In the search for new alternative biocontrol strategies, phytopathogenic fungi could represent a new frontier for weed management. In this respect, as part of our ongoing work aiming at using fungal pathogens as an alternative to common herbicides, the foliar pathogen Nigrospora sphaerica has been evaluated to control buffelgrass (Cenchrus ciliaris). In particular, in this work, the isolation and structural elucidation of two new biosynthetically related metabolites, named nigrosphaeritriol (3-(hydroxymethyl)-2-methylpentane-1,4-diol) and nigrosphaerilactol (3-(1-hydroxyethyl)-4-methyltetrahydrofuran-2-ol), from the phytotoxic culture filtrate extract were described, along with the identification of several known metabolites. Moreover, the absolute stereochemistry of (3R,4S,5S)-nigrosphaerilactone, previously reported as (3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone, was determined for the first time by X-ray diffraction analysis. Considering their structural relationship, the determination of the absolute stereochemistry of nigrosphaerilactone allowed us to hypothesize the absolute stereochemistry of nigrosphaeritriol and nigrosphaerilactol.

(10S,11S)-(-)-epi-pyriculol is a phytotoxic metabolite produced by Pyricularia grisea, a fungus identified as a foliar pathogen on the invasive weed species buffelgrass (Cenchrus ciliaris) in North America. The effective control of buffelgrass has not yet been achieved, and there is a need to develop effective and green solutions. Herbicides based on natural products and the use of phytopathogenic organisms could provide the most suitable tools for the control of weeds such as buffelgrass. Thus, one of the most relevant points to study about potential suitable phytotoxins such as (10S,11S)-(-)-epi-pyriculol is its production on a large scale, either by isolation from fungal fermentations or by synthesis. For these purposes, rapid and sensitive methods for the quantification of (10S,11S)-(-)-epi-pyriculol in complex mixtures are required. In this study, a high-pressure liquid chromatography (HPLC) method for its quantification was developed and applied to organic extracts from twelve P. grisea isolates obtained from diseased buffelgrass leaves and grown in potato dextrose broth (PDB) liquid cultures. The analysis proved that the production of (10S,11S)-(-)-epi-pyriculol is fungal-isolate dependent and strongly correlated with phytotoxic activity, shown by the P. grisea organic extracts in a buffelgrass radicle elongation test. The HPLC method reported herein allowed us to select the best strain for the production of (10S,11S)-(-)-epi-pyriculol and could be useful for selecting the best cultural conditions for its mass production, providing a tool for the use of this promising metabolite as a new bioherbicide for the control of buffelgrass.

Buffelgrass (Cenchrus ciliaris L.) is an invasive C4 perennial grass species that substantially reduces native plant diversity of the Sonoran Desert through fire promotion and resource competition. Broad-spectrum herbicides are essentially used for its control, but they have a negative environmental and ecological impact. Recently, phytotoxicity on C. ciliaris has been discovered for two metabolites produced in vitro by the phytopathogenic fungi Cochliobolus australiensis and Pyricularia grisea. They were identified as (10S,11S)-(—)-epi-pyriculol and radicinin and resulted in being potential candidates for the development of bioherbicides for buffelgrass biocontrol. They have already shown promising results, but their ecotoxicological profiles and degradability have been poorly investigated. In this study, ecotoxicological tests against representative organisms from aquatic ecosystems (Aliivibrio fischeri bacterium, Raphidocelis subcapitata alga, and Daphnia magna crustacean) revealed relatively low toxicity for these compounds, supporting further studies for their practical application. The stability of these metabolites in International Organization for Standardization (ISO) 8692:2012 culture medium under different temperatures and light conditions was also evaluated, revealing that 98.90% of radicinin degraded after 3 days in sunlight. Significant degradation percentages (59.51–73.82%) were also obtained at room temperature, 30 °C or under ultraviolet (254 nm) light exposure. On the other hand, (10S,11S)-epi-pyriculol showed more stability under all the aforementioned conditions (49.26–65.32%). The sunlight treatment was also shown to be most effective for the degradation of this metabolite. These results suggest that radicinin could provide rapid degradability when used in agrochemical formulations, whereas (10S,11S)-epi-pyriculol stands as a notably more stable compound.

