Recent Advances in the Use of Plant Pathogens as Biocontrol Agents of Weeds

Abstract

Among various organisms attacking skeleton weed, Chondrilla juncea, in Europe, a rust fungus, Puccinia chondrillina, and two powdery mildews, Erysiphe cichoracearum and Leveillula taurica f. sp. chondrillae were found to be the most damaging. P. chondrillina was shown to be specific to Chondrilla and has recently been introduced into Australia where skeleton weed is important in wheat cultivation. It has already spread throughout the weed infestations and is beginning to reduce plant populations. Encouraged by the result of the Chondrilla rust, the possible use of Puccinia xanthii for the control of Xanthium strumarium and X. spinosum has also been studied. Current research on the possible use of phytopathogens in the biological control of some other weeds is reviewed.     

A greenhouse screening technique to assess rust resistance in sorghum∗

Abstract

Studies were conducted to determine the influence of plant growth stage, inoculum density, temperature, and relative humidity (RH) on development of rust (Puccinia pupurea) in sorghum (Sorghum bicolor). Rust development was maximum (>80% severity), when plants of a susceptible sorghum genotype (IS 18420) were inoculated at the four‐ to five‐leaf stage with an inoculum concentration of 4 × 10⁶ urediniospores per ml and incubated at 20–25°C under high RH (>90%) for 24 h. Disease severity (percentage leaf area covered with rust pustules) scores were taken 2 weeks after inoculation. Using this technique, 29 sorghum genotypes were screened for rust resistance in a greenhouse. This technique proved effective In discerning resistant and susceptible genotypes, and IS 3979, ICSH 110, ICSH 86647 and ICSH 871035 were identified resistant (<20% rust severity) compared with a susceptible control IS 18420 (90% rust severity). This technique is simple and rapid, and can be used effectively and economically to screen, on a large scale, germplasm lines and breeding populations in the greenhouse.     

Pre-inoculation with Hyaloperonospora parasitica reduces incubation period and increases severity of disease caused by Albugo candida in a Brassica juncea variety resistant to downy mildew

Studies were undertaken to determine any interaction of an asymptomatic isolate of Hyaloperonospora parasitica (downy mildew) with a virulent isolate of Albugo candida (white rust) and Brassica juncea. White rust symptoms appeared 4 days earlier and were significantly more severe when a variety of B. juncea resistant to downy mildew but highly susceptible to white rust was first inoculated with A. candida followed 10 days later with H. parasitica. DNA extractions of tissues indicated H. parasitica had colonized the asymptomatic plants systemically. These are the first reports of (a) the systemic colonization by H. parasitica in a host resistant to downy mildew, and (b) the increase in susceptibility of a Brassica to white rust disease from asymptomatic colonization by H. parasitica.     

Effects of host plant genotype and seedbank density on Striga reproduction

Prevention of seed input to the seedbank of Striga hermonthica‐infested fields is an important objective of Striga management. In three consecutive years of field experimentation in Mali, Striga reproduction was studied for 10 sorghum genotypes at infestation levels ranging from 30 000 to 200 000 seeds m^?2. Host resistance was identified as an important determinant of Striga reproduction, with the most resistant genotypes (N13, IS9830 and SRN39) reducing Striga reproduction by 70–93% compared with the most susceptible genotype (CK60‐B). Seedbank density had a significant effect on Striga seed production. Higher seedbank density resulted in more Striga plants, which led to increased intra‐specific competition and consequently a reduced level of reproduction per plant. For the most susceptible sorghum genotypes, density dependence also occurred in the earlier belowground stages. Striga reproduction continued beyond harvest. At the high infestation level just 8% of the total reproduction was realised after harvest, whereas at the low infestation level 39% was attained after harvest. Even though host‐plant genotype plays a significant role in Striga reproduction, calculations indicated that only at very low infestation levels the use of the most resistant genotype was able to lower the Striga seedbank.     

