Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Alcohol acyl transferase 1 links two distinct volatile pathways that produce esters and phenylpropenes in apple fruit

Fruit accumulate a diverse set of volatiles including esters and phenylpropenes. Volatile esters are synthesised via fatty acid degradation or from amino acid precursors, with the final step being catalysed by alcohol acyl transferases (AATs). Phenylpropenes are produced as a side branch of the general phenylpropanoid pathway. Major quantitative trait loci (QTLs) on apple (Malus × domestica) linkage group (LG)2 for production of the phenylpropene estragole and volatile esters (including 2‐methylbutyl acetate and hexyl acetate) both co‐located with the MdAAT1 gene. MdAAT1 has previously been shown to be required for volatile ester production in apple (Plant J., 2014, https://doi.org/10.1111/tpj.12518 ), and here we show it is also required to produce p‐hydroxycinnamyl acetates that serve as substrates for a bifunctional chavicol/eugenol synthase (MdoPhR5) in ripe apple fruit. Fruit from transgenic ‘Royal Gala’ MdAAT1 knockdown lines produced significantly reduced phenylpropene levels, whilst manipulation of the phenylpropanoid pathway using MdCHS (chalcone synthase) knockout and MdMYB10 over‐expression lines increased phenylpropene production. Transient expression of MdAAT1, MdoPhR5 and MdoOMT1 (O‐methyltransferase) genes reconstituted the apple pathway to estragole production in tobacco. AATs from ripe strawberry (SAAT1) and tomato (SlAAT1) fruit can also utilise p‐coumaryl and coniferyl alcohols, indicating that ripening‐related AATs are likely to link volatile ester and phenylpropene production in many different fruit.     

The O‐methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit

Phenylpropenes, such as eugenol and trans‐anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non‐producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co‐located with seven candidate genes for phenylpropene O‐methyltransferases (MdoOMT1–7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over‐expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.     

Detection and Identification of ‘Candidatus Phytoplasma prunorum’, ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma pyri’ in Stone Fruit Trees in Poland

A survey was made to determine the incidence of phytoplasmas in 39 sweet and sour cherry, peach, nectarine, apricot and plum commercial and experimental orchards in seven growing regions of Poland. Nested polymerase chain reaction (PCR) using the phytoplasma‐universal primer pairs P1/P7 followed by R16F2n/R16R2 showed the presence of phytoplasmas in 29 of 435 tested stone fruit trees. The random fragment length polymorphism (RFLP) patterns obtained after digestion of the nested PCR products separately with RsaI, AluI and SspI endonucleases indicated that selected Prunus spp. trees were infected by phytoplasmas belonging to three different subgroups of the apple proliferation group (16SrX‐A, ‐B, ‐C). Nucleotide sequence analysis of 16S rDNA fragment amplified with primers R16F2n/R16R2 confirmed the PCR/Restriction Fragment Length Polymorphism (RFLP) results and revealed that phytoplasma infecting sweet cherry cv. Regina (Reg), sour cherry cv. Sokowka (Sok), apricots cv. Early Orange (EO) and AI/5, Japanese plum cv. Ozark Premier (OzPr) and peach cv. Redhaven (RedH) was closely related to isolate European stone fruit yellows‐G1 of the ‘Candidatus Phytoplasma prunorum’ (16SrX‐B). Sequence and phylogenetic analyses resulted in the highest similarity of the 16S rDNA fragment of phytoplasma from nectarine cv. Super Queen (SQ) with the parallel sequence of the strain AP15 of the ‘Candidatus Phytoplasma mali’ (16SrX‐A). The phytoplasma infecting sweet cherry cv. Kordia (Kord) was most similar to the PD1 strain of the ‘Candidatus Phytoplasma pyri’ (16SrX‐C). This is the first report of the occurrence of ‘Ca. P. prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ in naturally infected stone fruit trees in Poland.     

