Invasive Plant Relations in a Global Pandemic: Caring for a “Problematic Pesto”

In Spring 2020, amidst a COVID-19 state of emergency, the City of Toronto's Parks & Urban Forestry department posted signs in the city's remaining Black Oak Savannahs to announce the cancellation of the yearly ‘prescribed burn’ practice, citing fears it would exacerbate pandemic conditions. With this activity and other nature management events on hold, many invasive plants continued to establish and proliferate. This paper confronts dominant attitudes in invasion ecology with Indigenous epistemologies and ideas of transformative justice, asking what can be learned from building a relationship with a much-maligned invasive plant like garlic mustard. Written in isolation as the plant began to flower in the Black Oak savannahs and beyond, this paper situates the plant's abundance and gifts within pandemic-related ‘cancelled care’ and ‘cultivation activism’ as a means of exploring human-nature relations in the settler-colonial city. It also asks what transformative lessons garlic mustard can offer about precarity, non-linear temporalities, contamination, multispecies entanglements, and the impacts of colonial property regimes on possible relations. Highlighting the entanglements of historical and ongoing violences with invasion ecology, this paper presents ‘caring for invasives’ as a path toward more liveable futures.

First report of Erysiphe cruciferarum causing powdery mildew of Alliaria petiolata in Maryland

Alliaria petiolata (Bieb.) Cavara & Grande (garlic mustard) is a biennial crucifer native to Europe and invasive in North America, where it outcompetes native plants in deciduous forests. In July 2021, powdery mildew was observed on A. petiolata in Frederick County, Maryland. Signs of the disease included white, tomentose mycelium producing abundant conidia (Fig S1). A majority of plants were affected, and severity ranged from the presence of small, discrete infections to complete colonization of leaves, stems, and ripening seed pods. Conidia from field collected leaves were transferred to disease-free A. petiolata for maintenance in a growth chamber at 20°C and 80% RH with a 12 hr photoperiod. Fungal morphology was recorded 30 days after this transfer. Appressoria were irregularly lobed, and conidiophores were straight and composed of 2-3 cells. Cylindrical to oblong conidia were produced singly in pseudochains of 2-6 (x̄ = 3), measured 39-64 by 18-29 (x̄ = 52 by 24) μm, had a length to width ratio greater than two, and germinated at the ends. Fibrosin bodies were absent from conidia, and chasmothecia were not observed in the field or on inoculated plant material. Based on anamorphic characteristics, the pathogen was placed in the genus Erysiphe (Boesewinkel 1980). Species level identity was determined using DNA sequences. Conidia and mycelia were scraped from leaves and used for genomic DNA extraction with the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA). A portion of the internal transcribed spacer region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). Purified amplicons (PCR & DNA Cleanup Kit, New England BioLabs Inc., Ipswich, MA) were sequenced at Eurofins Genomics (Louisville, KY). The resulting sequence was compared to those in NCBI GenBank using the blastn algorithm (Altschul et al. 1990). The newly generated sequence (GenBank: OK157430) was identical (599/599 bp) to samples of E. cruciferarum from the United Kingdom (GenBank: KY660931.1, KY660879.1, KY660752.1). Because E. cruciferarum sensu lato is heterogeneous (Pastirčáková et al. 2016), additional sequence comparisons were made to the E. cruciferarum sensu stricto holotype (589/599 bp, GenBank: KU672364) and a vouchered E. cruciferarum s. lat. (596/599 bp, GenBank: LC009943). This supports identification of the pathogen as E. cruciferarum s. lat. and suggests the taxonomy of isolates from A. petiolata should be reassessed following any revision to E. cruciferarum. A modified Koch’s postulates procedure was followed to confirm pathogenicity. Leaves colonized by E. cruciferarum were briefly pressed against the leaves of three disease-free plants grown from seed in a greenhouse. After 14 days, inoculated plants showed signs of powdery mildew similar to those observed in the field, and a control treatment using pathogen-free leaves resulted in no disease. This inoculation experiment was performed twice, and the identity of the pathogen was reconfirmed based on morphology. This is the first report of powdery mildew on A. petiolata in Maryland. Erysiphe cruciferarum s. lat. is widely distributed on other hosts and has been found on A. petiolata throughout Europe and in Ohio and Indiana (Farr and Rossman 2021; Blossey et al. 2001; Enright and Cipollini 2007; Ciola and Cipollini 2011). This pathogen has been proposed as a biological control agent (Cipollini and Enright 2009; Cipollini et al. 2020), and the presence of disease in Maryland suggests the local population of A. petiolata is susceptible to E. cruciferarum and the environment there is favorable to disease development. References: Altschul, S. F., et al. 1990. J. Mol. Biol. 215:403. Blossey, B., et al. 2001. Nat. Areas J. 21:357. Boesewinkel, H. J. 1980. Bot. Rev. 46:167. Ciola, V., and Cipollini, D. 2011. Am. Midl. Nat. 166:40-52. Cipollini, D., and Enright, S. M. 2009. Invasive Plant Sci. Manag. 2:253. Cipollini, D., et al. 2020. Biol. Invasions. 22:1657-1668. Enright, S. M., and Cipollini, D. 2007. Am. J. Bot. 94:1813. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases, Syst. Mycol. Microbial. Lab., ARS, USDA. https://nt.ars-grin.gov/fungaldatabases/ Pastirčáková, K., et al. 2016. Mycol. Prog. 15:36 White, T. J., et al. 1990. Page 315 in: PCR Protocols. A Guide to Methods and Applications, Academic Press, San Diego. Keywords: Alliaria petiolata, Erysiphe cruciferarum, garlic mustard, powdery mildew, invasive plant, biocontrol Funding and Disclaimer: The author(s) declare no conflict of interest. This work was supported by USDA-ARS Appropriated Project Number 8044-22000-047-000-D. Mention of trade names or commercial products in this report is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity lender, provider, and employer.

