Summary: In 2014, the rust fungus Puccinia komarovii var. glanduliferae, native to the foothills of the Himalayas from Kashmir to Western Nepal, became the first fungal agent to be released into Europe for the classical biological control of a non‐native weed. The target, Impatiens glandulifera or Himalayan balsam, is a prolific invader of riparian habitats in Europe and North America. During the period 2015–2018, a strain of the rust from India was released at a total of 36 sites, in 17 counties in England and Wales. There was limited field infection in 2015 and inoculation experiments conducted under controlled conditions revealed significant variation in the susceptibility of plant populations to the rust, with some showing immunity. Subsequently, a second strain of the rust from Pakistan was released in 2017 and was found to infect a different cohort of Himalayan balsam populations. The rust mass production methodology, and field inoculation and monitoring protocol, are detailed, and plants were tested for susceptibility to both rust strains prior to field release. Levels of foliar infection at selected sites in 2017 and 2018 are presented, as well as seedling infection rates in the spring of 2019. The results show that the rust is able to overwinter and establish populations in stands of Himalayan balsam in England. The issues involved with measuring the impact of the biological control agent are discussed.

Matrix-assisted laser-desorption and ionisation time-of-flight mass spectroscopy (MALDI-TOF MS) is a powerful tool for the characterisation and/or identification of protein-containing samples. Several MALDI-TOF MS sample-preparation methods are currently available but few of these are well suited to the analysis of plant material. We have recently developed a simple, rapid, and relatively-cheap method for MALDI-TOF MS that is applicable to plant material (in addition to microbial and insect material), and our aim in this study was to distinguish between closely-related plant species and/or between regional biotypes within an invasive weed species using this method with a view to optimising the selection of biological control agents that can be used for weed management. We have employed a combination of principal-component analysis and closest-relatedness diagrams derived from MALDI-TOF MS spectral-comparison data to discriminate between the closely-related Impatiens spp. Impatiens noli-tangere, Impatiens parviflora, Impatiens scabrida, Impatiens balsamina, and two regional biotypes of the invasive weed Impatiens glandulifera. We have also developed a method for sample discrimination based upon comparison between blind-test MALDI-TOF MS spectra and reference-sample spectra. Using this latter method, we have been able to discriminate on the basis of the acid-soluble-protein mass spectra generated between four regional biotypes of I. glandulifera that differ in their susceptibility to the biological control agent Himalayan balsam rust (Puccinia komarovii var. glanduliferae) using mature leaf material. Using younger leaves, discrimination was not possible between these four regional biotypes. MALDI-TOF MS analysis is able to discriminate between closely-related plant species within the genus Impatiens and between regional biotypes of I. glandulifera. Because of this, MALDI-TOF MS holds great promise for improving weed biological control, a management technique which uses highly-specific co-evolved natural enemies for the control of an invasive non-native plant species, through the optimal matching of biological control agents with susceptible target species/regional biotypes.

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.

We have recently developed a simple, rapid, and relatively-cheap method for matrix-assisted laser-desorption and ionisation time-of-flight mass spectroscopy (MALDI-TOF MS) sample preparation that is applicable to plant material (in addition to microbial and insect material), and have used this to discriminate between closely-related Impatiens species and between regional biotypes of the invasive weed Impatiens glandulifera (commonly known as Himalayan balsam) using leaf samples. In the current paper, we have developed a complementary MALDI-TOF MS-based method for use with seeds. We have employed a combination of principal-component analysis and blind-tested comparison between reference-sample MALDI-TOF MS spectra and test-sample spectra to discriminate, on the basis of the acid-soluble seed-protein spectra generated by our method, between four regional biotypes of I. glandulifera from within the UK that differ in their susceptibility to the biological control agent Himalayan balsam rust (Puccinia komarovii var. glanduliferae). Peak-rich and highly-reproducible spectra were obtained and, in blind testing with test seeds collected in 2017 against reference seeds collected in 2017, we observed 100% identification accuracy in 12 blind tests. In blind testing with test seeds collected in 2016 against reference seeds collected in 2017, we observed 92% identification accuracy in 12 blind tests. MALDI-TOF MS analysis of seed material is able to discriminate between regional biotypes of I. glandulifera. MALDI-TOF MS therefore has the potential to improve the efficiency and efficacy of weed biological control using co-evolved natural enemies of invasive non-native plant species, through the matching of biological control agents with susceptible regional biotypes.

Impatiens glanduliferaor 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.