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Chemical agents are effective treatment methods for anthracnose induced by pathogenic Colletotrichum gloeosporioides on Stylosanthes. However, excess consumption of chemical agents destroys the environment, synthetic biology was capable of conquering the issue. The antifungal agent is developed by enclosing a bio-synthesized peptide aptamer with layered montmorillonite via electrostatic interaction. Compared with free peptide aptamer, the nanocomposite exhibits higher antifungal activity against Colletotrichum gloeosporioides, further improving the utilization of peptide aptamer. The nanocomposite killed Colletotrichum gloeosporioides by releasing peptide aptamer after they entered the spore. Moreover, montmorillonite enhances the adhesion ability of peptide aptamer via hydrophobic interactions between nanomaterials and leaves, prolonging the extension time of nanocomposite on leaves. Consequently, 0.1 mg of nanocomposite demonstrates a comparable effect to commercial carbendazim (1 %) to prevent anthracnose on leaves of Stylosanthes induced by HK-04 at room temperature. This work demonstrates an alternative to commercial antifungal agents and proposes a versatile approach to preparing environmental-friendly antifungal agents to inhibit fungal infections on crops.

Coffee leaf rust (CLR) caused by Hemileia vastatrix is a worldwide devastating disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, it is difficult to accurately identify CLR at the early stage via naked eye; and the detection of H. vastatrix using PCR-based methods were time-consuming, laborious, and sometimes low- sensitivity. LAMP technology is known for its speed, specificity, and sensitivity to accurately identify many different pathogens. Therefore, in this study, by comparing ITS sequences of H. vastatrix with other H. vastatrix and Uredinales strains using National Center for Biotec- hnology Information (NCBI) by BLASTn, we designed specific primers targeting the unique region and its flanking regions of ITS sequences for H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Furthermore, we optimized the LAMP protocol was to enhance both sensitivity and specificity for H. vastatrix detection. Under the optimized condition, the establised LAMP protocol could detect as low as 1pg/μl of H. vastatrix DNA in 60min at 63℃, which is ~100 times more sensitive than conventional PCR. This method can successfully detect H. vastatrix in the early CLR symptom on the coffee leaves in the field.

WRKY transcription factors are key regulators in regulating abiotic or biotic stress response in Arabidopsis. Previous studies showed that WRKY30 expression was induced by oxidative stress treatment, fungal elicitor, SA and ABA. However, functions of WRKY30 on viral defense are not well studied. Here, we found that Arabidopsis WRKY DNA binding protein 30 (WRKY30) plays essential roles in regulating Cucumber mosaic virus (CMV) resistance. The expression of WRKY30 was induced by CMV infection and wrky30 mutant displayed more susceptibility (including higher oxidative damages, induced reactive oxygen species synthesis and more PSII photochemistry compromise), while WRKY30 overexpression plants (WRKY30OX) exhibited more resistance to CMV infection. Moreover BRs-induced CMV tolerance is partly dependent on WRKY30. And WRKY30 expression increased after BL treatment. All these demonstrated that WRKY30 works as a positive regulator in plant CMV resistance process.

