Areca palm is an important tropical economic crop in Hainan Province. Areca palm yellow leaf disease (YLD) caused by phytoplasma infection is a devastating disease in the production of areca in China. In order to establish an accurate and efficient detection method for areca palm yellow leaf phytoplasma, this study designed and synthesized specific primers AMf/AMr and AM-Prode based on the 16S rDNA gene of areca palm yellow leaf phytoplasma. The method was used to test the accuracy, sensitivity, specificity and repeatability, and to detect phytoplasma diseases in other plants. The method could accurately detect the positive samples, and the healthy samples had no amplification curve. In the sensitivity test, the method could detect the sample concentration level of 1.16×10¹ copies/μL, and the standard curve equation was y=–3.4185x+43.624, the amplification efficiency was 96.12%, and the correlation coefficient R²=0.9833. In the specificity test, the method had good specificity for the detection of YLD, and the genomes of areca, other disease pathogens and the endophytes did not interfere with the method. In the repeatability test, the method had good repeatability for the detection of YLD. The detection method could be used to detect 8 phytoplasma diseases such as chinaberry yellow leaf disease, ‘Pericampylus glaucus’ witches' broom disease and pepper yellow leaf disease and the detection of phytoplasma has certain universality. The establishment of detection method is conducive to providing reliable technical means for the accurate diagnosis of YLD, pathogen monitoring and vector insect detection.

Hypothenemus hampei is a new invasive pest in China. It is a main coffee pest and poses a great threat to the coffee industry. The purpose of this study is to clarify the biological characteristics and field occurrence rules of the insect, and to provide a theoretical basis for later monitoring and forecasting and comprehensive management of the insect. In this study, the morphological characteristics, developmental duration and fecundity of various developmental stages of H. hampei were observed at each stage in the indoor feeding conditions. The annual life cycle of H. hampei was observed in the field. The population dynamics and spatial distribution patterns of H. hampei in Wanning city, Hainan province were measured through systematic field investigation from 2021 to 2022 to clarify the biological characteristics and field occurrence of H. hampei, and provide a theoretical basis for the subsequent integrated pest management of H. hampei. The results showed that the developmental duration of egg, larval, pupal and adult stage of H. hampei was (6.75±0.65) d, (17.41±1.63) d, (6.45±1.15) d, (42.74±9.55) d, respectively. The average generation duration was (73.35±11.26) d. The main morphological characteristics of each stage were as follows: egg, elliptic, milky white, with smooth and shiny surface; larval, usually “C” shaped, milky white, slightly transparent, with brown head and no feet; pupal, milky white at the initial stage, and later turn brown, the head hidden under the pronotum; adult body, cylindrical, dark brown to black, shiny; male smaller than the female. Female adults usually laid eggs under dark conditions, the number of eggs laid by a single female was 11.85±1.69, and the ratio of male to female was 10.25∶0.95. The results of indoor and field observations showed that H. hampei had seven generations a year in Hainan, and there was no phenomenon of overwintering. The results of the field survey in 2021—2022 showed that the population of H. hampei began to increase slowly from January, and the incidence of H. hampei increased sharply from April to May, reached a maximum of 60.15% and peak in May, and then declined rapidly. In October, it remained at a relatively stable level. The population increased again in November, with another small peak, reaching 19.87%. In December, it decreased, but it showed a slow increase until March of the following year related to the number of fruits. The survey results on space and orientation showed that during the fruit development period, H. hampei preferred the middle layer (0.5-1.0 m) fruit spatially, and preferred the east and north directions in orientation, which may be related to light and temperature. The results of the four aggregation parameters showed that H. hampei belonged to the aggregation distribution in both horizontal and vertical directions.