Functional allelic diversity of the apple alcohol acyl-transferase gene <Emphasis Type="Italic">MdAAT1</Emphasis> associated with fruit ester volatile contents in apple cultivars

Flavour is an important key factor of apple (Malus × domestica Borkh.) fruit quality, and its improvement is an important but complex breeding goal. Acetate esters are quantitatively the most important volatile compounds in apple fruit, and only a few of them dominate the typical aroma of a cultivar. Alcohol acyl-transferase (AAT) is a key enzyme involved in the last step of ester biosynthesis. The aim of this study was to target single nucleotide polymorphisms (SNPs) in an AAT candidate gene genetically associated with ester quantitative trait loci (QTL), to enable functional marker development for marker-assisted apple breeding programs. The AAT gene inventory of apple was characterized by in-silico mining of the assembled Golden Delicious genome, and 17 putative AAT genes in total were defined. MdAAT1 located on chromosome 2 was selected as the main candidate gene associated with QTL for different acetate esters, and its allelic diversity was assessed by direct amplicon sequencing in a collection of 102 apple cultivars characterized for ester volatile profiles. Sequencing a 468 bp nucleotide sequence of the MdAAT1 coding region resulted in the detection of four SNPs. In total, 18 different SNP haplotypes/heterozygous patterns were generated from the four SNPs identified within the apple collection. Association analyses resulted in highly significant associations of both individual SNPs and distinct haplotypes with the content of four acetate esters, including hexyl acetate, butyl acetate and 2-methyl-butyl acetate. About a third (31) of the 102 apple cultivars possessed the specific MdAAT1 haplotype H1 (C-A-C-A) and were characterized by strongly decreased ester concentrations. The contrasting H8 haplotype (T-G-T-G) was found in 28 varieties but was associated with normal to elevated ester concentrations. The observed association suggests a putative causal functional relationship between MdAAT1 and production of key apple esters.     

‘Cylindrocarpon’ and Ilyonectria Species Causing Root and Crown Rot Disease of Potted Laurustinus Plants in Italy

During the 2012 and 2013 growing seasons, a disease was detected on potted laurustinus (Viburnum tinus) plants in two nurseries located in the Catania province (eastern Sicily, Italy). ‘Cylindrocarpon’‐like species were consistently recovered from crown rot and stem rot tissues. Based on morphological characteristics, DNA sequencing and phylogenetic analysis of β‐tubulin (TUB), histone H3 (HIS3) and translation elongation factor‐1α (TEF‐1 α) gene sequences, the fungi associated with symptomatic tissues were identified as ‘Cylindrocarpon’ pauciseptatum, Ilyonectria novozelandica and I. torresensis. Koch's postulates were fulfilled by pathogenicity tests carried out on potted V. tinus cuttings. To our knowledge, this is the first report worldwide of ‘C.’ pauciseptatum, I. novozelandica and I. torresensis causing disease on V. tinus.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red‐leaf Disease of Salvia miltiorrhiza in China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma‐specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN‐1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII‐A), respectively. The phylogenetic trees showed that LN‐1 phytoplasma clustered with the members of 16SrXII‐A group, including Ca. P. solani. Computer‐simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.     

‘Collar propagation’ as an alternative propagation method for rhizomatous miscanthus

The demand for perennial nonfood crops, such as miscanthus, is increasing steadily, as fossil resources are replaced by biomass. However, as the establishment of miscanthus is very expensive, its cultivation area in Europe is still small. The most common propagation method for miscanthus is via rhizomes, the harvesting of which is very labour‐intensive. Seed propagation is promising, but not suitable for sterile genotypes. In this study, a new vegetative propagation method, ‘collar propagation’, was tested in field and controlled environment studies. Collars are built at the junction between rhizome and stem. They can be harvested in a less destructive way than rhizomes by pulling out the stems from winter‐dormant miscanthus plants. One genotype of each of the species M. sacchariflorus, M. × giganteus, M. sinensis in combination with three fragment types (collars, rhizomes, collars + rhizomes) were tested for establishment success and plant performance. The performance (e.g. dry matter yield) of collar‐propagated plants was either better than or not significantly different from rhizome‐propagated plants. Pregrown plantlets transplanted into the field showed no significant differences in establishment success between the fragments within a genotype. When directly planted into the field however, the fragment ‘rhizome+collar’ had a significantly better establishment success than the other two. The winter survival rate of the fragment ‘rhizome+collar’ was 70% for M. sacchariflorus and 75% for M. × giganteus. Emergence success from collar‐derived plants was not affected by harvest date (harvested monthly from November to February). This study showed that miscanthus propagation via collars is feasible and a promising alternative to rhizome propagation, as the multiplication rate of collars is comparable to that of rhizome propagation. Collar propagation is the more suitable method for the tested genotypes of the species M. sachariflorus and M. × giganteus, but not for M. sinensis genotypes, which may be better propagated by seeds.     

