Identification and antifungal activity analysis of two biocontrol antagonists to Colletotrichum musae

Postharvest anthracnose of banana caused by Colletotrichum musae is one of the major diseases resulting in huge economic losses worldwide. To control this disease using biocontrol agents, two antagonistic strains SD7 and NB20 with significant inhibitory effects on mycelial growth and conidial germination of C. musae were identified and evaluated in this study. The inhibitory effects of cell‐free culture filtrates of SD7 and NB20 on conidial germination of C. musae were both 100%, and those on mycelial growth of C. musae were 97.7 ± 0.9% and 95.0 ± 0.6%, respectively. The antifungal activities of cell‐free culture filtrates of both strains were still stable after they were stored at 4°C for 6 months. The control efficacies of cell‐free culture filtrates of SD7 and NB20 on postharvest anthracnose of banana were 55.9 ± 4.1% and 33.2 ± 3.9%, respectively. The disease severity (mean scale value) in banana fruit fingers was significantly lower after the treatment with a cultural suspension of the bacterial strain SD7 (1.4 ± 0.49) or actinomycete strain NB20 (2.0 ± 0.63), compared to that in the control (4.8 ± 0.40). After subculturing for 10 generations, the antifungal efficiency of NB20 remained stable, whereas that of strain SD7 declined obviously. Lastly, based on the morphological, physio‐biochemical and molecular characteristics, the bacterial strain SD7 was identified as Burkholderia cepacia, while the actinomycete strain NB20 was identified as Streptomyces katrae. The results from this study will provide the basis for developing an effective and novel biofungicide to control banana anthracnose disease.     

Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

The complete nucleotide sequence of an Indian isolate of Apple chlorotic leaf spot virus (ACLSV) was determined and found to be 7,525 nt in length. The genome organization was similar to known isolates of ACLSV, encoding three ORFs. Comparisons indicated high sequence variability among known isolates with overall nucleotide sequence identities of 80 to 84%. A striking variable region was identified among the replicase protein upstream of the RNA‐dependent RNA polymerase (aa 1510–1590), which showed a 41–43% match with the corresponding region in other isolates. Phylogenetic analysis at the nucleotide level clustered the isolates into three groups, without any relation to geographical origin. Recombination analysis showed that the isolate is a recombinant with recombination sites spread throughout the genome, especially in the polymerase gene region (nt 4700–5400). Most recombination sites were bordered by an upstream region (5′) of GC‐rich and downstream region (3′) of AU‐rich sequences of similar length. Correlation of recombination site with host type is discussed, and it was found that there were more interlineage recombinations in the apple host compared with intralineage recombinations.     

Genetic Diversities of Cymbidium mosaic virus and Odontoglossum ringspot virus Isolates Based on the Coat Protein Genes from Orchids in Guangdong Province,China

Orchids are some of the most important ornamental flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 CymMV and 28 ORSV isolates were selected for genetic diversity analysis. The CymMV isolates shared 84.6–100% and 89.5–100% identities of coat protein (CP) at the nucleotide (nt) and amino acid (aa) levels, respectively. The identities of ORSV isolates were 96.4–100% (nt) and 92.5–99.4% (aa). The CP genes of CymMV were found to have genetic diversity, and the CP genes of ORSV were genetically conservative. These results can aid in designing effective disease‐control strategies.     

Papaya Leaf Curl Guangdong Virus and Ageratum Yellow Vein Virus Associated with Leaf Curl Disease of Tobacco in China

Two samples (YC7, YC27) of Nicotiana tabacum showing leaf curling, vein swelling and enations on undersides of leaves were collected in the Fujian Province of China in 2007. Virus isolates YC7‐1 and YC7‐2 (associated with betasatellite, YC7‐2β) were detected in both samples. The complete DNA‐A sequence of YC7‐1 (FJ869907) comprised 2741 nucleotides (nt). The complete DNA‐A (FJ869908) and betasatellite (FJ869909) sequence of YC7‐2 consisted of 2754 and 1344 nt, respectively. YC7‐1 had the highest nucleotide sequence identity (97.3%) with Papaya leaf curl Guangdong virus (PaLCuGuV‐[CN:Gd2:02], AJ558122). YC7‐2 had the highest sequence identity (90.1%) with Ageratum yellow vein virus (AYVV‐TW[TW:Tai:99], AF307861) and its betasatellite (96.5%) with Ageratum yellow vein betasatellite (AYVB‐[TW:CHu:02], AJ542495). These indicate that YC7‐1 and YC7‐2 are isolates of PaLCuGuV and AYVV, respectively. Symptoms including leaf curling, vein swelling and enations on undersides of leaves were observed in N. tabacum and N. glutinosa when infected by whiteflies with sample YC7 as the viral source under greenhouse conditions. PCR results showed that these infected plants contained both YC7‐1 and YC7‐2/YC7‐2β. To our knowledge, this is the first report of PaLCuGuV and AYVV/AYVB co‐infecting N. tabacum in China.     