Plant species endemic to gypsum occur in arid and semiarid regions worldwide and are an important component of biodiversity conservation, but gypsophile habitat selection within more arid gypsum environments remains largely unexplored. Our goal in this study was to inform efforts to introduce new populations of the endangered Mojave Desert gypsophile Arctomecon humilis Coville (dwarf bear poppy) through direct seeding at more remote locations that are less impacted by threats associated with urban expansion. We characterized approximately 600 ‘safe sites’ where seedlings of this species had recently established and examined distribution of these safe sites across habitats on the gypsum cuesta landscape. Using drone imagery at the landscape scale, we found that juveniles showed an even stronger preference for the white surface cover class than adults and were also markedly under-represented in the dark crust cover class. Two-thirds of the juveniles were found on knoll tops and upper back slopes, while only 3% were found on bottoms. At the plot scale, most juveniles were associated with surfaces where lichen crust and white gravel occurred in a fine-scale mosaic. At the emergence microsite scale, two-thirds of the seedlings had emerged from narrow gaps between two or more contact substrates, most often lichen crust and gravel. Even when seedlings emerged from lichen crust alone, emergence was usually from a gap and not from the surface of intact lichen thallus. On bottoms, toe slopes, and lower back slopes where dark lichen crust is often heavy and continuous, very few juveniles were found. Fewer than 7% of juveniles were found in direct association with living adults, likely because of adaptations for both enhanced seed dispersal and a persistent seed bank. There was also no evidence for establishment facilitation by nurse plants of other species. These results are contrasted with gypsophile establishment ecology in less arid environments. They also enable drone-based screening at the landscape scale as an efficient first step in selection of introduction sites.

Blackbrush (Coleogyne ramosissima Torr.) is the dominant shrub on three million hectares across the transition zone between the western North American warm and cold deserts. This paper presents a study of blackbrush population structure at the stand level at sixteen sites across the species range. New stand-level information is then integrated with what is already known about blackbrush population ecology to explore the stand-level consequences of several unusual features of blackbrush life history, including its masting reproductive strategy, its interactions with heteromyid rodents that are both seed predators and dispersers, and its ability to form 'seedling banks' of growth-suppressed individuals, often within the crowns of adult conspecifics. It complements earlier work showing that blackbrush stands are organized at both the inter-clump and intra-clump levels. Each clump represents an establishment nexus where younger individual genets replace older genets over an extended time period. Inter-clump structure is thus determined by the rate of establishment of new clumps rather than new individuals. This has resulted in contrasting stand structures in response to rodent community composition, disturbance regimes, and climatic variability at the leading and trailing edges of current blackbrush distribution in the eastern Mojave Desert and Colorado Plateau regions. Because blackbrush likely disperses too slowly to track anthropogenic climate change, assisted migration with wild-collected seeds may be necessary to promote its continued survival and dominance.

Seed banks, the collection of viable seeds in the soil, are particularly important determinants of population survival in highly variable environments. Predictions of increased stochasticity in the amount and timing of precipitation in desert environments raise the question of how seed banks of desert species will respond to climate change, and ultimately, whether these species will persist. Here, we present data from our long-term studies of germination requirements and seed bank dynamics in a rare desert gypsophile perennial, Arctomecon californica (Las Vegas bearpoppy). Arctomecon californica is a relatively short-lived plant that recruits from seed in sequences of unusually favorable years. We used germination experiments, an in situ seed bank study, and a 15-year field seed retrieval study to examine factors affecting seed bank persistence. In the germination study, a majority of seeds remained dormant, despite a wide variety of treatments, suggesting that a large proportion of the seed dispersed each year has cue-nonresponsive dormancy. Our in situ seed bank study showed that seed density varied widely between sites, among transects, and among samples within a transect. The patchiness of seeds in the soil highlights the importance of protecting large areas where A. californica populations are known to have existed in the past. The seed retrieval study provided strong evidence that this species has a long-lived seed bank in which only a small fraction of seeds (roughly 5%) become nondormant each year, allowing seed banks of this species to last up to 20 years without a seed production event. Whether this impressive life-history strategy can maintain the species in the face of climate change depends on the future frequency of the well-timed precipitation that allows for the establishment of new cohorts of adult plants.