Coffee is a tropical plant with two widely cultivated species, namely Coffea arabica and Coffea canephora. A leaf spot disease causing brownish and necrotic lesions was broken out on the C. canephora coffee seedlings in a nursery in Ruili County, Yunnan Province, China, during 2018 to 2019. The incidence of the disease was 15% ~ 20%. Ten diseased leaf samples from five diseased plants were collected for pathogen isolation by tissue separation method. Leaf pieces were cut from the margin of the necrotic lesions (4 × 6 mm), surface-sterilized for 30 s in 75% ethanol, followed by 0.1% arsenic mercury solution for 15 s, then washed 3~4 times with sterilized distilled water and transferred onto potato dextrose agar (PDA) medium in petri plates. Four morphologically similar isolates were obtained from lesions and cultivated on PDA at 25°C. Initial colonies of isolates were round, neat edge, white, floccose mycelium and developed dark green-to-black concentric rings that were sporodochia bearing viscid spore masses after 5~7 days. Conidia were acetates, hyaline and cylindrical with both rounded ends and 4.8 to 6.4 µm long × 1.6 to 2.6 µm wide. Koch's test were conducted on three healthy plants leaves of original source variety C. canephora No.2 and C.arabica Catimor CIFC7963 (control plants) with spore suspension (1 × 106/mL), respectively. Meanwhile, equal numbers of healthy plants were inoculated with water as controls. After inoculation, the plants were transferred into an incubator at 25℃ with saturated humidity. After 10 days of inoculation, all the tested plants presented similar typical symptoms with the diseased leaves under natural conditions; whereas the controls remained healthy. Koch's postulates were performed by re-isolating the fungus from the inoculated leaves and verifying its colony and morphological characters. Two single spore isolates cultured on PDA medium were selected for DNA extraction. The ribosomal internal transcribed spacer (ITS) was PCR amplified by using primers ITS1 and ITS4 (White et al., 1990), β-tubulin gene by Bt2a and Bt2b (Glass and Donaldson, 1995), the RNA polymerase II second largest subunit (rpb2) by RPB2-5F2 and RPB2-7cR (O'Donnell et al, 2007), calmodulin (cmda) gene by CAL-228F and CAL2Rd (Groenewald et al., 2013). The sequences of ITS (MT853067 ~ MT853068), β-tubulin (MT897899 ~ MT897900), rpb2 (MW256264~ MW286265) and cmda (MT897897~ MT897898) were deposited in GenBank databases. BLAST analysis revealed that the representative isolates sequences shared 99.31%~99.65% similarities to the ITS sequence of Paramyrothecium breviseta (Accession Nos. NR_155670.1), 99.43% similarities to the β-tubulin sequence of P. breviseta (Accession Nos. KU846406.1), 98.98% similarities to the rpb2 sequence of P. breviseta (Accession Nos. KU846351.1), and 98.54%~98.71% similarities to the cmda sequence of P. breviseta (Accession Nos. KU846262.1). As it shown in the phylogenetic tree derived from combined ITS, β-tubulin, rpb2, and cmda gene sequences, the two representative isolates were clustered together with P. breviseta CBS 544.75 with 98% strong bootstrap support, which confirmed that P. breviseta is the causal agent of leaf spot of Coffea canephora. To our knowledge, this is the first report of a leaf spot disease caused by P. breviseta on C. canephora in China, which raised the caution that P. breviseta is also pathogenic to Coffea Arabica.

Agave angustifolia is commonly used for the production of bacanora, a kind of fermented and distilled beverage in Mexico. In the present study, we have successfully assembled its chloroplast genome...

Agave fourcroydes (henequen) is the only cultivated Agave species in the Yucatan Peninsula, which is mainly used for fiber production. In the present study, we have successfully assembled the chloroplast (cp) genome of A. fourcroydes. The full length of the cp genome is 157,291 bp with a GC content at 37.8%. The cp genome is constructed with an inverted repeat region a (IRa) of 26,573 bp, a small single copy region (SSC) of 18,230 bp, an inverted repeat region b (IRb) of 26,573 bp and a large single copy region (LSC) of 85,915 bp. The annotation result reveals 132 genes on the cp genome, including 86 protein-coding genes, 38 tRNAs and 8 rRNAs. Phylogenetic tree reveals that A. fourcroydes is closely related with A. sisalana.

Agave sisalana is one of the Agave cultivars for sisal fiber production around the tropical areas of the world. In the present study, we successfully sequenced and assembled its chloroplast genome. The full size of the genome is 157,268 bp with a GC content at 37.85%. The genome is constructed with a large single copy region (LSC, 85,894 bp), a pair of inverted repeat regions (IR, 26,573 bp) and a small single copy region (SSC, 18,228 bp). Besides, 86 protein-coding genes, 38 tRNAs and 8 rRNAs are annotated on the chloroplast genome. Phylogenetic result reveals that A. sisalana is closely related with A. americana and A. H11648.

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.

Agave species are an important family of crassulacean acid metabolism (CAM) plants with remarkable tolerance to heat and drought stresses (Agave deserti) in arid regions and multiple agricultural applications, such as spirit (Agave tequilana) and fiber (Agave sisalana) production. The agave genomes are commonly too large to sequence, which has significantly restricted our understanding to the molecular basis of stress tolerance and economic traits in agaves. In this study, we collected three transcriptome databases for comparison to reveal the phylogenic relationships and evolution patterns of the three agave species. The results indicated the close but distinctly domesticated relations between A. tequilana and A. sisalana. Natural abiotic and biotic selections are very important factors that have contributed to distinct economic traits in agave domestication together with artificial selection. Besides, a series of candidate unigenes regulating fructan, fiber, and stress response-related traits were identified in A. tequilana, A. sisalana, and A. deserti, respectively. This study represents the first transcriptome comparison within domesticated and wild agaves, which would serve as a guidance for further studies on agave evolution, environmental adaptation, and improvement of economically important traits.