Novel evidence suggests that a ‘Rickettsia felis‐like’ organism is an endosymbiont of the desert flea,Xenopsylla ramesis

Fleas are acknowledged vectors and reservoirs of various bacteria that present a wide range of pathogenicity. In this study, fleas collected from wild rodents from the Negev desert in southern Israel were tested for RickettsiaDNA by targeting the 16S rRNA (rrs) gene. Thirty‐eight Xenopsylla ramesis, 91 Synosternus cleopatrae and 15 Leptopsylla flea pools (a total of 568 fleas) were screened. RickettsiaDNA was detected in 100% of the X. ramesis and in one S. cleopatrae flea pools. None of L. algira flea pools was found positive. All positive flea pools were further characterized by sequencing of five additional genetic loci (gltA, ompB, ompA, htrA and fusA). The molecular identification of the positive samples showed all sequences to be closely related to the ‘Rickettsia felis‐like’ organisms (99–100% similarities in the six loci). To further investigate the association between ‘R. felis‐like’ and X. ramesis fleas, ten additional single X. ramesis adult fleas collected from the wild and five laboratory‐maintained X. ramesis imago, five larva pools (2–18 larvae per pool) and two egg pools (18 eggs per pool) were tested for the presence of ‘R. felis‐like’ DNA. All samples were found positive by a specific ompAPCR assay, confirming the close association of this Rickettsia species with X. ramesis in all its life stages. These results suggest a symbiotic association between ‘Rickettsia felis‐like’ and X. ramesis fleas.     

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.     

Molecular identification of diverse ‘Candidatus Phytoplasma’ species associated with grapevine decline in Iran

Grapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.     

Dynamic trajectories of volatile and non‐volatile specialised metabolites in ‘overnight’ fragrant flowers of Murraya paniculata

• Ephemeral flowers, especially nocturnal ones, usually emit characteristic scent profiles within their post‐anthesis lifespans of a few hours. Whether these flowers exhibit temporal variability in the composition and profile of volatile and non‐volatile specialised metabolites has received little attention.
• Flowers of Murraya paniculata bloom in the evenings during the summer and monsoon, and their sweet, intense fragrance enhances the plant's value as an ornamental. We aimed to investigate profiles of both volatile and non‐volatile endogenous specialised metabolites (ESM) in nocturnal ephemeral flowers of M. paniculata to examine whether any biochemically diverse groups of ESM follow distinct patterns of accumulation while maintaining synchrony with defensive physiological functions.
• Targeted ESM contents of M. paniculata flowers were profiled at ten time points at 2‐h intervals, starting from late bud stage (afternoon) up to the start of petal senescence (mid‐morning). Emitted volatiles were monitored continuously within the whole 20‐h period using headspace sampling. The ESM contents were mapped by time point to obtain a highly dynamic and biochemically diverse profile. Relative temporal patterns of ESM accumulation indicated that the active fragrance‐emitting period might be divided into ‘early bloom’, ‘mid‐bloom’ and ‘late bloom’ phases. Early and late bloom phases were characterised by high free radical generation, with immediate enhancement of antioxidant enzymes and phenolic compounds. The mid‐bloom phase was relatively stable and dedicated to maximum fragrance emission, with provision for strong terpenoid‐mediated defence against herbivores. The late bloom phase merged into senescence with the start of daylight; however, even the senescent petals continued to emit fragrance to attract diurnal pollinators.
• Our study suggests that dynamic relations between the different ESM groups regulate the short‐term requirements of floral advertisement and phytochemical defence in this ephemeral flower. This study also provided fundamental information on the temporal occurrence of emitted volatiles and internal pools of specialised metabolites in M. paniculata flowers, which could serve as an important model for pollination biology of Rutaceae, which includes many important fruit crops.

