Battling the biotypes of balsam: the biological control of Impatiens glandulifera using the rust fungus Puccinia komarovii var. glanduliferae in GB.

Impatiens glandulifera, or Himalayan balsam, is a prolific invader of riverine habitats. Introduced from the Himalayas for ornamental purposes in 1839, this annual species has naturalised across Great Britain (GB) forming dense monocultures with negative affects across whole ecosystems. In 2006 a programme exploring biocontrol as an alternative control method was initiated and to date, two strains of the rust fungus Puccinia komarovii var. glanduliferae have been released. To better understand the observed differences in susceptibility of GB Himalayan balsam stands to the two rust strains, inoculation studies were conducted using urediniospores and basidiospores. Experiments revealed large variation in the susceptibility of stands to urediniospores of the two rust strains, with some resistant to both. Furthermore, the infectivity of basidiospores was found to differ, with some stands fully susceptible to the urediniospore stage, being immune to basidiospore infection. Therefore, before further rust releases at new sites, it is necessary to ensure complete compatibility of the invasive stands with both urediniospores and basidiospores. However, for successful control across GB it is essential that plant biotypes are matched to the most virulent rust strains. This will involve additional strains from the native range to tackle those biotypes resistant to the strains currently released.

Chloroplast DNA analysis of the invasive weed, Himalayan balsam (Impatiens glandulifera), in the British Isles.

Impatiens glandulifera or Himalayan balsam (HB), is an invasive alien weed throughout the British Isles (BI). Classical biological control of HB in the BI using a rust fungus from the Himalayan native range was implemented in 2014. However, not all HB populations are susceptible to the two rust strains currently released. Additional strains are needed that infect resistant populations in order to achieve successful control. These are best sourced from the historical collecting sites. A molecular analysis was conducted using six chloroplast DNA sequences from leaf material from across the BI and the native range. Herbarium samples collected in the Himalayas between 1881 and 1956 were also included. Phylogenetic analyses resulted in the separation of two distinct groups, one containing samples from the BI and the native range, and the other from the BI only; suggesting that HB was introduced into the BI on at least two occasions. The former group is composed of two subgroups, indicating a third introduction. Ten and 15 haplotypes were found in the introduced and native range respectively, and with two of these found in both regions. Results show where to focus future surveys in the native range to find more compatible rust strains.

The oospore stage of Plasmopara obducens, impatiens downy mildew.

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.

Comparative Analysis of Impatiens Leaf Transcriptomes Reveal Candidate Genes for Resistance to Downy Mildew Caused by Plasmopara obducens.

Impatiens downy mildew (IDM) is a devastating disease to garden impatiens. A good understanding of IDM resistance in New Guinea impatiens is essential for improving garden impatiens resistance to this disease. The present study was conducted to sequence, assemble, annotate and compare the leaf transcriptomes of two impatiens cultivars differing in resistance to IDM, reveal sequence polymorphisms and identify candidate genes for IDM resistance. RNA-Seq was performed on cultivars Super Elfin® XP Pink (SEP) and SunPatiens® Compact Royal Magenta (SPR). De novo assembly of obtained sequence reads resulted in 121,497 unigenes with an average length of 1156 nucleotides and N50 length of 1778 nucleotides. Searching the non-redundant protein and non-redundant nucleotide, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes and Clusters of Orthologous Groups and Gene Ontology databases, resulted in annotation of 57.7% to 73.6% of the unigenes. Fifteen unigenes were highly similar to disease resistance genes and more abundant in the IDM-resistant cultivar than in the susceptible cultivar. A total of 22,484 simple sequence repeats (SSRs) and 245,936 and 120,073 single nucleotide polymorphisms (SNPs) were identified from SPR and SEP respectively. The assembled transcripts and unigenes, identified disease resistance genes and SSRs and SNPs sites will be a valuable resource for improving impatiens and its IDM resistance.

Transstadial transmission of Pythium in Bradysia impatiens and lack of adult vectoring capacity.

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

BACKGROUND: Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. RESULTS: In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. CONCLUSION: We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues.

Arbuscular mycorrhizal fungi protect a native plant from allelopathic effects of an invader.