Coffee leaf rust (CLR), caused by the obligate biotrophic fungus Hemileia vastatrix (Hv), is a devastating disease worldwide leading to severe reductions in yield and economic losses in coffee plan...

Agave hybrid 11648 is the most widely cultivated agave variety for sisal fiber production around the world. In the present study, we have successfully sequenced the chloroplast genome of A. H11648. The complete chloroplast genome size is 157,274 bp in length with a GC content of 37.8%. The genome contains a large single copy region (LSC) of 85,896 bp, a small single copy region (SSC) of 18,230 bp, and a pair of inverted repeat regions (IR) of 26,574 bp. 121 genes are annotated in the chloroplast genome. The numbers of protein-coding, tRNA and rRNA genes are 99, 40 and 8, respectively. Phylogenetic tree reveals that A. H11648 is closely related to A. americana.

Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear. We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana. Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET-induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ-treated and gene-silenced were completely eliminate this response. Our results suggest BR is involved in ET-induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR-dependent manner.

Asparagus officinalis L. is a dioecious perennial plant globally known for its fine flavor and high nutritional value. An evaluation of genetic diversity in 46 asparagus accessions was carried out based on morphological and inter-simple sequence repeat (ISSR) markers. The result show that the coefficient of variation for 20 morphological characteristics is between 12.45 and 62.22%. Factor analysis revealed that nine factors explained 83.37% of the total variance. At Euclidean distance of 135.7, 46 accessions were divided into two clusters. Genetic similarity coefficient (GSC) based on ISSR data ranged from 0.60 to 0.97, suggesting a relatively abundant genetic base. Furthermore, the 46 asparagus accessions could also be grouped into three major clusters at a GSC of 0.74. And there is no significant relation between the two marker systems using the Mantel test. Clustering based on morphological traits compared with that based on ISSR data was not consistent, however, some common groupings were observed between two dendrograms. Therefore the results elucidated asparagus germplasm genetic background and determined hybrid parents, which will facilitate optimal application of asparagus germplasm resources and provide additional data for genetic improvement.

Agave species are important crassulacean acid metabolism (CAM) plants and widely cultivated in tropical areas for producing tequila spirit and fiber. The hybrid H11648 of Agave ((A. amaniensis &times, A. angustifolia) &times, A. amaniensis) is the main cultivar for fiber production in Brazil, China, and African countries. Small Auxin Up-regulated RNA (SAUR) genes have broad effect on auxin signaling-regulated plant growth and development, while only few SAUR genes have been reported in Agave species. In this study, we identified 43, 60, 24, and 21 SAUR genes with full-length coding regions in A. deserti, A. tequilana, A. H11648, and A. americana, respectively. Although phylogenetic analysis revealed that rice contained a species-specific expansion pattern of SAUR gene, no similar phenomena were observed in Agave species. The in silico expression indicated that SAUR genes had a distinct expression pattern in A. H11648 compared with other Agave species, and four SAUR genes were differentially expressed during CAM diel cycle in A. americana. Additionally, an expression analysis was conducted to estimate SAUR gene expression during different leaf developmental stages, abiotic and biotic stresses in A. H11648. Together, we first characterized the SAUR genes of Agave based on previously published transcriptome datasets and emphasized the potential functions of SAUR genes in Agave&rsquo, s leaf development and stress responses. The identification of which further expands our understanding on auxin signaling-regulated plant growth and development in Agave species.