Effects of Intraspecific Density on Garlic Mustard (Alliaria Petiolata) Sinigrin Concentration

The production of secondary defense chemicals in plants represents a trade-off between defense and the primary functions of growth and reproduction, but the relative allocation to growth versus defense varies across species, types of defenses, ontogeny, and environment. Alliaria petiolata (garlic mustard) is a brassica that produces glucosinolates, a class of constituent secondary metabolites that defend against herbivores and pathogens. Sinigrin, a hydrolyzed product of glucosinolate present in garlic mustard, may aid in its success as an invasive species by disrupting native plant–mycorrhizae mutualisms and decreasing forest species diversity in North America. Here, we measured sinigrin concentration in garlic mustard populations of different field densities and in greenhouse experiments to evaluate the relationship between sinigrin concentration and growth in response to density and varying environmental conditions. We found clear evidence for growth vs. defense tradeoffs in both experimental and field settings, as well as higher levels of defense in more densely growing, smaller individual plants. However, sinigrin levels and tradeoffs were not explained by soil fertility or light, allowing us to conclude that sinigrin expression is not controlled by limitations in the measured abiotic factors. Our findings suggest sinigrin leaf concentration increases at high densities despite the pressures of intraspecific competition that demand allocation to growth.

Genome Report: A draft genome of Alliaria petiolata (garlic mustard) as a model system for invasion genetics

The emerging field of invasion genetics examines the genetic causes and consequences of biological invasions, but few study systems are available that integrate deep ecological knowledge with genomic tools. Here we report on the de novo assembly and annotation of a genome for the biennial herb Alliaria petiolata (M. Bieb.) Cavara & Grande (Brassicaceae), which is widespread in Eurasia and invasive across much of temperate North America. Our goal was to sequence and annotate a genome to complement resources available from hundreds of published ecological studies, a global field survey, and hundreds of genetic lines maintained in Germany and Canada. We sequenced a genotype (EFCC3-3-20) collected from the native range near Venice, Italy and sequenced paired-end and mate pair libraries at ∼70 × coverage. A de novo assembly resulted in a highly continuous draft genome (N50 = 121 Mb; L50 = 2) with 99.7% of the 1.1 Gb genome mapping to scaffolds of at least 50 Kb in length. A total of 64,770 predicted genes in the annotated genome include 99% of plant BUSCO genes and 98% of transcriptome reads. Consistent with previous reports of (auto)hexaploidy in western Europe, we found that almost one third of BUSCO genes (390/1440) mapped to two or more scaffolds despite < 2% genome-wide average heterozygosity. The continuity and gene space quality of our draft assembly will enable molecular and functional genomic studies of A. petiolata to address questions relevant to invasion genetics and conservation strategies.

Efficacy of some conventional fungicides and essential oils against tomato early blight disease

Fungicidal activity of nine selected conventional fungicides, identified five plant oils and their combined effect against A. solani, the causal pathogen of early blight on tomato. GC-MS analysis of the tested oils indicated that terpene hydrocarbons (97%) with cinnamaldehyde as the major compound; sulfur compounds; fatty acids and organo-sulfur-containing compounds were the main components in cinnamon, garlic, mustard and onion oils, respectively. In vitro, Speedcide® (difenoconazole) and Cabrioduo® (Dimethomorph + Pyraclostrobin) were the most effective against the A. solani fungus with EC50 values of 94.6 and 88.6 ppm respectively. Toledo® (Tebuconzole) achieved EC50 value of 631.2 ppm. Roxyl-M® was significantly more effective than Roxyl–plus® or Remik®. Speedcide® alone or mixed with mandipropamid or azoxystrobin in Revus-Top® or Cruze® were the lowest effective in vitro. Garlic and mustard oils were more effective in liquid media than solid one. Cinnamon, onion, garlic and bitter almond oils caused moderate fungi-toxicity against early blight pathogen and could be implemented in the IPM program. Lower EC50 values were obtained in case of combinations of fungicides with oils than separate treatments. Effect of the active conventional fungicides and plant oils on disease indices, relative disease control, chlorophyll content, sugar contents and poly phenol oxidase in tomato plants was also studied.

Do applications of systemic herbicides when green fruit are present prevent seed production or viability of Alliaria petiolata (garlic mustard)?

