually by sedges, while in plots subjected to chemical herbicide or hand weeding, broadleaf weeds remained dominant followed by grasses. The rice-wheat cropping system favoured the spread of paddy weed species; weeding had little effect on the composition of the dominant rice weeds but greatly influenced that of wheat weeds. Total seed density tended to decrease in both weeded and unweeded plots, but the species density and composition of the seed banks differed among plots treated differently. Weeding slightly increased weed species diversity and decreased weed community evenness and dominance in the first several years, but this scenario could have negative consequences in the long term; however, without weeding, stronger interspecific competition led to a decrease in weed species diversity whereas weed community evenness and dominance increased. CONCLUSION: Long-term and repeated application of pre-emergence chemical herbicides and hand weeding had similar effects on the weed community dynamics, indicating that exclusive application of pre-emergence herbicide could maintain the weed community at a durable relatively low infestation level. © 2019 Society of Chemical Industry.

Integration of Transcriptional and Post-transcriptional Analysis Revealed the Early Response Mechanism of Sugarcane to Cold Stress.

Cold stress causes major losses to sugarcane production, yet the precise molecular mechanisms that cause losses due to cold stress are not well-understood. To survey miRNAs and genes involved in cold tolerance, RNA-seq, miRNA-seq, and integration analyses were performed on Saccharum spontaneum. Results showed that a total of 118,015 genes and 6,034 of these differentially expressed genes (DEGs) were screened. Protein-protein interaction (PPI) analyses revealed that ABA signaling via protein phosphatase 2Cs was the most important signal transduction pathway and late embryogenesis abundant protein was the hub protein associated with adaptation to cold stress. Furthermore, a total of 856 miRNAs were identified in this study and 109 of them were differentially expressed in sugarcane responding to cold stress. Most importantly, the miRNA-gene regulatory networks suggested the complex post-transcriptional regulation in sugarcane under cold stress, including 10 miRNAs-42 genes, 16 miRNAs-70 genes, and three miRNAs-18 genes in CT vs. LT0.5, CT vs. LT1, and CT0.5 vs. LT1, respectively. Specifically, key regulators from 16 genes encoding laccase were targeted by novel-Chr4C_47059 and Novel-Chr4A_40498, while five LRR-RLK genes were targeted by Novel-Chr6B_65233 and Novel-Chr5D_60023, 19 PPR repeat proteins by Novel-Chr5C_57213 and Novel-Chr5D_58065. Our findings suggested that these miRNAs and cell wall-related genes played vital regulatory roles in the responses of sugarcane to cold stress. Overall, the results of this study provide insights into the transcriptional and post-transcriptional regulatory network underlying the responses of sugarcane to cold stress.

Molecular Characterization and Co-expression Analysis of the SnRK2 Gene Family in Sugarcane (Saccharum officinarum L.).

In plants, both abscisic acid (ABA) dependent and independent pathways form the basis for the response to environmental stresses. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) plays a central role in plant stress signal transduction. However, complete annotation and specific expression patterns of SnRK2s in sugarcane remain unclear. For the present study, we performed a full-length cDNA library survey of sugarcane, thus identifying ten SoSnRK2 genes via phylogenetic, local BLAST methods, and various bioinformatics analyses. Phylogenetic analysis indicated division of SoSnRK2 genes into three subgroups, similar to other plant species. Gene structure comparison with Arabidopsis suggested a unique evolutionary imprint of the SnRK2 gene family in sugarcane. Both sequence alignment and structural annotation provided an overview of the conserved N-terminal and variations of the C-terminal, suggesting functional divergence. Transcript and transient expression assays revealed SoSnRK2s to be involved in the responses to diverse stress signals, and strong ABA induction of SoSnRK2s in subgroup III. Co-expression network analyses indicated the existence of both conserved and variable biological functions among different SoSnRK2s members. In summary, this comprehensive analysis will facilitate further studies of the SoSnRK2 family and provide useful information for the functional validation of SoSnRK2s.

Genetic diversity analyses of Lasiodiplodia theobromae on Morus alba and Agave sisalana based on RAPD and ISSR molecular markers.

Genetic diversity of 23 Lasiodiplodia theobromae isolates on Morus alba and 6 isolates on Agave sisalana in Guangxi province, China, was studied by using random amplified polymorphic DNA and inter-simple sequence repeat molecular markers. Results of two molecular markers showed that the average percentage of polymorphic loci of all isolates was more than 93%. Both dendrograms of two molecular markers showed obvious relationship between groups and the geographical locations where those strains were collected, among which, the 23 isolates on M. alba were divided into 4 populations and the 6 isolates on A. sisalana were separated as a independent population. The average genetic identity and genetic distance of 5 populations were 0.7215, 0.3284 and 0.7915, 0.2347, respectively, which indicated that the genetic identity was high and the genetic distance was short in the 5 populations. Average value of the gene diversity index (H) and the Shannon's information index (I) of 29 isolates were significantly higher than 5 populations which showed that genetic diversity of those isolates was richer than the populations and the degree of genetic differentiation of the isolates was higher. The Gst and Nm of 29 isolates were 0.4411, 0.6335 and 0.4756, 0.5513, respectively, which showed that the genetic diversity was rich in those isolates.