     

The high‐quality genome of Brassica napus cultivar ‘ZS11’ reveals the introgression history in semi‐winter morphotype

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi‐winter oilseed rape cultivar ‘ZS11’ and its comprehensive genomic comparison with the genomes of the winter‐type cultivar ‘Darmor‐bzh’ as well as two progenitors. The integrated BAC‐to‐BAC and whole‐genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high‐quality genome assembly of B. napus ‘ZS11’. Within a short evolutionary period (~6700 years ago), semi‐winter‐type ‘ZS11’ and the winter‐type ‘Darmor‐bzh’ maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to ‘Darmor‐bzh’, both two subgenomes of ‘ZS11’ are closely related to its progenitors, and the ‘ZS11’ genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi‐winter‐type ‘ZS11’ underwent potential genomic introgressions with B. rapa (A_r). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization‐responsive flowering time in ‘ZS11’ was first experienced HE, and then underwent genomic introgression event with A_r, which potentially has led to genetic differences in controlling vernalization in the semi‐winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi‐winter oilseed rape in Asia.     

Testing parasite ‘intimacy’: the whipworm Trichuris muris in the European house mouse hybrid zone

Host‐parasite interaction studies across hybrid zones often focus on host genetic variation, treating parasites as homogeneous. ‘Intimately’ associated hosts and parasites might be expected to show similar patterns of genetic structure. In the literature, factors such as no intermediate host and no free‐living stage have been proposed as ‘intimacy’ factors likely constraining parasites to closely follow the evolutionary history of their hosts. To test whether the whipworm, Trichuris muris, is intimately associated with its house mouse host, we studied its population genetics across the European house mouse hybrid zone (HMHZ) which has a strong central barrier to gene flow between mouse taxa. T. muris has a direct life cycle and nonmobile free stage: if these traits constrain the parasite to an intimate association with its host we expect a geographic break in the parasite genetic structure across the HMHZ. We genotyped 205 worms from 56 localities across the HMHZ and additionally T. muris collected from sympatric woodmice (Apodemus spp.) and allopatric murine species, using mt‐COX1, ITS1‐5.8S‐ITS2 rDNA and 10 microsatellites. We show four haplogroups of mt‐COX1 and three clear ITS1‐5.8S‐ITS2 clades in the HMHZ suggesting a complex demographic/phylogeographic history. Microsatellites show strong structure between groups of localities. However, no marker type shows a break across the HMHZ. Whipworms from Apodemus in the HMHZ cluster, and share mitochondrial haplotypes, with those from house mice. We conclude Trichuris should not be regarded as an ‘intimate’ parasite of the house mouse: while its life history might suggest intimacy, passage through alternate hosts is sufficiently common to erase signal of genetic structure associated with any particular host taxon.     

Winter hardiness of Miscanthus (I): Overwintering ability and yield of new Miscanthus ×giganteus genotypes in Illinois and Arkansas