Garlic mustard [Alliaria petiolata (M. Bieb.) Cavara & Grande] is a biennial invasive plant commonly found in the northeastern and midwestern United States. Although it is not recommended to apply herbicides after flowering, land managers frequently desire to conduct management during this timing. We applied glyphosate and triclopyr (3% v/v and 1% v/v using 31.8% and 39.8% acid equivalent formulations, respectively) postemergence to established, second-year A. petiolata populations at three locations when petals were dehiscing, and evaluated control, seed production and seed viability. Postemergence glyphosate applications at this timing provided 100% control of A. petiolata by 4 weeks after treatment at all locations whereas triclopyr efficacy was variable, providing 38-62% control. Seed production was only reduced at one location, with similar results regardless of treatment. Percent seed viability was also reduced, and when combined with reductions in seed production, we found a 71-99% reduction in number of viable seed produced plant-1 regardless of treatment. While applications did not eliminate viable seed production, our findings indicate that glyphosate and triclopyr applied while petals were dehiscing is a viable alternative to cutting or hand-pulling at this timing as it substantially decreased viable A. petiolata seed production. Management Implications Postemergence glyphosate and triclopyr applications in the early spring to rosettes are standard treatments used to manage A. petiolata. However, weather and other priorities limit the window for management, forcing field practitioners to utilize more labor-intensive methods such as hand-pulling. It is not known how late in the development of A. petiolata these herbicides can be applied to prevent viable seed production. Since prevention of soil seedbank replenishment is a key management factor for effective long-term control of biennial invasive species, we hypothesized late spring foliar herbicide applications to second year A. petiolata plants when flower petals were dehiscing could be an effective management tool if seed production or viability is eliminated. Our study indicated that glyphosate applications at this timing provided 100% control of A. petiolata plants by 4 weeks after treatment at all locations, whereas triclopyr efficacy was inconsistent. Although both glyphosate and triclopyr decreased viable seed production to nearly zero at one of our three study locations, the same treatments produced significant amounts of viable seed at the other two locations. Our findings suggest late spring glyphosate and triclopyr applications should not be recommended over early spring applications to rosettes for A. petiolata management, as our late spring application timing did not prevent viable seed production, and may require multiple years of implementation to eradicate populations. Nonetheless, this application timing holds value in areas devoid of desirable understory vegetation compared to no management practices or mechanical management options including hand-pulling when fruit are present, as overall viable seed production was reduced to similar levels as these treatments.

A draft genome of Alliaria petiolata (garlic mustard) as a model system for invasion genetics

The emerging field of invasion genetics examines the genetic causes and consequences of biological invasions, but few study systems are available that integrate deep ecological knowledge with genomic tools. Here we report on the de novo assembly and annotation of a genome for the biennial herb Alliaria petiolata (M. Bieb.) Cavara & Grande (Brassicaceae), which is widespread in Eurasia and invasive across much of temperate North America. Our goal was to sequence and annotate a genome to complement resources available from hundreds of published ecological studies, a global field survey, and hundreds of genetic lines maintained in Germany and Canada. We sequenced a genotype (EFCC-3-20) collected from the native range near Venice, Italy and sequenced paired-end and mate pair libraries at ~70× coverage. A de novo assembly resulted in a highly continuous draft genome (N50 = 121Mb; L50 = 2) with 99.7% of the 1.1Gb genome mapping to contigs of at least 50Kb in length. A total of 64,770 predicted genes in the annotated genome include 99% of plant BUSCO genes and 98% of transcriptome reads. Consistent with previous reports of (auto)hexaploidy in western Europe Almost, we found that almost one third of BUSCO genes (390/1440) mapped to two or more scaffolds despite a genome-wide average of < 2% heterozygosity. The continuity and gene space quality of our draft genome assembly will enable genomic studies of A. petiolata to address questions relevant to invasion genetics and conservation efforts.

Invasive garlic mustard demonstrates stronger relationships with pathotrophic than mycorrhizal fungi

Garlic mustard (Alliaria petiolata) has long been known to degrade mycorrhizal mutualisms in soils it invades and may also promote the abundance of microbial pathogens harmful to native plants or alter saprotrophic communities to disrupt nutrient cycling. Phenology of other invasive species, like Lepidium latifolium and Lonicera maackii, plays a role in their interactions with soil microbial communities, and so we may expect garlic mustard phenology to influence its effects on native soil microbiomes as well. Here, we investigate differences in fungal, bacterial, and archaeal community structure, as well as the abundance of key functional groups, between garlic mustard present, absent, and removed treatments in central-Illinois forest soils across different stages of the garlic mustard life cycle. Across its phenology, garlic mustard present soils had different overall fungal community structure and greater abundance of pathotrophic fungi than soils where garlic mustard was absent or removed. However, abundance of ectomycorrhizal and saprotrophic fungi as well as bacterial and archaeal community structure were similar between treatments and did not interact with garlic mustard phenology. The most abundant overall fungal taxon was a plant pathogen, Entorrhiza aschersoniana, that was greatest in garlic mustard present soils, particularly while the plants were flowering. These results support the hypothesis that invasive plants form active relationships with microbial pathogens that could contribute to their overall success in invading ecosystems.