Comparative Genomics of 12 Strains of Erwinia amylovora Identifies a Pan-Genome with a Large Conserved Core

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1_Ea and a putative secondary metabolite pathway only present in Rubus-infecting strains.     

Detection and Partial Characterization of Milk vetch dwarf virus Isolates from Faba Bean (Vicia faba L.) in Yunnan Province, China

A virus disease of faba bean ( Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus . The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.     

EPG monitoring of the probing behaviour of the common brown leafhopper Orosius orientalis on artificial diet and selected host plants

The common brown leafhopper Orosius orientalis (Hemiptera: Cicadellidae) is a polyphagous vector of a range of economically important pathogens, including phytoplasmas and viruses, which infect a diverse range of crops. Studies on the plant penetration behaviour by O. orientalis were conducted using the electrical penetration graph (EPG) technique to assist in the characterisation of pathogen acquisition and transmission. EPG waveforms representing different probing activities were acquired from adult O. orientalis probing in planta, using two host species, tobacco Nicotiana tabacum and bean Phaseolus vulgaris, and in vitro using a simple sucrose-based artificial diet. Five waveforms (O1?CO5) were evident when O. orientalis fed on bean, whereas only four waveforms (O1?CO4) and three waveforms (O1?CO3) were observed when the leafhopper fed on tobacco and on the artificial diet, respectively. Both the mean duration of each waveform and waveform type differed markedly depending on the food substrate. Waveform O4 was not observed on the artificial diet and occurred relatively rarely on tobacco plants when compared with bean plants. Waveform O5 was only observed with leafhoppers probing on beans. The attributes of the waveforms and comparative analyses with previously published Hemipteran data are presented and discussed, but further characterisation studies will be needed to confirm our suggestions.     

Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of Erwinia amylovora

Comparative genomic analysis revealed differences in the lipopolysaccharide (LPS) biosynthesis gene cluster between the Rubus‐infecting strain ATCC BAA‐2158 and the Spiraeoideae‐infecting strain CFBP 1430 of Erwinia amylovora. These differences corroborate rpoB‐based phylogenetic clustering of E. amylovora into four different groups and enable the discrimination of Spiraeoideae‐ and Rubus‐infecting strains. The structure of the differences between the two groups supports the hypothesis that adaptation to Rubus spp. took place after species separation of E. amylovora and E. pyrifoliae that contrasts with a recently proposed scenario, based on CRISPR data, in which the shift to domesticated apple would have caused an evolutionary bottleneck in the Spiraeoideae‐infecting strains of E. amylovora which would be a much earlier event. In the core region of the LPS biosynthetic gene cluster, Spiraeoideae‐infecting strains encode three glycosyltransferases and an LPS ligase (Spiraeoideae‐type waaL), whereas Rubus‐infecting strains encode two glycosyltransferases and a different LPS ligase (Rubus‐type waaL). These coding domains share little to no homology at the amino acid level between Rubus‐ and Spiraeoideae‐infecting strains, and this genotypic difference was confirmed by polymerase chain reaction analysis of the associated DNA region in 31 Rubus‐ and Spiraeoideae‐infecting strains. The LPS biosynthesis gene cluster may thus be used as a molecular marker to distinguish between Rubus‐ and Spiraeoideae‐infecting strains of E. amylovora using primers designed in this study.     

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Seasonal activity and abundance of Orosius orientalis (Hemiptera: Cicadellidae) at agricultural sites in Southeastern Australia

Orosius orientalis is a leafhopper vector of several viruses and phytoplasmas affecting a broad range of agricultural crops. Sweep net, yellow pan trap and yellow sticky trap collection techniques were evaluated. Seasonal distribution of O. orientalis was surveyed over two successive growing seasons around the borders of commercially grown tobacco crops. Orosius orientalis seasonal activity as assessed using pan and sticky traps was characterised by a trimodal peak and relative abundance as assessed using sweep nets differed between field sites with peak activity occurring in spring and summer months. Yellow pan traps consistently trapped a higher number of O. orientalis than yellow sticky traps.     