Genome Characteristics,Phylogeny and Varying Host Specificity of Polish Kra and Ros Isolates of Tomato torrado virus

The genome sequences of two Polish Kra and Ros isolates of Tomato torrado virus (ToTV) were determined and compared with data of previously described ToTV isolates and other Torradovirus members. Whole‐genome sequence comparisons revealed 97.0–99.6% nucleotide sequence identities and close relatedness, with other known ToTV isolates. The high homology between Kra, Ros and Wal'03 ToTVs is likely responsible for the similar symptoms observed on infected plants. However, the symptoms differed in intensity and various host specificity. We report that Kra ToTV caused a milder expression of symptoms on Solanum tuberosum than Wal'03. We hypothesize this may be a result of the significant variability observed within the 3′‐UTR of RNA1 of Kra as well as of Ros ToTV isolates. In the light of this fact, potato may be considered an indicator plant for distinguishing Kra and Wal'03 ToTV isolates.     

The Complete Nucleotide Sequence of the RNA 1 of a Chinese Isolate of Tomato chlorosis virus

Interveinal leaf chlorosis, brittleness, limited necrotic flecking or bronzing developed on greenhouse‐grown tobacco and tomato plants at Nanjing Agricultural University from 2010 to 2013. A positive RT‐PCR using a pair of degenerate primers for Crinivirus confirmed the diseased plants were infected with Tomato chlorosis virus (ToCV). The complete RNA 1 genomic sequence of this ToCV isolate was determined; it comprises of 8596 nucleotides with four open reading frames. Phylogenetic analysis of ToCV isolates from diverse geographical regions categorized the ToCV isolates into two main groups. Group one consisted of Chinese, American‐Florida, Greek and Brazilian isolates, while Group two contained only the Spanish isolate. The first group had two subgroups, one of Chinese and American‐Florida isolates, while the other subgroup had Greek and Brazilian isolates. This is the first study of the complete nucleotide sequence of the RNA 1 of ToCV isolated from China.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Plum Pox Virus in Japanese Plum from Argentina: Serological Detection and Molecular Characterization of an Isolate from cv. Red Beauty

A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina.     

Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

Characterization of Bacillus altitudinis as a New Causative Agent of Bacterial Soft Rot

A total of 67 bacterial isolates were obtained from apple and pear fruits with signs of soft rot collected from Egyptian markets. Pathogenicity tests showed that 25 isolates (37%) were pathogenic to apple and pear fruits, with considerable variation of virulence. Among these isolates, 16 (64%) were Gram‐positive, motile, spore‐forming long rods and were identified as members of the genus Bacillus based on an API test. In addition, five isolates (20%) were Gram‐negative, non‐spore‐forming, motile, oxidase and catalase‐positive short rods and were identified as members of the genus Pseudomonas. Furthermore, four isolates (16%) were Gram‐negative, non‐spore‐forming, motile, catalase‐positive and oxidase negative short rods and were identified as belonging to the genus Erwinia. All selected isolates showed a wide host range and could cause soft rot of all representative fruits and vegetables tested. The three most virulent isolates, AB4, AB6 and PB6, exhibited the highest soft rot severity on different apple and pear cultivars, and apple cv. Anna (116) was the most susceptible to infection by isolates AB4 and AB6, with soft rot severities of 63.33 and 60.67%, respectively. Also, pear cv. Le‐Conte was most susceptible to infection by isolate AB6, with a soft rot severity of 89.9%. A phylogenetic tree based on 16S rRNA gene sequences indicated that strains AB4 and AB6 were very similar to one another and also showed a similarity of 99% to Bacillus altitudinis, and strain PB6 revealed a similarity of 99% to Bacillus pumilus. To our knowledge, this is the first report of B. altitudinis as a soft rot pathogen for both apple and pear fruits.     