Unique infection structures produced by Pseudocercosporella capsellae on oilseed crops Brassica carinata,B. juncea and B. napus in Western Australia

White leaf spot disease (Pseudocercosporella capsellae) is widespread across oilseed, vegetable and forage brassicas. Light (LM) and scanning electron (SEM) microscope studies were undertaken to investigate host–pathogen interactions on cotyledons of resistant and susceptible Brassica carinata, B. juncea and B. napus. Under LM, unique brown structures were present, particularly on susceptible genotypes, in two morphologically distinct forms: first, as thread‐like structures within cortical tissue by 24 h post‐inoculation (hpi) and secondly, as brown ropy strand structures either within cortical tissues (internal ropy strands), or extruded out through stomatal pores (ropy strand extrusions). Under LM, these brown structures were most prevalent in highly susceptible B. juncea ‘Vardan’ that had both a high incidence within cortical tissue (70%) and of ropy strand extrusions (73%), as did susceptible B. napus ‘Trilogy’ within cortical tissue (60%). Under SEM, both these genotypes showed thread‐like structures smaller than hyphae forming highly branched networks and ropy strand‐like structures. While there were fewer brown structures in susceptible B. carinata UWA #012 (35%), fine, thread‐like structures forming networks were again prominent (SEM). In contrast, for resistant genotypes, brown structures (LM) were of very low frequency or absent; only 5% in resistant B. juncea ‘Dune’ and none in resistant B. napus ‘Hyola 42’ or highly resistant B. carinata ATC94129P. Under SEM, fine, thread‐like structures were present in the resistant B. juncea ‘Dune’ and B. napus ‘Hyola 42’. Liquid chromatographic analyses of brown structures revealed that both internal ropy strands within cortical tissues and ropy strand extrusions contained the mycotoxin cercosporin.     

First release of a fungal classical biocontrol agent against an invasive alien weed in Europe: biology of the rust,Puccinia komarovii var. glanduliferae

The rust fungus Puccinia komarovii var. glanduliferae was first identified infecting Impatiens glandulifera in its native range (western Himalayas) between 2006 and 2010. Subsequently, it was imported into quarantine in the UK for evaluation as a classical biocontrol agent. To assess the safety of the rust, plant species relevant to Europe were tested for susceptibility. To confirm the life cycle, all infective spore stages were inoculated on I. glandulifera to follow disease progression. Teliospores were primed using bleaching and low temperatures to break dormancy. Temperature and dew period experiments using urediniospores were conducted to assess the parameters required for infection. Of the 74 plant species tested, only I. balsamina, an ornamental species, was fully susceptible to urediniospore inoculum. The life cycle of the rust – an autoecious, full‐cycled species with five spore stages – was confirmed. Urediniospores were infective between 5 and 25°C, with an optimum at 15°C. A minimum of 8 h dew period was required to achieve consistent infection. Based on a pest risk assessment, the rust poses no threat to native biodiversity within EU Member States; making P. komarovii var. glanduliferae a suitable candidate as the first fungal classical biocontrol agent against an exotic weed in the region.     

Infection process of Phoma koolunga on stem and leaf tissue of resistant and susceptible field pea (Pisum sativum)

This study investigates the infection process of Phoma koolunga on field pea (Pisum sativum) stems and leaves using different susceptible and resistant pea genotypes for each tissue, viz. 05P778‐BSR‐701 (resistant) and 06P830‐(F5)‐BSR‐5 (susceptible) for stems and ATC 866 (resistant) and ATC 5347 (susceptible) for leaves. On both resistant and susceptible genotypes, light and scanning electron microscopy showed P. koolunga conidia infect stem and leaf tissues directly via appressoria or stomatal penetration, but with more infections involving formation of appressoria on stems than on leaves. On leaves of the resistant genotype, at 72 h post‐inoculation, P. koolunga penetrated more frequently via stomata (5.2 conidia per 36 893 μm²) than by formation of appressoria (1.8 conidia); yet no such difference was observed on stems of the resistant genotype. In contrast, at the same time point, the number of conidia infecting the susceptible genotype by formation of appressoria on either stems (135 conidia) or leaves (11.3 conidia) was significantly greater than via stomata (8 and 7.3 conidia, stems and leaves, respectively). Mean germ tube length of germinating P. koolunga conidia on both stems (29.8 μm) and leaves (32.9 μm) of the resistant genotype was less than on the susceptible genotype (40.5 and 63.7 μm, stem and leaves, respectively). In addition, there were differences in the number of germ tubes emerging from conidia on resistant and susceptible genotypes. These are the first insights into the nature of leaf and stem resistance mechanisms operating in field pea against P. koolunga.     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

Selection pressure effects on the proportion and movement of resistance alleles introgressed from wheat in Aegilops cylindrica

