During the past 30 yr an impasse has developed in the discovery and commercialization of synthetic herbicides with new molecular targets and novel chemistries. Similarly, there has been little success with bioherbicides, both microbial and chemical. These bioherbicides are needed to combat fast-growing herbicide resistance and to fulfill the need for more environmentally and toxicologically safe herbicides. In response to this substantial and growing opportunity, numerous start-up companies are utilizing novel approaches to provide new tools for weed management. These diverse new tools broaden the scope of discovery, encompassing advanced computational, bioinformatic, and imaging platforms; plant genome–editing and targeted protein degradation technologies; and machine learning and artificial intelligence (AI)-based strategies. This review contains summaries of the presentations of 10 such companies that took part in a symposium held at the WSSA annual meeting in 2024. Four of the companies are developing microbial bioherbicides or natural product–based herbicides, and the other six are using advanced technologies, such as AI, to accelerate the discovery of herbicides with novel molecular target sites or to develop non-GMO, herbicide-resistant crops.

A fungal pathogen, Colletotrichum gloeosporioides (Penz.) Sacc. f. sp. malvae, isolated from anthracnose symptoms of round-leaved mallow (Malva pusilla Sm.), was shown in greenhouse tests to be host specific to Malva spp. and velvetleaf (Abutilon theophrasti Medic. #3 ABUTH), and only with slight attack on hollyhock [Althaea rosea (L.) Cav. # ALGRO], Malope trifida Cav., and Venice mallow (Hibiscus trionum L. # HIBTR). Round-leaved mallow plants inoculated with a spore suspension of the fungus were killed after 17 to 20 days. It was less pathogenic on velvetleaf with 60 to 70% attack. The fungus can readily be cultured and field tests from 1982 to 1987 resulted in excellent control of round-leaved mallow under natural conditions. Therefore, it has good potential for biological control of round-leaved mallow in field crops.

Rust-specific polymerase chain reaction (PCR) primers selectively amplified ribosomal DNA of a rust fungus from infected dyers woad. PCR enabled DNA of the fungus to be detected in symptomatic plants as well as in asymptomatic parts of diseased plants. The use of PCR enabled early detection of rust infections in dyers woad plants during their first season when they are often asymptomatic Dried plant samples stored at room temperature for several months worked as well as lyophilized material for DNA extraction prior to PCR. The PCR detection method should greatly facilitate further studies on the biology and inoculation of this and other systemic rusts that have potential for use in biocontrol of weeds.

Sixty isolates of the plant pathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary and six isolates of Sclerotinia minor Jagger were evaluated in a controlled environment for virulence on leaves excised from 8-week-old dandelion plants. Significant negative correlations were obtained between the relative virulence of the isolates and the dry weights of dandelion plants inoculated in a controlled environment; and positive correlations were detected between the relative virulence of isolates and reduction in number of dandelion plants in turfgrass swards infested with inoculum of the isolates. In August 1988, an 80.7% reduction in the number of dandelion plants was detected in a turfgrass sward treated in 1987 with four applications of heat-killed seed of perennial ryegrass (100 g m−2 application–1) infested with isolate R30 of S. sclerotiorum, followed by six applications at the same rate in 1988. Populations of dandelions in untreated swards increased by 22.2% during the same period. Heat-killed seed of perennial ryegrass (100 g m−2) infested with isolate R30 applied simultaneously with dandelion seed (25 g m−2) onto a sward of Kentucky bluegrass reduced the establishment of dandelion seedlings by 85.5%. Necrosis or discoloration did not develop on Kentucky bluegrass, creeping bentgrass, annual bluegrass, or quackgrass treated with inoculum of Sclerotinia. Sclerotia of Sclerotinia spp. were found, on occasion, in crowns but not on the foliage of diseased dandelions.

The determination of host range is an important component in developing a plant pathogen for use as a bioherbicide. The safety of non-target economic and wild plants must be assured before experimental release and commercial use. In contrast to other methods of weed control, the genetic variability and genetic stability of both the weed and the biological control agent must be considered. Schemes to determine host range generally assume a close phylogenetic relationship between the weed host and its co-evolved pathogens. Therefore, testing generally is based on inoculation of genetically related plant species and progresses to more distantly related species until the host range is circumscribed. Several potential weaknesses in these schemes will be illustrated with examples using specific biological control agents. Future tests must place greater emphasis on testing taxa representing the full range of genetic diversity within the biogeographic area of intended use.

Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) was evaluated under greenhouse conditions for round-leaved mallow control in combination with the following herbicides: bromoxynil plus MCPA (1:1), diclofop, imazethapyr, metribuzin, sethoxydim, and 2,4-DB. Treatments consisted of C.g.m. and herbicide applied alone, tank mixed, and C.g.m. applied 48 h before, and 1, 24, and 72 h after herbicide. Split applications with bromoxynil plus MCPA, imazethapyr, metribuzin, (C.g.m. before or after) and sethoxydim (C.g.m. before) enhanced round-leaved mallow control over C.g.m. applied alone. However, diclofop or 2,4-DB tank mixed or applied in a split application with C.g.m. did not improve round-leaved mallow control over C.g.m. applied alone. Bromoxynil plus MCPA, metribuzin, 2,4-DB, and imazethapyr may be tank mixed with C.g.m. without significantly reducing round-leaved mallow control, unlike sethoxydim or diclofop which inhibited C.g.m. development. Dew period did not influence the effectiveness of bromoxynil plus MCPA applied 30 h before C.g.m., but round-leaved mallow control increased with increasing dew period for metribuzin applied 30 h before C.g.m.

R230, an isolate of Rhizoctonia solani anastomosis group 5 from leafy spurge, caused root and crown rots on leafy spurge when inoculated via soil in greenhouse experiments. The fungus grew through soil over a wide range of matric potentials (–0.01 to −1 MPa). When applied to leafy spurge foliage, the fungus blighted young adventitious shoots, but not mature stems. Foliar infection required high relative humidity. Mycelial growth on plant surfaces stopped at below 92% relative humidity. Pathogenicity of R230 was not limited to leafy spurge. The fungus caused stem and foliar lesions on all crops tested (alfalfa, smooth bromegrass, corn, Kentucky bluegrass, soybean, tall fescue, and wheat) when plants were kept under high humidity. It reduced seedling emergence and survival in soybean, Kentucky bluegrass, and tall fescue. R230 also caused a foliar blight on spotted spurge. The potential for R230 to damage crops will limit application of the fungus to noncultivated areas and to sites where it is endemic.

In greenhouse experiments, conidia of Colletotrichum truncatum applied in an invert emulsion formulation controlled hemp sesbania 100% in the absence of a dew treatment. In field experiments, hemp sesbania control averaged 95 and 97% in 1989 and 1990, respectively, when this formulation was applied to hemp sesbania seedlings using tractor-mounted, air-assist nozzles. This level of weed control was comparable to that achieved from the herbicide acifluorfen. These results indicate that C. truncatum has excellent potential as a mycoherbicide for controlling hemp sesbania and that this potential can be augmented by formulating the pathogen as an invert emulsion.

Johnsongrass seedlings were inoculated with conidia of four fungi, Exserohilum turcicum (Et), Colletotrichum graminicola (Cg), Gloeocercospora sorghi (Gs), and/or Bipolaris halepense (Bh). Leaf injury from Et was the most extensive. Johnsongrass was equally or more susceptible to these fungi 7 days after emergence (DAE) than at 14, 21, or 28 DAE. The greatest leaf injury, more than 90%, was induced by Et with 2 × 105 conidia/ml and a 24-h dew period. Sequential inoculation with Et at 15 and 20 DAE resulted in injury to more leaves than a single inoculation 15 DAE. No synergistic or antagonistic effects were observed in combinations of Et with Gs or Cg. Injury was limited to exposed leaves and sheaths, injured seedlings resumed growth and developed new leaves. Development of new leaves from inoculated plants was reduced by 30% or less at 14 days after inoculation (DAI) compared to uninoculated plants. Inoculation with Et or Bh was not as effective as paraquat or sethoxydim, since treatment with both herbicides resulted in seedling death. Seedling kill was not observed in any fungal tests.

Latent infections of Colletotrichum gloeosporioides f.sp. malvae (C. g. malvae) in plants and seed were determined in several crops under field conditions after application of C. g. malvae conidia at 12 times the recommended rate. Colletotrichum g. malvae was isolated in trace amounts from Brassica napus/campestris, Linum usitatissimum, Lens culinaris, Brassica juncea/hirta, Helianthus annuus, and Triticum spp. only at the 2-wk isolation. Colletotrichum g. malvae was never isolated from Beta vulgaris. After 5 wk, C. g. malvae was not detected in any of the crops, except for Carthamus tinctorius at harvest, and was not detected from wash water of plant tissue or seed after 72 h. Colletotrichum g. malvae recovery in the field, as in controlled environmental conditions, decreased with time, which indicates that buildup of inoculum did not occur. Fungi other than C. g. malvae and bacteria isolated from seed and from wash water of plant tissue increased with time throughout the growing season. Colletotrichum g. malvae was not recovered from seed or crowns of any of the crop cultivars at either location in either growing season, except for C. tinctorius. Only one seed of the C. tinctorius cultivar ‘S-208’ out of 17,280 C. tinctorius seed analyzed over three growing seasons tested positive for C. g. malvae. Colletotrichum g. malvae was found only on the seed coat; the germinated seedling was not infected. Colletotrichum g. malvae was not recovered from any of the wash water from seed. These results should alleviate any of the concerns regarding the fate of the millions of conidia applied during a normal spray operation on nontarget plants, the potential infectivity of nontarget plants, the potential buildup of C. g. malvae in the field, and the utilization of any of the C. g. malvae-treated crops for food or feed.