Evolution of Chlorophyll Degradation: The Significance of RCC Reductase

Abstract: In angiosperms the key process of chlorophyll breakdown in senescing leaves is catalyzed by pheophorbide a oxygenase and RCC reductase which, in a metabolically channeled reaction, cleave the porphyrin macrocycle and produce a colourless primary catabolite, pFCC. RCC reductase is responsible for the reduction of the C20/C1 double bond of the intermediary catabolite, RCC. Depending on plant species, RCC reductase produces one of the two C1 stereoisomers, pFCC-1 or pFCC-2. Screening of a large number of taxa for the type of RCCR revealed that the isomer produced is uniform within families. It also revealed that type RCCR-2 is predominant; RCCR-1 seems to represent a recent derivation which in unrelated lineages has evolved independently from RCCR-2. A third type of pFCC was produced by RCCR from basal pteridophytes and some gymnosperms; its structure is unknown. Collectively, the data suggest that the pathway of chlorophyll breakdown is very conserved in vascular plants. RCCR appears to represent a decisive addition to the catabolic pathway: it allows terrestrial plants to metabolize the porphyrin part of the chlorophyll molecule to photodynamically inactive final products that are stored in the vacuoles of senescing mesophyll cells.     

Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato (Lycopersicon esculentum Mill.)

The gf tomato mutant, which retains chlorophyll during ripening, has been found to be affected in leaf senescence. The leaves of the gfmutant show an absolute stay-green phenotype. As leaf senescence and fruit ripening proceed, there is a marked difference in chlorophyll content between wild-type and gf. In both attached and detached leaf studies, or after treatment with ethylene, the leaves withered and abscised in gf with only slight loss of chlorophyll and carotenoids. Total protein content declined and free amino acids increased during leaf senescence in wild-type and gf, but Western analysis showed that LHCII polypeptides were retained at higher levels in gf. Expression of senescence-related mRNAs increased normally in gf whereas those for cab, rbcS and rbcL declined in both mutant and wild-type. The mutant possesses enzyme activity for chlorophyllase, the formation of phaeophorbide a by the action of Mg-dechelatase and the oxygenolytic opening of the porphyrin macrocycle. Analysis of chlorophyll breakdown products in fruit indicated that gf, like other stay-green mutants, accumulates chlorophyllides a and b, but phaeophorbide a does not accumulate in vivo. This may indicate that, in the mutant, in vivo the action of phaeophorbide a-oxygenase is somehow presented, either by altered accessibility or transport of components required for thylakoid disassembly or the absence of another factor.     

Genome sequence of an Erwinia amylovora strain with pathogenicity restricted to Rubus plants

Here, we present the genome of a strain of Erwinia amylovora, the fire blight pathogen, with pathogenicity restricted to Rubus spp. Comparative genomics of ATCC BAA-2158 with E. amylovora strains from non-Rubus hosts identified significant genetic differences but support the inclusion of this strain within the species E. amylovora.     

Virus disease in wheat predicted to increase with a changing climate

Current atmospheric CO₂ levels are about 400 μmol mol^?1 and are predicted to rise to 650 μmol mol^?1 later this century. Although the positive and negative impacts of CO₂ on plants are well documented, little is known about interactions with pests and diseases. If disease severity increases under future environmental conditions, then it becomes imperative to understand the impacts of pathogens on crop production in order to minimize crop losses and maximize food production. Barley yellow dwarf virus (BYDV) adversely affects the yield and quality of economically important crops including wheat, barley and oats. It is transmitted by numerous aphid species and causes a serious disease of cereal crops worldwide. This study examined the effects of ambient (aCO₂; 400 μmol mol^?1) and elevated CO₂ (eCO₂; 650 μmol mol^?1) on noninfected and BYDV‐infected wheat. Using a RT‐qPCR technique, we measured virus titre from aCO₂ and eCO₂ treatments. BYDV titre increased significantly by 36.8% in leaves of wheat grown under eCO₂ conditions compared to aCO₂. Plant growth parameters including height, tiller number, leaf area and biomass were generally higher in plants exposed to higher CO₂ levels but increased growth did not explain the increase in BYDV titre in these plants. High virus titre in plants has been shown to have a significant negative effect on plant yield and causes earlier and more pronounced symptom expression increasing the probability of virus spread by insects. The combination of these factors could negatively impact food production in Australia and worldwide under future climate conditions. This is the first quantitative evidence that BYDV titre increases in plants grown under elevated CO₂ levels.