The genus Pyrenophora includes two important cereal crop foliar pathogens and a large number of less well-known species, many of which are also grass pathogens. Only a few of these have been examined in terms of secondary metabolite production, yet even these few species have yielded a remarkable array of bioactive metabolites that include compounds produced through each of the major biosynthetic pathways. There is little overlap among species in the compounds identified. Pyrenophora tritici-repentis produces protein toxin effectors that mediate host-specific responses as well as spirocyclic lactams and at least one anthraquinone. Pyrenophora teres produces marasmine amino acid and isoquinoline derivatives involved in pathogenesis on barley as well as nonenolides with antifungal activity, while P. semeniperda produces cytochalasans and sesquiterpenoids implicated in pathogenesis on seeds as well as spirocyclic lactams with phytotoxic and antibacterial activity. Less well-known species have produced some unusual macrocyclic compounds in addition to a diverse array of anthraquinones. For the three best-studied species, in silico genome mining has predicted the existence of biosynthetic pathways for a much larger array of potentially toxic secondary metabolites than has yet been produced in culture. Most compounds identified to date have potentially useful biological activity.

Cheatgrass (Bromus tectorum L.) is an invasive annual grass (Poaceae) that has colonized large portions of the Intermountain West. Cheatgrass stand failures have been observed throughout the invaded region, the cause of which may be related to the presence of several species of pathogenic fungi in the soil or surface litter. In this metabarcoding study, we compared the fungal communities between sites that have and have not experienced stand failure. Samples were taken from the soil and surface litter near Winnemucca, Nevada, and in Skull Valley, Utah. Our results show distinct fungal communities associated with stand failure based on both geography and sample type. In both the Winnemucca and Skull Valley surface litter, there was an elevated abundance of the endophyte Ramimonilia apicalis in samples that had experienced a stand failure. Winnemucca surface litter stand failure samples had an increased abundance of a potential pathogen in the genus Comoclathris. Skull Valley surface litter stand failure samples had an increased abundance of an undescribed new species in the Rustroemiaceae family which is responsible for the so-called bleach blonde syndrome in cheatgrass, while the soils had an increased abundance of potential pathogens in the genera Olpidium and Monosporascus.

AbstractMuch of the literature dealing with the biology and management of invasive species has focused on the damaging ecological and economic consequences of invasions (see Chaps.2,3, and14of this volume for review). In this chapter, we shift the focus to the causes of invasion, with the goal of proactively limiting or preventing invasions rather than reacting to them once they have occurred. Preventing the introduction of invasive species is one key element in this proactive approach (Chap.6, this volume). Here, we specifically focus on ecosystem attributes that affect whether or not an ecosystem is vulnerable to invasion, that is, the features that affect its invasibility (Lonsdale 1999), with particular emphasis on the role of natural and anthropogenic disturbance.

A census of endangered plant populations is critical to determining their size, spatial distribution, and geographical extent. Traditional, on-the-ground methods for collecting census data are labor-intensive, time-consuming, and expensive. Use of drone imagery coupled with application of rapidly advancing deep learning technology could greatly reduce the effort and cost of collecting and analyzing population-level data across relatively large areas. We used a customization of the YOLOv5 object detection model to identify and count individual dwarf bear poppy (Arctomecon humilis) plants in drone imagery obtained at 40 m altitude. We compared human-based and model-based detection at 40 m on n = 11 test plots for two areas that differed in image quality. The model out-performed human visual poppy detection for precision and recall, and was 1100× faster at inference/evaluation on the test plots. Model inference precision was 0.83, and recall was 0.74, while human evaluation resulted in precision of 0.67, and recall of 0.71. Both model and human performance were better in the area with higher-quality imagery, suggesting that image quality is a primary factor limiting model performance. Evaluation of drone-based census imagery from the 255 ha Webb Hill population with our customized YOLOv5 model was completed in <3 h and provided a reasonable estimate of population size (7414 poppies) with minimal investment of on-the-ground resources.