Nucleotide‐binding resistance gene signatures in sugar beet,insights from a new reference genome

Nucleotide‐binding (NB‐ARC), leucine‐rich‐repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long‐read sequencing, are improving resolution of repeat‐rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB‐ARC domain as a model, 231 tentative NB‐ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB‐ARC signatures as well as full‐length sequences. The NB‐ARC‐associated proteins contained NLR resistance gene domains, including TIR, CC and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB‐ARC domains to validated R‐genes from monocots and eudicots suggested extensive Beta vulgaris‐specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR‐like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic‐acid‐based method for NLR prediction in non‐model plant species.     

Identification and Characterization of the Causal Agent of Gummy Stem Blight from Muskmelon and Watermelon in East China

Gummy stem blight (GSB) is one of the most destructive foliar diseases of cucurbits, worldwide. To identify and characterize the pathogen which causes GSB on watermelon and muskmelon in East China, morphological characteristics, pathogenicity assays as well as sequence characterization of the rDNA internal transcribed spacer (ITS) were performed on 41 isolates collected from Jiangsu, Zhejiang, Anhui and Jiangxi provinces. The mycelia of all the isolates were white on top and olivaceous green to black with concentric circles at the bottom on potato‐dextrose agar medium. The isolates differed significantly on aggressiveness based on pathogenicity assays. rDNA‐ITS sequences and phylogenetic analysis confirmed the isolates as Didymella bryoniae. The isolates were found to be highly identical with the exception of 13 isolates, which had a guanine substitution instead of adenine at position 131 of the ITS.     

Genetic Diversity and Pathogenic Variability of Colletotrichum truncatum Causing Anthracnose of Pepper in Malaysia

Colletotrichum truncatum was initially described from pepper and has been reported to infect 180 host genera in 55 plant families worldwide. Samples were collected from pepper plants showing typical anthracnose symptoms. Diseased samples after isolation were identified as C. truncatum based on morphological characters and ITS‐rDNA and β‐tubulin sequence data. Intersimple sequence repeat (ISSR) markers were used to estimate genetic diversity in C. truncatum from Malaysia. A set of 3 ISSR primers revealed a total 26 allele from the amplified products. Cluster analysis with UPGMA method clustered C. truncatum isolates into two main groups, which differed with a distance of 0.64. However, the genetic diversity of C. truncatum isolates showed correlation between genetic and geographical distribution, but it failed to reveal a relationship between clustering and pathogenic variability. Phylogenetic analyses discriminated the C. truncatum isolates from other reference Colletotrichum species derived from GenBank. Among the morphological characters, shape, colour of colony and growth rate in culture were partially correlated with the ISSR and phylogenetic grouping. Pathogenicity tests revealed that C. truncatum isolates were causal agents for pepper anthracnose. In the cross‐inoculation assays, C. truncatum isolates were able to produce anthracnose symptoms on tomato, eggplant, onion, lettuce and cabbage. A pathogenicity and cross‐inoculation studies indicated the potential of C. truncatum for virulence and dominancy on plant resistance.     

Genetic Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

A missense mutation in CHS1, a TIR‐NB protein,induces chilling sensitivity in Arabidopsis

Low temperature is an environmental factor that affects plant growth and development and plant–pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling‐sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling‐sensitive phenotype, and also displayed defense‐associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map‐based cloning analysis revealed that CHS1 encodes a TIR‐NB‐type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1–conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR‐NB‐type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR‐NB protein in Arabidopsis.     

Directed dimerization: an in vivo expression system for functional studies of type II phytochromes

Type II phytochromes (phy) in Arabidopsis form homodimers and heterodimers, resulting in a diverse collection of light‐stable red/far‐red (R/FR) sensing photoreceptors. We describe an in vivo protein engineering system and its use in characterizing the activities of these molecules. Using a phyB null mutant background, singly and doubly transgenic plants were generated that express fusion proteins containing the phyB–phyE N–terminal photosensory regions (NB–NE PSRs), a nuclear localization sequence, and small yeast protein domains that mediate either homodimerization or heterodimerization. Activity of NB/NB homodimers but not monomeric NB subunits in control of seedling and adult plant responses to R light is demonstrated. Heterodimers of the NB sequence with the chromophoreless NB^C357S sequence, which mimic phyB Pfr/Pr photo‐heterodimers, mediate R sensitivity in leaves and petioles but not hypocotyls. Homodimerization of the NC, ND and NE sequences and directed heterodimerization of these photosensory regions with the NB region reveal form‐specific R‐induced activities for different type II phy dimers. The experimental approach developed here of directed assembly of defined protein dimer combinations in vivo may be applicable to other systems.