Miscanthus ×giganteus (M×g) is an important bioenergy feedstock crop. However, biomass production of Miscanthus has been largely limited to one sterile triploid cultivar, M×g ‘1993‐1780’, which we demonstrate can have insufficient overwintering ability in temperate regions with cold winters. Key objectives for Miscanthus breeding include greater biomass yield and better adaptation to different production environments than M×g ‘1993‐1780’. In this study, we evaluated 13 M×g genotypes, including ‘1993‐1780’, in replicated field trials conducted for three years at Urbana, IL; Dixon Springs, IL; and Jonesboro, AR. Entries were phenotyped for first‐winter overwintering ability and plant hardiness (ratio of new tillers to old), yield in years 2 and 3, and first heading date, plant height, and culm number in years 1 and 2. We observed substantial variation for overwintering ability and biomass yield among the M×g genotypes tested and identified ones with better overwintering ability and/or higher biomass yield than ‘1993‐1780’. Most entries at Urbana were damaged during the first winter, whereas few or no entries were damaged at Dixon Springs or Jonesboro. However, M×g ‘Nagara’ was entirely undamaged during the first winter and produced high biomass yields at Urbana (19.7 Mg/ha in year 2 and 20.9 Mg/ha in year 3), whereas M×g ‘1993‐1780’ exhibited an overwintering loss of 29%, had severely damaged survivors (hardiness score of 25%), and reduced biomass yield (8.1 Mg/ha in year 2 and 16.2 Mg/ha in year 3), indicating that M×g ‘Nagara’ could be a better choice in hardiness zone 5 (average annual minimum air temperature of ?23.3 to ?28.9°C) or lower. In Dixon Springs, where M×g ‘1993‐1780’ was undamaged by the first winter, it yielded highest among all the entries (21.6 Mg/ha in year 3), though not significantly higher than M×g ‘Nagara’ (18.2 Mg/ha in year 3).     

Genome of ‘Charleston Gray’, the principal American watermelon cultivar,and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection

Years of selection for desirable fruit quality traits in dessert watermelon (Citrullus lanatus) has resulted in a narrow genetic base in modern cultivars. Development of novel genomic and genetic resources offers great potential to expand genetic diversity and improve important traits in watermelon. Here, we report a high‐quality genome sequence of watermelon cultivar ‘Charleston Gray’, a principal American dessert watermelon, to complement the existing reference genome from ‘97103’, an East Asian cultivar. Comparative analyses between genomes of ‘Charleston Gray’ and ‘97103’ revealed genomic variants that may underlie phenotypic differences between the two cultivars. We then genotyped 1365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping‐by‐sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25 000 high‐quality single nucleotide polymorphisms (SNPs) were derived from the GBS data using the ‘Charleston Gray’ genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome‐wide association studies. The high‐quality ‘Charleston Gray’ genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.     

‘Fukusensor:’ a genetically engineered plant for reporting DNA damage in response to gamma radiation

Transgenic plants can be designed to be ‘phytosensors’ for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)‐transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co‐60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.     

Non‐host plant essential oil volatiles with potential for a ‘push‐pull’ strategy to control the tea green leafhopper,Empoasca vitis

The tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), is an economically important pest of tea crops, Camellia sinensis (L.) O. Kuntze (Theaceae), in China. The use of non‐host plant essential oils for manipulation of E. vitis was investigated for potential incorporation into a ‘push‐pull’ control strategy for this pest. The effectiveness of 14 plant essential oils in repelling E. vitis was investigated in laboratory assays. Rosemary oil, geranium oil, lavender oil, cinnamon oil, and basil oil repelled leafhoppers in a Y‐shaped olfactometer. We also compared the efficacy of these five plant essential oils to repel E. vitis in the presence of a host plant volatile‐based leafhopper attractant, (Z)‐3‐hexenyl acetate, in a tea plantation. In the treatment combination, four plates (north, south, east, and west) treated with an essential oil surrounded a central sticky plate treated with (Z)‐3‐hexenyl acetate. Fewer E. vitis were found on the plates treated with rosemary oil (12.5% reduction) than on the four water‐sprayed control treatment plates surrounding a central plate with (Z)‐3‐hexenyl acetate. We compared the distribution of E. vitis on the plates, and the relative numbers of E. vitis on each plate were compared with similar plates in the control treatment. When four plates treated with rosemary oil surrounded a central (Z)‐3‐hexenyl acetate‐treated plate, the distribution of E. vitis on the different plates changed significantly compared with that of the control. Relatively fewer E. vitis were found on the east (13.0% reduction) rosemary oil‐treated plates and more E. vitis (11.3% increase) were found on the central attractant‐treated plate. Our findings indicate that rosemary oil is a promising leafhopper repellent that should be tested further in a ‘push‐pull’ strategy for control of E. vitis.