Abstract This study of Astragalus holmgreniorum examines its adaptations to the warm desert environment and whether these adaptations will enable it to persist. Its spring ephemeral hemicryptophyte life‐history strategy is unusual in warm deserts. We used data from a 22‐year demographic study supplemented with reproductive output, seed bank, and germinant survival studies to examine the population dynamics of this species using discrete‐time stochastic matrix modeling. The model showed that A. holmgreniorum is likely to persist in the warm desert in spite of high dormant‐season mortality. It relies on a stochastically varying environment with high inter‐annual variation in precipitation for persistence, but without a long‐lived seed bank, environmental stochasticity confers no advantage. Episodic high reproductive output and frequent seedling recruitment along with a persistent seed bank are adaptations that facilitate its survival. These adaptations place its life‐history strategy further along the spectrum from “slower” to “faster” relative to other perennial spring ephemerals. The extinction risk for small populations is relatively high even though mean λs > 1 because of the high variance in year quality. This risk is also strongly dependent on seed bank starting values, creating a moving window of extinction risk that varies with population size through time. Astragalus holmgreniorum life‐history strategy combines the perennial spring ephemeral life form with features more characteristic of desert annuals. These adaptations permit persistence in the warm desert environment. A promising conclusion is that new populations of this endangered species can likely be established through direct seeding.

For plant species of conservation concern, knowledge of changes in abundance through time is a minimum requirement for informed management. This information is usually acquired through on-the-ground monitoring, which entails counting individuals in defined areas over multiple years. Demographic studies, which involve tracking individual plants through time, are usually carried out at limited spatial scales and over shorter time frames than monitoring, but are more useful to management. In this study we explored the use of drone (UAV or unmanned aerial vehicle) imagery analysis as a tool for collecting demographic data for dwarf bear poppy (Arctomecon humilis), an endangered species restricted to gypsum outcrops in the northeastern Mojave Desert, USA. We obtained imagery at 15 m altitude during peak flowering at four populations in spring 2019. Each poppy plant in the imagery was georeferenced, measured and scored for flowering. To estimate reproductive output, we developed independent data sets relating plant diameter to flower number, then sampled to determine mean fruit set per flower and seeds per fruit. We used these relationships along with plant diameter and reproductive status for each plant in the drone imagery to estimate seed rain on an area basis across nine 0.6 ha demography plots at each population. This method enabled us to collect demographic data on >3,000 plants, including estimated production of ca. 3.7 million seeds, across >20 ha of habitat. We also analyzed imagery acquired in both 2018 and 2019 at two of the four populations and quantified recruitment, growth, and mortality of individual georeferenced plants. Our study is among the first to demonstrate the utility of drone imagery analysis in plant demographic studies. The method is most applicable for non-clonal perennial species with distinctive morphology that occur in habitats with low vegetative cover.

Arctomecon humilis (dwarf bear poppy) is an endangered gypsophile restricted to the St. George area in southern Utah, USA. Extirpation of the rare oligolectic bee Perdita meconis and decline of other native pollinators within the last 20 years has been largely attributed to the advent of invasive Africanized honeybees (AHBs), which have become the predominant pollinator for A. humilis. A three-year study was carried out to determine if reliance on AHBs had a negative effect on reproductive output. In the first year, pollen addition experiments were carried out in a small, isolated population to determine if pollinator scarcity or genetic factors affected reproductive output. In the subsequent two years, reproductive output studies were conducted across four populations. In all years, fruit set across study sites was exceptionally high. There was no consistent effect from flower number or interplant distance, which have historically been shown to have a significant impact on reproductive output prior to AHB arrival. The consequences and sustainability of this relatively young relationship between A. humilis and the AHB remain unclear. However, high reproductive success shows that the AHB is currently capable of adequately fulfilling pollinator services for A. humilis.