Sporisorium scitamineum, the pathogen of destructive disease smut, usually causes abnormal growth of meristematic tissue and forms a “black whip” in meristem tissues. In some cases, it induces formation of flowering structures in some sugarcane cultivars (genotypes), but the exact mechanism remains unclear. In order to explore flowering related genes and the expression patterns in sugarcane in response to infection by S. scitamineum, the healthy and S. scitamineum infected ‘guitang42’ sugarcane cultivar samples were analyzed by transcriptome sequencing. A total of 3276 differentially expressed genes (DEGs) were identified, of which 1677 genes were up-regulated (P<0.05) and 1599 genes down-regulated (P<0.05). The DEGs were mostly enriched in the biosynthesis process, ribosome, ribonucleoprotein complex, ribosome structural components, etc. according to GO enrichment analysis. The KEGG analysis showed that the DEGs were mostly enriched in amino acid biosynthesis, purine metabolism, carbon metabolism, phagocytosis, and other metabolic pathways. Also, out of 30 DEGs were identified to be associated with flowering, and the representative genes such as FT1, PIE1, GID1, GA20ox-1, GA20ox-2 were validated by qRT-PCR. The qRT-PCR results were consistent with the transcriptomic data, confirming the dependability of the transcriptome sequencing results. The findings would present essential candidate genes for revealing the mechanism of early flowering of sugarcane plants caused by S. scitamineum, and lay the foundation for disease resistance breeding.

Hevea brasiliensis is an important natural rubber-producing plant. Rubber particles (RP) in latex are important organelles for the synthesis of natural rubber, which can be divided into large rubber particles (LRP) and small rubber particles (SRP) according to the diameter. Although the specific mechanism of its regulation of natural rubber synthesis in response to exogenous ethylene stimulation has been studied. However, the specific regulatory mechanism of LRP and SRP in regulating natural rubber synthesis in response to ethylene stimulation is still unclear. In order to clarify the role of RP with different diameters in the synthesis of natural rubber, LRP and SRP stimulated by ethylene were isolated and the proteins of corresponding samples were extracted for differential protein analysis. 37 differential proteins in response to ethylene stimulation were identified in the LRP, which were involved in natural rubber biosynthesis, glycolysis/gluconeogenesis, carbon metabolism and amino acid biosynthesis, and some other metabolic pathways, including four members of the REF/SRPP family. 56 differential proteins were identified in SRP, which were involved in protein processing, endocytosis, splicing in endoplasmic reticulum, and some other metabolic pathways, including five members of the REF/SRPP family. The key differential accumulation protein REF138 had many isoforms with different isoelectric points (pI) and molecular weights (MW). REF138 isoforms below the standard pI (4.80) on LRP were down-regulated in response to ethylene stimulation, while isoforms above the standard pI were up-regulated to ethylene stimulation. Different from LRP, more isoforms of REF138 on SRP changed in response to ethylene stimulation, and isoforms with standard MW (14.7 kDa) were up-regulated in response to ethylene stimulation, while isoforms with higher than standard MW were down-regulated in response to ethylene stimulation. The function analysis of key differential proteins showed that there were interactions among the members of the REF/SRPP family. REF138, REF175, REF258, SRPP117 and SRPP204 may form protein complexes and bind to RP. In addition to the members of the REF/SRPP family, 20 proteins interacting with REF138 were mainly involved in the spliceosome and endocytic metabolic pathways, and 50 proteins interacting with REF258 were involved in the regulation of lipid metabolism and the synthesis of secondary metabolites. In conclusion, functional analysis of differential proteins and the interaction proteins responsive to ethylene stimulation on LRP and SRP might preliminarily reveal the metabolic regulatory mechanism of LRP and SRP in regulating natural rubber synthesis in response to exogenous ethylene stimulation.

The quality and performance of natural rubber (NR) are closely related to Hevea brasiliensis (rubber tree) varieties, tapping systems, seasonal variation and phenology etc. In order to uncover the annual change rules from April to December of NR qualities among different rubber tree varieties, we investigated the NR qualities from five clones including PR 107, RRIM 600, Reyan 917, Reyan 73397 and Reyan 879 cultivated in Danzhon of Hainan, China, and analysed the vulcanization characteristics and the mechanical properties of the vulcanized NR prepared from the latex harvested in May. The results showed that the mooney viscosity, protein percentage, volatile percentage, Wallace plasticity (P₀) and plasticity retention index (PRI) presented a downward or upward trend in an annual tapping cycle from April to December, which indicates that the NR quality parameters are affected by both genotypes and environmental factors such as weather and sunshine etc. The protein percentage in NR from the latex of the four rubber tree clones was more than 3.0% in December. The cure characteristics of NR from Reyan 917 were totally different from those of the other four rubber trees. Under the same tapping system of S/2 d/3, the tensile strength and the elongation at break of the vulcanized rubber from Reyan 879 was the smallest compared with the other four clones. The results would contribute to instructing the new variety breeding and rubber production on the one hand, and to providing scientific supports for high-performance NR manufacturing on the other hand.

