A new endophytic Penicillium oxalicum with aphicidal activity and its infection mechanism.

BACKGROUND: Aphid infestation adversely affects the yield and quality of crops. Rapid reproduction and insecticidal resistance have made controlling aphids in the field challenging. Therefore, the present study investigated the insecticidal property of Penicillium oxalicum (QLhf-1) and its mechanism of action against aphids, Hyalopterus arundimis Fabricius. RESULTS: Bioassay revealed that the control efficacy of the spores against aphids (86.30% and 89.05% on the third day and fifth day after infection, respectively) were higher than other components, such as the mycelium. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that QLhf-1 invaded the aphid cuticle through spores and used the aphid tissues as a nutrient source for growth and reproduction, causing stiffness and atrophy and a final death. Three extracellular enzymes, lipase, protease, and chitinase had a synergistic effect with spores, and they acted together to complete the infection process by degrading the aphid body wall and accelerating the infection process. CONCLUSION: The newly discovered endophytic penicillin strain P. oxalicum 'QLhf-1' can effectively kill aphids. The results provided strong evidence for the biological control of aphids, and lay a foundation for the development and utilization of QLhf-1. © 2024 Society of Chemical Industry.

First Report of Nigrospora oryzae Causing Leaf Spot Disease on Amorphophallus albus in Shaanxi Province of China.

Amorphophallus albus P. Y. Liu & J. F. Chen is a typical cash crop widely planted in southwest China (Gao et al., 2022). In early August of 2021, a peculiar leaf spot disease was first detected on A. albus in Ankang Academy of Agricultural Sciences manufacturing base (32°69'N, 109°02'E), Shaanxi, China. Small irregular yellow-brown spots (1 to 2 mm) were observed on the surface of A. albus leaf. Following infection of the leaf, it expanded (3 to 5 mm) and became necrotic. Nine planting bases were investigated, and approximately 75% of plants were symptomatic during the rapid expansion period of bulb growth in Hanyin, Langao and Hanbin counties, Ankang City, Shaanxi, China. Higher disease incidence was observed at temperatures above 30℃ and humidity above 80%. Twenty-seven symptomatic tissues of infected leaves were first surface sterilized by immersion in 75% ethanol for 1 minute, followed by rinsing three times in sterile distilled water. The tissues were then cut into 4-5 mm pieces, plated on 1.5% potato dextrose agar (PDA), and incubated at 28±2°C. The hyphal tip from the growing edge of colonies cultured for three days at 28±2℃ was transferred to PDA to obtain pure cultures. Fungal colonies were white, then grey to black with an unevenly distributed, fast-growing aerial mycelium covering the petri dish within five days at 28±2℃. The colony turned dark brown when maintained in the dark at 28±2℃ after seven days, then grayish brown upon sporulation after 15 days (Fig.1f-g). Conidia were brown or black, smooth, spherical to sub-spherical, single-celled (8-12 µm × 10-13µm, average 9-11.5 µm in diameter, n=5µm). The nutritional hyphae exhibited septa, and a portion of the aerial hyphae formed a long, rough conidium, giving rise to a nearly spherical apical sac (Fig.1h). The surface gave rise to several small peduncles bearing clusters of surfaced spherical conidia (Fig.1i). Surfaced spherical conidia were generated on the surface of the small peduncle (Fig.1j). These morphological features were consistent with Nigrospora oryzae (Li et al., 2017). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), LSU (28S) and BenA gene of the isolate were amplified by PCR (Wang et al., 2017) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, LSU, BenA are OR723825, OR775345, OR277316, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of N.oryzae (GeneBank ID of N.oryzae strain LC2707 ITS, LSU, BenA are KX985954, KY806242, KY019481, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that A-pb169 had the closest match with N. oryzae (Fig. 2). For pathogenicity testing, fifty plants in a period of rapid expansion of bulb growth were selected. Four leaves per plant were inoculated by sprayed till runoff with a conidial suspension of the pathogen (50 µL, 1×106 conidia/ml sterile water), and incubated at 30±2℃ and 80 ± 5% humidity. Control plants received sterile water. On the third day after inoculation, a yellow-brown spot appeared on leave surfaces, the spot gradually expanded; the infection rate was 90 to 95%. Fifteen days after inoculation, infected leaves showed symptoms like those observed in the field, whereas 100 control leaves sprayed with sterile water remained symptomless (Fig.1 a-e). The pathogen was reisolated from infected leaves and confirmed as N. oryzae by morphology and molecular identification. To our knowledge, this is the first report of leaf spot disease of A. albus caused by N. oryzae in China. Since its one of the major cash crops of the southeastern China, further work is necessary to determine its spread and economic impact as well as developing sustainable disease management options.