Teliospores of Sphacelotheca holci Jack. [= S. cruenta (Kuhn.) Potter] infected johnsongrass [Sorghum halepense (L.) Pers. # SORHA) systemically after inoculation of cut stems in greenhouse studies. A preliminary host range test in the greenhouse revealed that no infection occurred on four varieties of sorghum (S. vulgare L.) and three cultivars of sudangrass (S. Sudanese L.). Infected johnsongrass plants grown in the greenhouse exhibited reduced vegetative growth and altered morphological characteristics compared to healthy johnsongrass plants. The aboveground biomass and lateral rhizome expansion of smutted johnsongrass field transplants were significantly lower than that of uninfected plants. Significant correlations between fraction of johnsongrass stems infected and plant weight, and basal area of plants were observed in field studies. Corn (Zea mays L.) and alfalfa (Medicago sativa L.) grown in competition with johnsongrass, and johnsongrass infected with S. holci reduced the aboveground biomass and rhizome length of johnsongrass plants significantly, compared to that of uninfected johnsongrass plants grown alone in greenhouse studies. Uninfected johnsongrass plants also exhibited higher plant weight than infected plants when both were grown in competition with alfalfa, regardless of the alfalfa planting density in the greenhouse or when measured after 3 months in field trials.

A method is described for the large-scale production of a granular formulation of Alternaria macrospora Zimm. The granular preparation consisted of spores, mycelia, and vermiculite. This formulation of A. macrospora, applied preemergence or postemergence, controlled spurred anoda (Anoda cristata Schlecht.) plants in greenhouse and field tests. The pathogen did not significantly affect emergence or dry weight production of cotton (Gossypium hirsutum L. ‘Stoneville 213’) seedlings. The fungicides PCNB (pentachloronitrobenzene) and ETMT [5-ethoxy-3-(trichloromethyl)-1,2,4-thiadiazole] did not significantly affect spurred anoda control with A. macrospora. The method for production and formulation of A. macrospora was modified to produce spores and granular formulations of other Fungi Imperfecti: Drechslera sp. and Fusarium spp. (Moniliales), Colletotrichum spp. (Melanconiales), and Phyllosticta sp. (Sphaeropsidales).

An indigenous soil-borne fungus was isolated from infected seeds and seedlings of Texas gourd [Cucurbita texana (A.) Gray] and evaluated as a mycoherbicide. The pathogen was identified as Fusarium solani App. & Wr. f. sp. cucurbitae Snyd. & Hans. Only Cucurbita species were susceptible in host range tests. Ample quantities of microconidia for greenhouse and field plot inoculum were produced in shake culture on Richards' solution. Granular inoculum for field plot tests was produced in 5% (w/w) cornmeal/sand medium. In controlled-environment experiments, inoculated Texas gourd seedlings were killed over a range of air temperatures from 16 to 40 C. Optimum air temperature for disease development was 26 to 30 C. Complete kill of 4- and 8-cm-tall seedlings was achieved after 3 weeks when sprayed until runoff with a microconidial suspension containing 2.0 × 106 spores/ml. Texas gourd seedlings were effectively controlled in field plots (99% maximum) with liquid or granular inoculum. Soil samples from infested plots indicated that the fungus persists up to 12 months in a fine sandy loam, but after 12 months inoculum levels were insufficient to cause disease on seedlings grown under optimum conditions for disease development.

Formulations of Fusarium oxysporum Schlechtend:Fr. f.sp. erythroxyli isolate EN-4, pathogenic to Erythroxylum coca var. coca (coca) were evaluated in greenhouse and field studies to determine their relative ability to enhance pathogen populations in the soil or cause disease in coca. The formulations rice-alginate prill, Pesta, and C6 were most thoroughly tested and are products of three formulation processes. The formulations were applied in the greenhouse and field at 33.6 kg ha−1. All the formulations tested enhanced the population of EN-4 in the soil during greenhouse (> 6 wk) and field (> 7 mo) experiments. EN-4 was present in the upper 1 cm of formulation-treated soil at > 200-fold higher populations than established below a depth of 7 to 10 cm in both greenhouse and field experiments. This population distribution was maintained throughout the 7-mo sampling period for field experiments. The enhanced soil populations of EN-4 were associated with an increase in root colonization by the pathogen EN-4 in the field 5 to 7 mo after treatment. The formulated F. oxysporum began to have a significant effect on plant death 100 to 200 d after application in two of three field experiments, based on repeated measures analysis. The various formulations tested tended to establish similar pathogen populations in soil and subsequently caused similar levels of disease. The primary factors influencing formulation performance may be environmental, since inoculum production is dependent on sporulation of the formulation, which, although rapid, may continue over several weeks subsequent to application.