In winter wheat in the USA, Aegilops cylindrica is one of the most troublesome weeds, while the pathogen Oculimacula spp. causes foot rot disease. Imazamox‐resistant (IR) and foot rot‐resistant (FR) wheat cultivars represent effective tools to control the weed and prevent disease infection. However, resistance allele (RA) movement between wheat and A. cylindrica facilitates the introgression process under herbicide and disease selection pressure. Field experiments using IR and FR A. cylindrica plants intermixed with susceptible plants were conducted to measure the proportion of the RAs in the progeny and RA movement with and without herbicide and disease selection. Yield components of A. cylindrica plants were determined across treatments. The herbicide RA proportion in the progeny was greater when plants were treated with the herbicide imazamox in both years. Disease RA proportion was greater with disease occurrence only in one year. Herbicide RA movement from resistant to susceptible plants was greater with herbicide than without it only in one year. Plants carrying the RAs had greater total spikelet weight and 1000‐spikelet weight compared with susceptible plants with or without selection. However, susceptible plants produced more spikelets than the resistant ones in the absence of selection. If plants within an A. cylindrica population acquire the herbicide RA, its proportion will increase each generation under selection. These findings contribute to the understanding of crop allele introgression into related species and the evolution of increased weediness, with weed management implications.     

Quantitative reproductive potential of Heterodera schachtii on weeds typical for late summer fallow in sugar beet rotations

In Europe, sugar beet is often produced in a 3‐year rotation with cereals, leaving stubble fields fallow from cereal harvest until primary tillage in autumn in the year prior to sugar beet production. The weed flora on such fields could include host plants of Heterodera schachtii that is one of the most important pests of sugar beet. Crop sequences with non‐hosts and cover cropping with resistant cruciferous hosts during this period have been crucial for its management. Availability of resistant and tolerant sugar beet cultivars could entice growers to forego cover cropping, exacerbating weed problems during the fallow period. The objective of this study was to determine the reproductive potential of H. schachtii on weeds that develop during this period. Under glasshouse conditions, reproduction on 39 plant species was compared with that on oilseed radish and sugar beet of differing nematode host status. In 2 years in field microplots, 18 previously tested species were grown in H. schachtii‐infested soil during the typical fallow period at 60 plants m^?2, and nine of these species were also grown at 180 plants m^?2. There were variable results between years after 8 weeks of growth, but most weeds allowed lower reproduction (<10%) than the susceptible sugar beet; only Stellaria media at 180 plants m^?2 and Thlaspi arvense at both plant densities increased nematodes. Such weed densities may seldom occur under commercial conditions; thus, weed management for nematological considerations during the stubble period may have limited importance.     

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas

BACKGROUND: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non‐crop systems. A glyphosate‐resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS: The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6‐fold higher basal EPSPS enzyme activities than the susceptible plants, but their I₅₀ values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION: Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate‐resistant Italian ryegrass. Other resistance mechanism(s) may also be involved. Copyright © 2012 Society of Chemical Industry     

Enhanced weed‐crop competition effects on growth and seed production of herbicide‐resistant and herbicide‐susceptible annual sowthistle (Sonchus oleraceus)

Enhanced crop competition could aid in the management of annual sowthistle (Sonchus oleraceus L.), a dominant weed of Australian cropping systems. A two‐year pot study was conducted to evaluate the effect of wheat (Triticum aestivum L.) planting densities (0, 82, and 164 wheat plants/m²) on growth and seed production of glyphosate‐resistant (GR) and glyphosate‐susceptible (GS) biotypes of annual sowthistle. Without competition, both biotypes produced a similar number of leaves and biomass, but the GS biotype produced 80% more seeds (46,050 per plant) than the GR biotype. In competition with 164 wheat plants/m², the number of leaves in the GR and GS biotypes was reduced by 62 and 61%, respectively, in comparison with the no‐competition treatment, and similarly, weed biomass was reduced by 78 and 77%, respectively. Compared to no‐competition treatment, the seed production of GR and GS biotypes was reduced by 33 and 69%, respectively, when grown with 82 wheat plants/m², but increasing wheat density from 82 to 164 plants/m² reduced the number of seeds only in the GS biotype (81%). Both biotypes produced greater than 6,000 seeds per plant when grown in competition with 164 plants/m², suggesting that increased crop density should be integrated with other weed management strategies for efficient control of annual sowthistle.     

