Exploring cadmium stress responses in sisal roots: Insights from biochemical and transcriptome analysis.

Sisal is a leaf fiber crop with a high integrated value and a wide range of uses in the application of soil remediation of heavy metal contamination. This study provides a preliminary understanding of how sisal responds to Cd stress and presents a theoretical basis for exploring the potential of sisal in the remediation of Cd-contaminated soils. In this work, the activities of the antioxidant enzymes (SOD, POD, and CAT) of sisal were measured by hydroponics with the addition of CdCl2·2.5H2O and different concentrations of Cd stress. Whole transcriptome sequencing (RNA-Seq) analysis was performed with lllumina sequencing technology, and qRT-PCR was conducted to verify the differential genes. The results obtained were as follows: (1) Short-term low concentration of Cd stress (20 mg/kg) had a transient promotion effect on the growth of sisal roots, but Cd showed a significant inhibitory effect on the growth of sisal roots over time. (2) Under different concentrations of Cd stress, the Cd content in sisal root was greater than that in sisal leaf, and Cd accumulated mainly in sisal roots. (3) With the increase of Cd stress concentration, the antioxidant enzyme catalase activity increased, peroxidase activity showed a decreasing trend, and superoxide dismutase showed a trend of increasing and then decreasing. (4) Transcriptome sequencing analysis detected 123 differentially expressed genes (DEGs), among which 85 genes were up-regulated and 38 genes were down-regulated. The DEGs were mainly concentrated in flavonoid biosynthesis and glutathione metabolism, and both processes had some regulatory effects on the Cd tolerance characteristics of sisal. This study elucidated the physiological, biochemical and transcriptomic responses of sisal under cadmium stress, and provided a theoretical basis for the ecological restoration function of sisal.

First Report of Nigrospora hainanensis Causing Leaf Blight on Brachiaria Griseb in China.

Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 µL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.

The Lesson Learned from the Unique Evolutionary Story of Avirulence Gene AvrPii of Magnaporthe oryzae.

Blast, caused by Magnaporthe oryzae, is one of the most destructive diseases affecting rice production. Understanding population dynamics of the pathogen's avirulence genes is pre-required for breeding and then deploying new cultivars carrying promising resistance genes. The divergence and population structure of AvrPii was dissected in the populations of southern (Guangdong, Hunan, and Guizhou) and northern (Jilin, Liaoning, and Heilongjiang) China, via population genetic and evolutionary approaches. The evolutionary divergence between a known haplotype AvrPii-J and a novel one AvrPii-C was demonstrated by haplotype-specific amplicon-based sequencing and genetic transformation. The different avirulent performances of a set of seven haplotype-chimeric mutants suggested that the integrity of the full-length gene structures is crucial to express functionality of individual haplotypes. All the four combinations of phenotypes/genotypes were detected in the three southern populations, and only two in the northern three, suggesting that genic diversity in the southern region was higher than those in the northern one. The population structure of the AvrPii family was shaped by balancing, purifying, and positive selection pressures in the Chinese populations. The AvrPii-J was recognized as the wild type that emerged before rice domestication. Considering higher frequencies of avirulent isolates were detected in Hunan, Guizhou, and Liaoning, the cognate resistance gene Pii could be continuously used as a basic and critical resistance resource in such regions. The unique population structures of the AvrPii family found in China have significant implications for understanding how the AvrPii family has kept an artful balance and purity among its members (haplotypes) those keenly interact with Pii under gene-for-gene relationships. The lesson learned from case studies on the AvrPii family is that much attention should be paid to haplotype divergence of target gene.

Development of a Specific Nested PCR Assay for the Detection of 16SrI Group Phytoplasmas Associated with Sisal Purple Leafroll Disease in Sisal Plants and Mealybugs.

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/µL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

The complete chloroplast genome of Coffea liberica (Gentianales: Rubiaceae).

Coffee is one of the most popular beverages around the world. As one of the best-known coffee species, Liberian coffee (Coffea liberica Bull ex Hiern 1876) has a high resistance to leaf rust, a devasting disease caused by Hemileia vastatrix. However, there are few reports on the systematic position and phylogenetic relationship of C. liberica at the chloroplast (cp) genome level. Thus, we successfully assembled its cp genome. The full length is 154,799 bp with a GC content of 37.48%. We have further annotated the cp genome and predicted 85 protein-coding genes together with 8 rRNAs and 37 tRNAs. Furthermore, a large single copy region (LSC), a small single copy region (SSC), an inverted repeat region a (IRa) and an inverted repeat region b (IRb) are identified with lengths of 84,868 bp, 18,121 bp, 25,905 bp and 25,905 bp, respectively. The phylogenetic tree indicates that C. liberica is closely related to C. canephora, which is consistent with a previous result obtained from genotyping-by-sequencing.