A method is described to produce inoculum of Alternaria cassiae Jurair and Khan for biocontrol studies. Approximately 8 g of a conidial preparation containing 1 × 108 conidia/g were produced per liter of growth medium. In greenhouse studies the effectiveness of A. cassiae as a biocontrol agent for sicklepod (Cassia obtusifolia L.) was affected by dew-period temperature, dew-period duration, inoculum concentration, and the stage of growth of sicklepod seedlings at the time of inoculation. Optimum environmental conditions included at least 8 h of free moisture at 20 to 30C. Spray mixtures containing approximately 5 × 104 or more conidia/ml gave maximum control when sicklepod seedlings were sprayed to runoff. Sicklepod seedlings in the cotyledon to first-leaf stage were most susceptible to the pathogen.

With few exceptions, north temperate North American weeds are native species that have proliferated following site disturbance. Social pressures are rapidly eroding the availability of essential silvicultural tools such as chemical herbicides and prescribed burning. Biological control with microorganisms in forestry is at an early stage of development. An inoculation strategy involving a fungus pathogen (PFC-082: Nectria ditissima Tul./ALDERKILL™), its formulation for biological control of Alnus rubra Bong. in the form of the PFC-MYCOCHARGE™ and a newly devised instrument for bioherbicide delivery into woody stems (PFC-ALDERWAK™) are described. The overall strategy and implements are potentially useful for the delivery of any biological or translocatable material into amenity or orchard trees as well. PFC-082 was the single isolate of N. ditissima tested that produced 100% infection and incited the formation of red alder cankers longer than 0.5 m in 30 mo when inoculated by the method described herein. Natural infections by this pathogen occur on fewer than 1% of red alder stems in the forest.

A water-corn oil (unrefined) emulsion has promise as a carrier for application of the mycoherbicide, Colletotrichum truncatum. We investigated this formulation (normal emulsion; oil in water) for influences on conidia germination, appressoria formation, and mycoherbicidal activity of C. truncatum. Germination of conidia after application to hemp sesbania leaves was increased from 30% in water alone to 92% in the water-corn oil emulsion (water:oil, 1:1, by vol). The emulsion did not affect appressoria formation. of the several other oils tested, safflower seed oil enhanced in vitro germination but did not enhance germination and mycoherbicide activity when the conidia-emulsion formulation was applied to hemp sesbania leaves. Application of C. truncatum conidia in the unrefined corn oil emulsion to hemp sesbania seedlings resulted in 89 to 97% control of the weed after a 24 to 72 h dew delay. The emulsion enhanced mycoherbicide efficacy by stimulating conidia germination by protecting the conidia during a dew-free period, hence, increasing weed infection when a dew occurred.

The fungus Colletotrichum coccodes (Wallr.) Hughes was isolated from diseased plants of eastern black nightshade (Solanum ptycanthum Dun. ♯ SOLPT) growing in the field. In greenhouse experiments, the isolate was highly pathogenic to eastern black nightshade and less so to seven other weedy species within the Solanaceae. No disease symptoms were observed on seedlings of tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), or 10 other species inoculated with the isolate. Dew periods of 16 h or longer were required for maximum disease development. This requirement could limit the practical use of this pathogen as a my coherbicide.

A native fungus, Botryosphaeria ribis, was evaluated under field conditions to determine its potential to control melaleuca. Applications consisted of either wound inoculations of trees with B. ribis or fresh stump treatments with B. ribis alone or mixed with imazapyr herbicide. There was no mortality among nondefoliated trees inoculated with B. ribis. Mortality of B. ribis-inoculated trees was increased by three complete defoliation cycles. Defoliated trees inoculated with isolate BR-5 exhibited 100% mortality compared to 17% for defoliated but noninoculated trees. Wounds inoculated with B. ribis during winter produced longer cankers than did noninoculated wounds. Stump regrowth reduction by treatment with B. ribis alone was less effective than treatment with imazapyr alone. Mixtures of B. ribis with imazapyr or imazapyr alone at comparable concentrations did not differ in stump regrowth control.

A strain of Fusarium oxysporum was isolated from infected sicklepod stems in Stoneville, MS, in 1989. When formulated as granules either in a fungus-infested rice preparation or encapsulated in a wheat-gluten matrix called ‘Pesta’, biocontrol of sicklepod, coffee senna, and hemp sesbania was achieved with preemergence or preplant-incorporated treatments. Liquid conidial applications were less effective. Plants were killed by a preemergence damping-off. Applications made postemergence were significantly less effective. Results of these tests suggest that this fungus has potential as a mycoherbicide for controlling sicklepod, coffee senna, and hemp sesbania.