Agave plants are important crassulacean acid metabolism (CAM) plants with multiple agricultural uses, such as being used in tequila and fiber production. Agave hybrid H11648 ((A. amaniensis Trel. and Nowell &times, A. angustifolia Haw.) &times, A. amaniensis) is the main cultivated Agave species for fiber production in large tropical areas around the world. In this study, we conducted a transcriptome analysis of A. H11648. About 49.25 million clean reads were obtained by Illumina paired-end sequencing. De novo assembly produced 148,046 unigenes with more than 40% annotated in public databases, or matched homologs in model plants. More homologous gene pairs were found in Asparagus genome than in Arabidopsis or rice, which indicated a close evolutionary relationship between Asparagus and A. H11648. CAM-related gene families were also characterized as previously reported in A. americana. We further identified 12 cellulose synthase genes (CesA) in Asparagus genome and 38 CesA sequences from A. H11648, A. americana, A. deserti and A. tequilana. The full-length CesA genes were used as references for the cloning and assembly of their homologs in other Agave species. As a result, we obtained CesA1/3/4/5/7 genes with full-length coding region in the four Agave species. Phylogenetic and expression analysis revealed a conserved evolutionary pattern, which could not explain the distinct fiber traits in different Agave species. We inferred that transcriptional regulation might be responsible for Agave fiber development. This study represents the transcriptome of A. H11648, which would expand the number of Agave genes and benefit relevant studies of Agave fiber development.

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the species ‘Candidatus Liberibacter asiaticus (Las)’ in China. All currently popular cultivars are susceptible to HLB, and there are no effective approaches to control the disease once the plants have been infected. Therefore, rapid and easy diagnostic tools are needed for maintaining clean nursery stock and inoculum removal strategies to control HLB. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) targeting16S rDNA was developed for the rapid and quantitative detection of Las in China. The detection sensitivity of the RealAmp assay was approximately 1 pg/μl template DNA (equal to 2.4 × 105 target DNA copies/μl) and no cross-reaction was observed with other pathogens or virus-free seedlings. Besides the quantitative detection using SYTO-9 fluorescent dye, an improved closed-tube visual inspection technique using SYBR Green I staining was developed for rapid screening of samples and minimize the risk of cross-contamination. The RealAmp assay is an alternative quantitative method, which would be used as a routine detection service for nursery materials in China.

Coffee is one of the most popular beverages around the world, which is mainly produced from the allopolyploid Coffea arabica. The genomes of C. arabica and its two ancestors C. canephora and C. eugenioides have been released due to the development of next generation sequencing. However, few studies on C. arabica are related to the PIN-FORMED (PIN) auxin efflux transporter despite its importance in auxin-mediated plant growth and development. In the present study, we conducted a genome-wide analysis of the PIN gene family in the three coffee species. Totals of 17, 9 and 10 of the PIN members were characterized in C. Arabica, C. canephora and C. eugenioides, respectively. Phylogenetic analysis revealed gene loss of PIN1 and PIN2 homologs in C. arabica, as well as gene duplication of PIN5 homologs during the fractionation process after tetraploidy. Furthermore, we conducted expression analysis of PIN genes in C. arabica by in silico and qRT-PCR. The results revealed the existence of gene expression dominance in allopolyploid coffee and illustrated several PIN candidates in regulating auxin transport and homeostasis under leaf rust fungus inoculation and the tissue-specific expression pattern of C. arabica. Together, this study provides the basis and guideline for future functional characterization of the PIN gene family.

Tetraploid induction is an effective way to generate new genotypes. In this study, tetraploid inductions of asparagus (Asparagus officinalis L.) were obtained by applying several treatments with colchicine. Optimal results (31 %) were obtained by applying 0.15 % colchicine plus 2 % dimethyl sulphoxide (DMSO) to recently germinated seeds (5 days old) for 16 h. Sixty-five tetraploid plants were recovered from this treatment and diploid and tetraploid plants showed significant differences in morphology. The stomatal density of tetraploid asparagus was approximately half than that of diploid plants, whereas the chloroplast number of tetraploid plants was twice that of diploid plants; moreover, the stomatal length and breadth were slightly larger in tetraploid plants than in diploid plants. Furthermore, in tetraploid genotypes the chlorophyl A content was double than that of diploid plants, whereas chlorophyl B and total chlorophyl contents were three times higher than in diploid plants. The tetraploid plants showed better growth, more flowering flourish, deeper color, and bulkier leaves in comparison to the diploid plants. Moreover, tetraploid plants had lower malonaldehyde (MDA) and higher activity of superoxide dismutase (SOD), peroxidase (POD) and free proline (Pro), the differences were all significant, indicating that tetraploid plants had increased heat resistance however there was no significant difference in the heat tolerance between plants of the same ploidy. Tetraploid plants with strong heat resistance broaden the asparagus gene pool and could aid future breeding work in asparagus.