Conflicting priorities for livestock grazing management in the context of rare plant conservation are common worldwide, especially in semiarid and arid ecosystems, where open-range livestock grazing is a primary land use. Detailed information on the interaction between livestock impacts and rare plant demography would facilitate improved management, yet this information is most often not available. In this study we examined the direct impacts of cattle trampling on dormant season survival of Astragalus holmgreniorum (Fabaceae), a globally endangered perennial spring ephemeral of the northeastern Mojave Desert, USA. Current management restricts livestock access to the dormant season, when plants are not active above-ground, but monitoring indicates possible population decline under this regime. We tagged > 2100 A. holmgreniorum plants to document dormant season survival and quantified cattle trampling and other disturbance impacts in small plots at six sites with contrasting disturbance regimes. We documented disturbance impacts on survival at the site, plot, and individual plant level across two dormant season iterations. We consistently found significant negative relationships between plant dormant season survival and cattle impacts. Plants at sites with heavy cattle trampling were up to five times more likely to experience mortality than those at sites with low cattle impact. Other surface disturbances (rodent activity, human impacts, water erosion) were also quantified but contributed substantially to reduced survival in only one case of water erosion disturbance. We conclude that grazing management in A. holmgreniorum critical habitat on federal land may require modification to reduce extinction risk for this federally listed endangered species.

The fungal pathogen Pyricularia grisea has been studied to evaluate its production of phytotoxins for the biocontrol of the buffelgrass ( Cenchrus ciliaris L.) weed. A first investigation allowed to isolate several new and known phytotoxic metabolites. However, the further investigation on the organic extract obtained from the fungus liquid culture showed the presence of other metabolites possibly contributing to its phytotoxicity. Thus, four known metabolites were isolated and identified by spectroscopic (nuclear magnetic resonance [NMR] and high‐resolution electrospray ionization mass spectrometry [HRESIMS]) methods as dihydropyriculol ( 1 ), epi ‐dihydropyriculol ( 2 ), 3‐methoxy‐6,8‐dihydroxy‐3‐methyl‐3,4‐dihydroisocoumarin ( 3 ), and ( R )‐mevalonolactone ( 4 ). The absolute configuration of 1 – 3 was determined for the first time by a computational analysis of their electronic circular dichroism (ECD) spectra. When the isolated compounds were bioassayed at a concentration of 5 × 10 –3 M in a buffelgrass coleoptile and radicle elongation test no toxicity was detected. On the contrary, compounds 1 and 3 showed a significant stimulating effect of radical elongation. Furthermore, the difference in growth stimulation between 1 and its epimer 2 highlights the tight relationship between absolute configuration and biological activity of these fungal metabolites.

Premise Adaptive seed dispersal mechanisms are fundamental to plant fitness, but dispersal advantage is scale-dependent. We tested the hypothesis that informed dispersal in response to an environmental cue enables dispersal by wind on a local scale for Astragalus holmgreniorum, a desert species restricted to swales and wash skirts with overland flow, but prevents longer-distance dispersal by water into unfavorable wash habitats. Methods Pod biomechanics in A. holmgreniorum lead to major shape modifications with changes in moisture content. We performed laboratory experiments to examine the interaction of pod shape with wind and water, and conducted field experiments in A. holmgreniorum habitat evaluating the roles of wind, water, and seed predators on dispersal. Results Dry pods exhibit a flattened crescent shape with partial dehiscence that facilitated wind dispersal by ground tumbling and seed scattering in laboratory experiments. Rain simulation experiments showed that even small precipitation events returned wetted pods to their cylindrical shape and opened the dorsal suture, exposing the seeds. In the field experiments, dry pods were moved locally by wind, whereas rain caused pod opening and washing out of seeds in place. Seed predators had minimal effect on pod movement. Conclusions Astragalus holmgreniorum exhibits pod structural remodeling in response to environmental change in a striking and novel demonstration of informed dispersal. Wind-driven movement of dry pods facilitates local seed dispersal, but rain causes pods to open and release seeds, ensuring that they are not transported out of suitable habitats and into active washes where they would be lost from the seed bank.