First Report of Paramyrothecium breviseta Causing Leaf Spot Disease of Coffea canephora in China.

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), ß-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), ß-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 ß-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, ß-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.

Genome-Wide Analysis of the PIN Auxin Efflux Carrier Gene Family in Coffee.

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.

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

De Novo Transcriptome Assembly of Agave H11648 by Illumina Sequencing and Identification of Cellulose Synthase Genes 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 × 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.

Transcriptome Comparison Reveals Distinct Selection Patterns in Domesticated and Wild Agave Species, the Important CAM Plants.

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.

Function and evolution of Magnaporthe oryzae avirulence gene AvrPib responding to the rice blast resistance gene Pib.

Magnaporthe oryzae (Mo) is the causative pathogen of the damaging disease rice blast. The effector gene AvrPib, which confers avirulence to host carrying resistance gene Pib, was isolated via map-based cloning. The gene encodes a 75-residue protein, which includes a signal peptide. Phenotyping and genotyping of 60 isolates from each of five geographically distinct Mo populations revealed that the frequency of virulent isolates, as well as the sequence diversity within the AvrPib gene increased from a low level in the far northeastern region of China to a much higher one in the southern region, indicating a process of host-driven selection. Resequencing of the AvrPiballele harbored by a set of 108 diverse isolates revealed that there were four pathoways, transposable element (TE) insertion (frequency 81.7%), segmental deletion (11.1%), complete absence (6.7%), and point mutation (0.6%), leading to loss of the avirulence function. The lack of any TE insertion in a sample of non-rice infecting Moisolates suggested that it occurred after the host specialization of Mo. Both the deletions and the functional point mutation were confined to the signal peptide. The reconstruction of 16 alleles confirmed seven functional nucleotide polymorphisms for the AvrPiballeles, which generated three distinct expression profiles.

A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight.

BACKGROUND: Rice is a major crop worldwide. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has become one of the most devastating diseases for rice. It has been clear that phosphorylation plays essential roles in plant disease resistance. However, the role of phosphorylation is poorly understood in rice-Xoo system. Here, we report the first study on large scale enrichment of phosphopeptides and identification of phosphosites in rice before and 24 h after Xoo infection. RESULTS: We have successfully identified 2367 and 2223 phosphosites on 1334 and 1297 representative proteins in 0 h and 24 h after Xoo infection, respectively. A total of 762 differentially phosphorylated proteins, including transcription factors, kinases, epi-genetic controlling factors and many well-known disease resistant proteins, are identified after Xoo infection suggesting that they may be functionally relevant to Xoo resistance. In particular, we found that phosphorylation/dephosphorylation might be a key switch turning on/off many epi-genetic controlling factors, including HDT701, in response to Xoo infection, suggesting that phosphorylation switch overriding the epi-genetic regulation may be a very universal model in the plant disease resistance pathway. CONCLUSIONS: The phosphosites identified in this study would be a big complementation to our current knowledge in the phosphorylation status and sites of rice proteins. This research represents a substantial advance in understanding the rice phosphoproteome as well as the mechanism of rice bacterial blight resistance.

Stepwise arms race between AvrPik and Pik alleles in the rice blast pathosystem.

A stepwise mutation that occurred in both pathogens and their respective hosts has played a seminal role in the co-evolutionary arms race evolution in diverse pathosystems. The process driven by rice blast AvrPik and Pik alleles was investigated through population genetic and evolutionary approaches. The genetic diversity of the non-signal domain of AvrPik was higher than that in its signal peptide domain. Positive selection for particular AvrPik alleles in the northeastern region of China was stronger than in the south. The perfect relationship between the functional lineages and AvrPik allele-specific pathotypes was established by ruling out the nonfunctional lineages derived from additional copies. Only four alleles conditioning stepwise pathotypes were detected in natural populations, which were likely created by only one evolutionary pathway with three recognizable mutation steps. Two non-stepwise pathotypes were determined by two blocks in a network constructed by all 16 possible alleles, indicating that a natural evolution process can be artificially changed by a combination of specific single-nucleotide polymorphisms. Assuming that AvrPik evolution has been largely driven by host selection, the co-evolutionary stepwise relationships between AvrPik and Pik was established. The experimental validation of stepwise mutation is required for the development of sustainable management strategies against plant disease.

The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast.

We report the isolation of Pi1, a gene conferring broad-spectrum resistance to rice blast (Magnaporthe oryzae). Using loss- and gain-of-function approaches, we demonstrate that Pi1 is an allele at the Pik locus. Like other alleles at this locus, Pi1 consists of two genes. A functional nucleotide polymorphism (FNP) was identified that allows differentiation of Pi1 from other Pik alleles and other non-Pik genes. A extensive germplasm survey using this FNP reveals that Pi1 is a rare allele in germplasm collections and one that has conferred durable resistance to a broad spectrum of pathogen isolates.