The aim of this study was to explore the protective effect of arecoline on the cell model of H₂O₂-induced oxidative stress damaged model in SH-SY5Y cells and elucidate its mechanism. Cell viability was detected by CCK-8 assay. Spectrophotometry was used to detect LDH release, MDA, SOD, and CAT contents. Flow cytometry was used to detect cell apoptosis and mitochondrial membrane potential. Western blot was used to detect the expressions of Nrf2, HO-1, Keap1, Bcl-2, Bax and Caspase-3. Arecoline could reduce cell apoptosis and significantly increase mitochondrial membrane potential. The level of SOD elucidateand CAT increased while the level of MDA decreased. Arecoline 140 μmol/L group could significantly up-regulate the protein expression of Nrf2, HO-1 and Bcl-2 (P<0.001, P<0.0001), and down-regulate the protein expression of Keap1, Bax and Caspase-3 (P<0.001, P<0.0001). Conclusions: arecoline can effectively improve H₂O₂-induced oxidative stress injury in SH-SY5Y cell by activating the Nrf2/HO-1 signaling pathway, enhancing the activities of antioxidant enzymes and regulating the oxidation reduction system of cells, as well as regulating the Bcl-2/Bax/Caspase-3 signaling pathway and inhibiting cell apoptosis.

The present work was conducted to identify the pathogen causing anthracnose of Liberica coffee (Coffea liberica) plants and to screen fungicides against the disease pathogen. The leaves of C. liberica with anthracnose symptoms were sampled from fields for fungal isolation and purification. Four pure fungal isolates were obtained and only KFTJ01 was verified pathogenic to the coffee leaves by tests following Koch’s Postulates. The rDNA-ITS sequence (554 bp), beta-tubulin gene (tub2) and calmodulin gene (CAL) were successfully amplified via PCR from genomic DNA of KFTJ01 and sequenced. Blastn analysis of the the three DNA sequences showed that KFTJ01 was identical to the strain WZ-135 (99.62%, MN856281), CREADC-ER2212 (100%, MT409131) and YMTJ4 (100%, MK569149) of Colletotrichum kahawae. KFTJ01 was also clustered with C. kahawae WZ-135 strain on the same end-branch of the phylogenic tree (bootstrap confidence is 98%). Therefore, the pathogen of Liberica coffee was identified as C. kahawae which is a quarantine species and has never been recorded in China. Fungicide selection experiments showed that among the 10 chemicals, methionine-acetazolyl and methyl thiobacillam almost completely (100%) suppressed the colony growth of KFTJ01. The median effective concentration calculated from the functions was 0.0607 mg/mL and 0.0809 mg/mL, respectively, indicating that both the chemicals were very effective against the anthracnose pathogen. The results are important references for the field diagnosis of the coffee plant anthracnose and for controlling the disease epidemics.

Potted bitter melon was treated with Bacillus velezensis N46 to explore the mechanism of preventing powdery mildew of bitter melon. The prevention mechanism of N46 was studied by detecting the spore germination rate of powdery mildew pathogen (Podosphaera xanthii), changes in leaf defense enzyme activity, accumulation of reactive oxygen species, cell allergic necrosis, lignin accumulation, changes in the expression of disease-resistance related genes and disease-resistance pathways. The control effect of high concentration (6×10⁸ CFU/mL) of B. velezensis N46 on powdery mildew of bitter melon reached 59.05%. In the N46 treatment group, the range and peak value of the activity of defense enzymes POD, CAT and SOD increased significantly, the accumulation of reactive oxygen species (ROS) increased significantly, the rate of anaphylactic necrosis increased significantly, and the rate of lignin accumulation in cell wall was significantly enhanced. N46 had a significant inhibitory effect on the conidial germination of pathogen powdery mildew. The expression levels of JA reactive marker lipoxygenase gene (LOC111018837), SA reactive marker gene (LOC111017362) and peroxidase gene (LOC111021192) associated with allergic reaction and cell wall strengthening were detected in the N46 treated bitter melon. Compared with the control group, the expression levels of the genes increased significantly when bitter melon was infected with the powdery mildew bacteria, and the expression levels of JA reactive marker genes in the treatment group were also higher than those in the control group when there was no contact with pathogenic bacteria. The results indicated that N46 treated bitter melon maintained a certain level of “preactivation” of resistance genes when it was not exposed to pathogenic bacteria, but showed a higher expression level when it was exposed to pathogenic bacteria. Finally, the JA response pathway was inhibited by ibuprofen (IBU), and the control effect of B. velesiensis N46 on powdery mildew of bitter melon was almost disappeared, suggesting that the generation of N46 induced powdery mildew resistance of bitter melon depended on JA pathway. In conclusion, B. velezensis N46 has a good control effect on powdery mildew of bitter melon by inhibiting the conidium germination of megalomycesfoliata and inducing JA pathway dependent resistance of bitter melon. This study would provide a new method for the biological control of powdery mildew of bitter melon and a theoretical basis for the effect of B. velezensis on the control of powdery mildew of bitter melon.