Agave plants are important crassulacean acid metabolism (CAM) plants with multiple agricultural uses, such as being used in tequila and fiber production. Agave hybrid H11648 ((A. amaniensis Trel. and Nowell × A. angustifolia Haw.) × A. amaniensis) is the main cultivated Agave species for fiber production in large tropical areas around the world. In this study, we conducted a transcriptome analysis of A. H11648. About 49.25 million clean reads were obtained by Illumina paired-end sequencing. De novo assembly produced 148,046 unigenes with more than 40% annotated in public databases, or matched homologs in model plants. More homologous gene pairs were found in Asparagus genome than in Arabidopsis or rice, which indicated a close evolutionary relationship between Asparagus and A. H11648. CAM-related gene families were also characterized as previously reported in A. americana. We further identified 12 cellulose synthase genes (CesA) in Asparagus genome and 38 CesA sequences from A. H11648, A. americana, A. deserti and A. tequilana. The full-length CesA genes were used as references for the cloning and assembly of their homologs in other Agave species. As a result, we obtained CesA1/3/4/5/7 genes with full-length coding region in the four Agave species. Phylogenetic and expression analysis revealed a conserved evolutionary pattern, which could not explain the distinct fiber traits in different Agave species. We inferred that transcriptional regulation might be responsible for Agave fiber development. This study represents the transcriptome of A. H11648, which would expand the number of Agave genes and benefit relevant studies of Agave fiber development.

A rod-shaped bacterium was isolated from the soil in a karst area of Guilin, China and its biotransformation of steroidal saponins in sisal (Agave sisalana Perrine) to tigogenin was presented for the first time. A total of 22 strains for the degradation of steroidal saponins in sisal were isolated from 48 soil samples, and the isolated rod-shaped, bacterial strain ZG-21 was used for the production of tigogenin due to its highest degradation efficiency of steroidal saponins in sisal. The parameters affecting biotransformation by strain ZG-21 were optimized. Under the optimized conditions of temperature (30 °C), pH (6), time (5 days) and substrate concentration (5 mg/mL), a maximum tigogenin yield of 26.7 mg/g was achieved. Compared with the conventional method of acid hydrolysis, the biotransformation method provided a clean and eco-friendly alternative for the production of tigogenin.

Zebra disease, which is caused by Phytophthora nicotianae, is a serious global threat to the main cultivar Agave hybrid No. 11648 (H.11648). This disease requires a long period to obtain anti-P. nicotianae variants by crossbreeding, given that 10 years are needed for this plant to bloom. Therefore, transgenic technology could be useful for the production of new agave lines that might develop significant tolerance to P. nicotianae within a relatively short period. In this study, a tissue culture system was tested on H.11648. Results showed that the optimum culture medium for callus induction was SH + 3 mg L−1 6-BA + 0.5 mg L−1 NAA + 0.1 mg L−1 2,4-D + 30 g L−1 sucrose + 6.5 g L−1 carrageenan, with pH of 5.8. The induction medium for budding was SH + 1.5 mg L−1 6-BA + 0.5 mg L−1 NAA + 30 g L−1 sucrose + 6.5 g L−1 carrageenan at pH 5.8. The induction medium for rooting was SH + 0.1 mg L−1 IAA + 30 g L−1 sucrose + 6.5 g L−1 carrageenan. The primary factors influencing plant transformation efficiency were also investigated. Results showed that the effective time for infection was 10 min, the optimum concentration of acetosyringone was 200 μM, the optimum time for pre-culture of sisal callus was 3 days, and the optimum co-culture time for calli and Agrobacterium was 4 days. Sisal calli will die when phosphinothricin concentration reaches 2.0 mg L−1. Furthermore, a hevein-like protein gene from Pharbitis nil was introduced to H.11648. The integration stability and expression level of the transgene in the H.11648 genome were analyzed by using Southern blot and reverse transcription polymerase chain reaction. The mycelial growth rate of P. nicotianae was significantly inhibited by the crude leaf extracts from the transgenic H.11648 plants. After further study in vivo, the resistance of transgenic H.11648 plants to P. nicotianae infection was enhanced.