Pyrenophoric acid (P-Acid), P-Acid B, and P-Acid C are three phytotoxic sesquiterpenoids produced by the ascomycete seed pathogen Pyrenophora semeniperda, a fungus proposed as a mycoherbicide for biocontrol of cheatgrass, an extremely invasive weed. When tested in cheatgrass bioassays, these metabolites were able to delay seed germination, with P-Acid B being the most active compound. Here, we have investigated the cross-kingdom activity of P-Acid B and its mode of action, and found that it activates the abscisic acid (ABA) signaling pathway in order to inhibit seedling establishment. P-Acid B inhibits seedling establishment in wild-type Arabidopsis thaliana, while several mutants affected in the early perception as well as in downstream ABA signaling components were insensitive to the fungal compound. However, in spite of structural similarities between ABA and P-Acid B, the latter is not able to activate the PYR/PYL family of ABA receptors. Instead, we have found that P-Acid B uses the ABA biosynthesis pathway at the level of alcohol dehydrogenase ABA2 to reduce seedling establishment. We propose that the fungus P. semeniperda manipulates plant ABA biosynthesis as a strategy to reduce seed germination, increasing its ability to cause seed mortality and thereby increase its fitness through higher reproductive success.

Radicinin (1), is a fungal dihydropyranopyran-4,5-dione isolated together with some analogues, namely 3-epi-radicinin, radicinol, 3-epi-radicinol, and cochliotoxin (2-5), from the culture filtrates of the fungus Cochliobolus australiensis, a foliar pathogen of buffelgrass (Cenchrus ciliaris), an invasive weed in North America. Among the different metabolites 1 showed target-specific activity against the host plant and no toxicity on zebrafish embryos, promoting its potential use to develop a natural bioherbicide formulation to manage buffelgrass. These data and the peculiar structural feature of 1 suggested to carry out a structure-activity relationship study, preparing some key hemisynthetic derivatives and to test their phytotoxicity. In particular, p-bromobenzoyl, 5-azidopentanoyl, stearoyl, mesyl and acetyl esters of radicinin were semisynthesized as well as the monoacetyl ester of 3-epi-radicinin, the diacetyl esters of radicinol and its 3 epimer, and two hexa-hydro derivatives of radicinin. The spectroscopic characterization and the activity by leaf puncture bioassay against buffelgrass of all the derivatives is reported. Most of the compounds showed phytotoxicity but none of them had comparable or higher activity than radicinin. Thus, the presence of an α,β unsaturated carbonyl group at C-4, as well as, the presence of a free secondary hydroxyl group at C-3 and the stereochemistry of the same carbon proved to be the essential feature for activity.

Radicinin is a phytotoxic fungal dihydropyranopyran-4,5-dione under evaluation for the development of a target-specific bioherbicide for invasive buffelgrass (Cenchrus ciliaris) control. It has already demonstrated high toxicity on host plants, low toxicity to native plants and no negative effects on zebrafish embryos. To continue these studies at the whole-plant level there is a need to obtain much larger quantities of radicinin, either by optimizing its large-scale production by fungal fermentation or through its total stereoselective synthesis. A rapid and sensitive HPLC method for quantification of radicinin in complex mixtures has been developed in order to evaluate its production by different Cochliobolus australiensis strains and in different cultural conditions. The analysis proved that radicinin is not produced by all the strains tested and its synthesis is strongly affected by cultural conditions. The HPLC method could be useful in selecting the best fungal source for the production of this promising potential bioherbicide.

The fungal pathogens Cochliobolus australiensis and Pyricularia grisea have recently been isolated from diseased leaves of buffelgrass (Cenchrus ciliaris) in its North American range, and their ability to produce phytotoxic metabolites that could potentially be used as natural herbicides against this invasive weed was investigated. Fourteen secondary metabolites obtained from in vitro cultures of these two pathogens were tested by leaf puncture assay on the host plant at different concentrations. Radicinin and (10S, 11S)-epi-pyriculol proved to be the most promising compounds. Thus, their phytotoxic activity was also evaluated on non-host indigenous plants. Radicinin demonstrated high target-specific toxicity on buffelgrass, low toxicity to native plants, and no teratogenic, sub-lethal, or lethal effects on zebrafish (Brachydanio rerio) embryos. It is now under consideration for the development of a target-specific bioherbicide to be used against buffelgrass in natural systems where synthetic herbicides cause excessive damage to native plants.