Tigernut (Cyperus esculentus L.), which belongs to the Cyperaceae family within Poales, is a novel herbaceous oil crop producing high amounts of oil in underground tubers. Like other tuber and tuberous root crops, water accounts for approximately 85% of immature tubers, implying a crucial role of water balance for tuber development in tigernut. Plasma membrane intrinsic proteins (PIPs), which include two groups (i.e. PIP1 and PIP2) typically localized to the cell membrane, constitute a subfamily of aquaporin facilitating the passive transport of water. Based on one PIP identified in the proteomes of tigernut tubers, its coding gene CePIP1;1 was cloned using the RT-PCR technique, followed by analyzing the exon-intron structure, sequence features, evolutionary relationships, expression profiles, and protein subcellular localization. CePIP1;1 was shown to contain three introns with a coding sequence of 867 bp, putatively encoding 288 amino acids with the theoretical molecular weight of 30.76 kDa, the isoelectric point of 8.82, the instability index of 32.95, the grand average of hydropathicity of 0.384, and the aliphatic index of 95.28, implying that it is stable, basic, and hydrophobic. The protein was predicted to harbor one conserved MIP (major intrinsic protein) domain, including six transmembrane helices, two half helices, and dual NPA motifs located at the N-termini of two half helices. Sequence alignment and phylogenetic analysis revealed that CePIP1;1 clustered with Oryza sativa PIP1s and possesses an extended N-terminus but a short C-terminus relative to Spinacia oleracea PIP2;1, supporting its classification. Transient over-expression in Nicotiana benthamiana leaves supported the plasma membrane localization of CePIP1;1, which is consistent with the bioinformatics prediction. Further expression analysis showed that CePIP1;1 was constitutively expressed in all tissues examined in this study. During tuber development, CePIP1;1 exhibited a typical bell-shaped expression pattern, which is consistent with that of the moisture content. These results lay a solid foundation for further functional analysis and genetic improvement in tigernut.

Oncidium is an important fresh-cut flower in the market because of its unique flower pattern and brilliant color. The petals of Oncidium are highly susceptible to flower diversity. RNA extraction and transcriptome analysis were performed on the lateral petals of O. flexuosum ‘Honey Angel’ and variants with two lateral petals converted into lips to explore the molecular mechanism regulating the labialization of Oncidium lateral petals. A total of 45 525 415 high-quality reads were obtained from the sequencing results and 52 253 unigenes were identified. The seven databases of NR, Swissprot, PFAM, GO, NT, KO and KOG were used to annotate gene functions and annotated to 31 828, 23 174, 21 369, 21 367, 20 830, 10 523, 6465 unigenes. Upon expression comparison,3120 differential genes were obtained between O. flexuosum ‘Honey Angel’ and the mutant. A total of 1537 differentially expressed genes were annotated into three functional groups: biological processes, cellular components and molecular functions. KEGG functional enrichment analysis enriched 690 differentially expressed genes into different metabolic pathways, with the most genes enriched by metabolic pathway. To date, the study of lip formation in orchids has focused more on the MADS-box gene family, not only five significantly differentially expressed MADS-box genes were screened in this transcriptome data, but also other transcription factor such as NAC, TCP, MYB and WRKY that may be related to the lip formation in Oncidium were also discovered. RT-qPCR was used to verify the expression of key genes, and the quantitative results were consistent with RNA-seq data, most of the genes were highly expressed in the lip-like lateral petals, indicating that the genes may positively regulate the development of the lip of Oncidium. In summary, this study excavated a large number of key transcription factors involved in the variation of labialization in Oncidium, which would provide a certain reference for the study of the molecular mechanism and regulatory network of lip formation of orchid.