The allelopathic potential of the Eurasian invasive plant Alliaria petiolata has been well documented, with the bulk of the effects believed to be mediated by arbuscular mycorrhizal fungi (AMF). We exposed the herbaceous annual Impatiens pallida, which is native to North America, to fractionated A. petiolata extracts at four developmental stages (germination, presymbiosis growth, symbiosis formation, and symbiosis growth) by using exposure levels expected to be similar to field levels. Surprisingly, we found strong direct effects on I. pallida germination and growth, but no indirect effects on I. pallida growth mediated by AMF. We also observed strong synergistic effects with a complete A. petiolata extract that inhibited I. pallida germination and presymbiosis root growth more than either a glucosinolate or flavonoid enriched fraction alone. In fact, the flavonoid enriched fraction tended to stimulate germination and presymbiosis root growth. In contrast to these strong direct effects, I. pallida plant growth during both the symbiosis formation and symbiosis growth phases was unaffected by A. petiolata extracts. We also found no inhibition of AMF colonization of roots or soils by A. petiolata extracts. We show that AMF can actually ameliorate allelopathic effects of an invasive plant, and suggest that previously observed allelopathic effects of A. petiolata may be due to direct inhibition of plant and fungal growth before symbiosis formation.

Endophytic fungi diversity of aquatic/riparian plants and their antifungal activity in vitro.

Two hundred and fourteen endophytic fungi were isolated from 500 segments of aquatic/riparian plants Ottelia acuminata, Myriophyllum verticillatum, Equisetum arvense, Cardamine multijuga, and Impatiens chinensis. They were identified to 31 taxa in which Cladosporium, Fusarium, and Geotrichum were the dominant genera. Among all isolates, 169 (79%) were anamorphic fungi, 1 (0.5%) was an teleomorphic ascomycete and 44 (21%) were sterile mycelia. There were significant differences in the colonization frequency of endophytes between the five plant species (X~2=51.128, P<0.001, Chi-square test). The riparian plants harboured more endophytes than the submerged plants. The antifungal activity of these isolates against Fusarium solani and Phytophthora nicotianae in vitro were tested and 28 (13.1%) isolates showed antifungal activities with more than 30% growth inhibition rate against the two pathogens.

The mixed mating system of Impatiens capensis and infection by a foliar rust pathogen: patterns of resistance and fitness consequences.

Outcrossing by hosts may offer protection from natural enemies adapted to parental genotypes by creating diverse progeny that differ from their parents through genetic recombination. However, past experimental work addressing the relationship between mating system and disease in offspring has given conflicting results, suggesting that outcrossing might also cause the dissolution of resistant genotypes. To determine if selfed progeny are more susceptible to disease caused by the heteroecious rust, Puccinia recondita, or if selfing preserves existing resistant genotypes, we used a factorial design to compare levels of infection of selfed and outcrossed progeny of Impatiens capensis, a woodland annual with a mixed mating system. We compared the level of host infection when exposed to three pathogen sources in the field: the sympatric rust population, and two allopatric rust populations. Outcrossed progeny exposed to sympatric rust had higher infection scores than selfed progeny exposed to the same rust, suggesting that outcrossing breaks up resistant genotypes. In addition, there was a trend for the rust to be more infective on sympatric rather than allopatric hosts. We also examined whether rust infection differentially alters the fitness of selfed and outcrossed progeny. Outcrossed plants that escaped infection had higher fitness, as measured by fruit production, than selfed plants, but there was no difference in fitness between infected selfed and infected outcrossed plants. Thus, outcrossing was advantageous in the absence of disease, but there was no fitness difference between selfed and outcrossed progeny in the presence of disease. In sum, our results indicate that interactions with pathogens can eliminate or reverse the advantage of outcrossing.

A tritrophic effect of host plant on susceptibility of western flower thrips to the entomopathogenic fungus Beauveria bassiana.

Adult female western flower thrips (Frankliniella occidentalis) were exposed 12-24h to bean (Phaseolus vulgaris) and impatiens (Impatiens wallerana) leaf disks treated with Beauveria bassiana conidia and then transferred to clean bean or impatiens at various times post-treatment. Significantly greater levels of fungal infection were observed when thrips were treated on bean versus impatiens, but exposure to impatiens following treatment had no effect on fungal infection (percent mortality). This result, combined with observations of no inhibition of germination of conidia exposed to intact or macerated impatiens foliage, indicated that the negative effect of the impatiens host plant was not due to plant chemical compounds (antibiosis). Further observations revealed that insects acquired (picked-up) 75% more conidia from treated bean disks than from treated impatiens disks. This difference in dose acquisition was determined to account for the observed difference in percent mortality (15%) following treatment on the two host plants. Median lethal doses (LD(50)) of B. bassiana were not significantly different on the two host plants, but median lethal concentrations were nearly 7-fold greater on impatiens. This difference was explained by disproportionate rates of conidial acquisition at measured rates of conidial deposition (an inverse relationship was observed between application rate expressed as conidia/mm(2) and the number of conidia acquired). The mechanism underlying the differential rates of conidial acquisition from bean versus impatiens was not determined.