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus.

Diosgenin is an important raw material used in the synthesis of steroid drugs, and it is widely used in the pharmaceutical industry. The traditional method of producing diosgenin is through using raw materials provided via the plant Dioscorea zingiberensis C. H. Wright (DZW), which is subsequently industrially hydrolyzed using a high quantity of hydrochloric and sulfuric acids at temperatures ranging from 70 °C to 175 °C. This process results in a significant amount of unmanageable wastewater, creates issues of severe environmental pollution and consumes high quantities of energy. As an alternative, the enzymolysis of DZW to produce diosgenin is an environmentally and friendly method with wide-ranging prospects for its application. However, there are still only a few enzymes that are suitable for production on an industrial scale. In this study, three new key enzymes, E1, E2, and E3, with a high conversion stability of diosgenin, were isolated and identified using an enzyme-linked-substrate autography strategy. HPLC-MS/MS identification showed that E1, a 134.45 kDa protein with 1019 amino acids (AAs), is a zinc-dependent protein similar to the M16 family. E2, a 97.89 kDa protein with 910 AAs, is a type of endo-ß-1,3-glucanase. E3, a 51.6 kDa protein with 476 AAs, is a type of Xaa-Pro aminopeptidase. In addition, the method to immobilize these proteins was optimized, and stability was achieved. The results show that the optimal immobilization parameters are 3.5% sodium alginate, 3.45% calcium chloride concentration, 1.4 h fixed time, and pH 8.8; and the recovery rate of enzyme activity can reach 43.98%. A level of 70.3% relative enzyme activity can be obtained after employing six cycles of the optimized technology. Compared with free enzymes, immobilized enzymes have improved stability, acid and alkaline resistance and reusability, which are conducive to large-scale industrial production.

[Relationship Between Macrophyte Communities and Macroinvertebrate Communities in an Urban Stream].

The channelization has caused severe degradation of aquatic ecosystems during the past decades of rapid urbanization in metropolitan areas of China. The re-construction of the in-stream habitat of aquatic organisms and the restoration of aquatic ecosystems were more difficult in the urban stream than in the natural stream with the effluence of nutrient enrichment, water contamination, and in-stream habitat loss. Considering the ecological effects of the submerged macrophyte replantation on macroinvertebrate communities, one of the urban streams that used replantation restoration strategy was seasonally monitored from October 2012 to July 2013. There were four sampling sites, two located at the upper region and two in the middle region. Due to the relatively high levels of organic matter contamination, there are no sites in the downstream region of Qinghe River. Four types of submerged macrophyte were planted at each site to restore the in-stream habitat of two years ago, including Potamogeton pectinatus, Potamogeton crispus, Hydrilla verticillate, and Ceratophyllum demersum. Because of the unexpected development, Myriophyllum spicatum and Acorus calamus appeared at the upper reach of Qinghe River. Considering the average water depth of 30-40 cm, the revised Surber net with the enlarged net was used to collect macroinvertebrate samples. At each sampling site, three replicates of macroinvertebrates were carefully collected for each type of macrophyte. Three replicates were sampled for the comparison at the imperviously concrete sections of each site. The community composition of the macroinvertebrate was determined by cluster analysis and ordination analysis. The density, biodiversity, and community stability were higher in the sampling sections with the replantation of macrophyte than in impervious concrete sections. The occurrence of intermediate tolerant taxa such as Ephemera sp., Caenis sinensis Gui, Ecnomus sp., and Hydropsyche sp. indicated the recovery and restoration of macroinvertebrates in Qinghe River. However, the differences in the community structure, density, taxa richness, biodiversity index, and the composition of functional feeding groups of macroinvertebrates among different types of submerged macrophyte were not identified. Unlike providing extra habitats for macroinvertebrates in the vertical direction in natural streams and lakes, the submerged macrophyte provided more stable benthic habitats in urban streams. The stem and leaf of macrophytes could adjust the hydraulics and continually absorb, fix, and accumulate the suspended solids in the sediment and water interface. The root systems could stabilize the microhabitat of the sediment for macroinvertebrates, benthic algae, and microorganisms. Those aquatic organisms played an important role in the decomposition, transformation, and mineralization of nutrients and organic matter in urban streams. Following the recovery of stabilized microhabitats, community restoration and secondary succession of macroinvertebrates could continually and slowly happen. This study improved the understanding of the ecological effects of macrophytes on the restoration of aquatic organisms and ecosystems in urban streams.