Bioactive natural compounds from the mangrove endophytic fungi.

In recent years, mangrove endophytic fungi are increasingly attracting attention of the pharmaceutical community as they produce a wide variety of metabolites that are structurally unique and pharmacologically active. Previous chemical investigation of mangrove fungi resulted in the discovery of various bioactive secondary metabolites including terpenes, chromones, coumarins, polyketides, alkaloids and peptides with diverse structural features. The present report reviews the papers, which have appeared in the literature till now, concerning the isolation, structural elucidation, and biological activities of the secondary metabolites from mangrove endophytic fungi.

Effects of external potassium (k) supply on drought tolerances of two contrasting winter wheat cultivars.

BACKGROUND: Drought is a common stress limiting crops growth and productivities worldwide. Water deficit may increase cellular membrane permeability, resulting in K outflow. Internal K starvation may disorder plant metabolism and limit plant growth. However, it is seldom reported about the effects of external K on drought tolerance of contrasting wheat cultivars. METHODOLOGY/PRINCIPAL FINDINGS: A hydroponics experiment was carried out in a non-controlled greenhouse. Seedlings of drought-tolerant SN16 and intolerant JM22 were simultaneously treated by five levels of K2CO3 (0, 2.5, 5, 7.5, 10 mM) and two levels of PEG6000 (0, 20%) for 7 days. External K2CO3 significantly increased shoot K(+) content, water potential, chlorophyll content as well as gas exchange, but decreased electrolyte leakage (EL) and MDA content in both cultivars under PEG6000 stress. Antioxidant enzymes activities were up-regulated by PEG6000 while external K2CO3 reduced those changes. Molecular basis was explained by measuring the expression levels of antioxidant enzymes related genes. Shoot and root biomass were also increased by K2CO3 supply under drought stress. Although adequate K2CO3 application enhanced plant growth for both cultivars under drought stress, SN16 was better than JM22 due to its high drought tolerance. CONCLUSIONS/SIGNIFICANCE: Adequate external K may effectively protect winter wheat from drought injuries. We conclude that drought-tolerant wheat combined with adequate external K supply may be a promising strategy for better growth in arid and semi-arid regions.

Bioactive natural compounds from the plant endophytic fungi Pestalotiopsis spp.

The plant-endophytic strains of the fungus Pestalotiopsis (Amphisphaeriaceae) are distributed throughout the world. Previous chemical investigation of members of the genus resulted in the discovery of various bioactive secondary metabolites including chromones, cytosporones, polyketides, terpenoids and coumarins with diverse structural features. The present report reviews the papers, which have appeared in the literature till now, concerning the isolation, structural elucidation, and biological activities of the secondary metabolites from Pestalotiopsis species.

Entomopathogenic fungi on Hemiberlesia pitysophila.

Hemiberlesia pitysophila Takagi is an extremely harmful exotic insect in forest to Pinus species, including Pinus massoniana. Using both morphological taxonomy and molecular phylogenetics, we identified 15 strains of entomogenous fungi, which belong to 9 genera with high diversities. Surprisingly, we found that five strains that were classified as species of Pestalotiopsis, which has been considered plant pathogens and endophytes, were the dominant entomopathogenic fungus of H. pitysophila. Molecular phylogenetic tree established by analyzing sequences of ribosomal DNA internal transcribed spacer showed that entomopathogenic Pestalotiopsis spp. were similar to plant Pestalotiopsis, but not to other pathogens and endophytes of its host plant P. massoniana. We were the first to isolate entomopathogenic Pestalotiopsis spp. from H. pitysophila. Our findings suggest a potential and promising method of H. pitysophila bio-control.

[Control effects of rice-duck farming and other weed management strategies on weed communities in paddy fields].

By the methods of community ecology, field studies were conducted to evaluate the control effects of three weed management strategies, i. e., rice-duck farming (RD), manual weeding (MW) and chemical weeding (CW), on the weed communities in paddy fields. The results showed that under rice-duck farming, the weed density in paddy fields decreased significantly, and the control effects on dominant weed species such as Monochoria vaginalis, Cyperus difformis, Sagittaria pygmaea were all above 95%, with an overall effect higher than CW and MW. Under RD, the species richness and Shannon-Wiener diversity indices decreased slightly, while Pielou community evenness indices increased markedly, indicating that the species composition of weed community was greatly improved, and the infestation of former dominant weed species was reduced. The structure of weed communities in paddy fields varied with different weed management strategies, e. g., under RD, Lindernia procumbens, Cyperus difformis and Fimbristylis miliacea constituted the major weed community, and the Whittaker index was significant higher than that of CW, MW and CK, which indicated that rice-duck farming had a greater effect on the structure of the weed communities. The same conclusion could be drawn from Sorensen's similarity indices and cluster analysis with Sorensen's index as the distance measurement.