Three-way fungal interactions affect the potential biological control of Himalayan balsam,Impatiens glandulifera

Himalayan balsam (Impatiens glandulifera) is one of the most invasive weeds across Europe. The rust fungus, Puccinia komarovii var. glanduliferae has been introduced as a biological control agent, but success has been patchy. Here, we investigated whether mycorrhizal and endophytic fungi can affect rust efficacy and plant growth. Over three experiments, we found that AM fungi and the rust alone or together consistently reduced plant growth, but this depended on the identity of species in the AM inoculum. Meanwhile, AM fungi increased infection frequency of the endophyte Colletotrichum acutatum. Rust inoculation had no detrimental effects on mycorrhizal colonisation or C. acutatum infection, but the latter two fungi reduced rust sporulation. However, plant size was reduced when all three fungal types were present, suggesting that a combined fungal inoculum offers a promising approach for the control of this weed.     

Control by glyphosate and its alternatives of glyphosate‐susceptible and glyphosate‐resistant Echinochloa colona in the fallow phase of crop rotations in subtropical Australia

Echinochloa colona is the most common grass weed of summer fallows in the grain‐cropping systems of the subtropical region of Australia. Glyphosate is the most commonly used herbicide for summer grass control in fallows in this region. The world's first population of glyphosate‐resistant E. colona was confirmed in Australia in 2007 and, since then, >70 populations have been confirmed to be resistant in the subtropical region. The efficacy of alternative herbicides on glyphosate‐susceptible populations was evaluated in three field experiments and on both glyphosate‐susceptible and glyphosate‐resistant populations in two pot experiments. The treatments were knockdown and pre‐emergence herbicides that were applied as a single application (alone or in a mixture) or as part of a sequential application to weeds at different growth stages. Glyphosate at 720 g ai ha^?1 provided good control of small glyphosate‐susceptible plants (pre‐ to early tillering), but was not always effective on larger susceptible plants. Paraquat was effective and the most reliable when applied at 500 g ai ha^?1 on small plants, irrespective of the glyphosate resistance status. The sequential application of glyphosate followed by paraquat provided 96–100% control across all experiments, irrespective of the growth stage, and the addition of metolachlor and metolachlor + atrazine to glyphosate or paraquat significantly reduced subsequent emergence. Herbicide treatments have been identified that provide excellent control of small E. colona plants, irrespective of their glyphosate resistance status. These tactics of knockdown herbicides, sequential applications and pre‐emergence herbicides should be incorporated into an integrated weed management strategy in order to greatly improve E. colona control, reduce seed production by the sprayed survivors and to minimize the risk of the further development of glyphosate resistance.     

Host specificity of Puccinia canaliculata,a potential biocontrol agent for Cyperus esculentus

Aim was to clarify the rust fungusPuccinia canaliculata for release in a new environment where it may be utilized as a microbial herbicide. A strain of the rust was introduced from the USA into the Netherlands and kept in quarantaine. The susceptibility ofCyperus esculentus and several other species of the Cyperaceae was tested. C. esculentus leptostachyus from five locations was susceptible toP. canaliculata. C. esculentus of a yet unidentified biotype from two locations was moderately susceptible (fewer and smaller pustules than on leptostachyus), whereas plants of the same biotype from a third location were resistant to the rust.C. esculentus esculentus (from one location) andC. esculentus sativus (a crop) were also resistant to the rust. C. albostriatus, C. alternifolius, C. flavescens, C. rotundus, Carex hirta, Eleocharis palustris, andScirpus maritimus were resistant toP. canaliculata. OnCyperus fuscus, P. canaliculata produced very small urediniosori (less then 1 mm in diameter); the sori were surrounded by a zone of necrotic plant tissue.From the observations onC. fuscus we concluded that the (potential) host range ofP. canaliculata is wider than we originally expected. For safety reasons, it was decided not to release it in the Netherlands.     

Endophytic fungi in the invasive weed Impatiens glandulifera: a barrier to classical biological control?

The rust fungus, Puccinia komarovii var. glanduliferae, has been introduced into the UK for biological control of the invasive weed, Impatiens glandulifera (Himalayan balsam). However, establishment of the pathogen has differed across the country, which may be partly explained by variation in plant genotype. The aim of this study was to examine whether there is a further layer of phenotypic resistance, provided by indigenous foliar endophytic fungi. Culturable endophytes were isolated from a number of different balsam populations, and the commonest species were inoculated into ‘clean’ balsam plants, to test their interactions with the rust. We found that endophyte communities within balsam are low in diversity and become more dissimilar with increasing distance between populations. Three endophytes (Colletotrichum acutatum, Alternaria alternata and Cladosporium oxysporum) were common and appeared to be antagonistic to the rust, reducing pustule number and mitigating the effect of the pathogen on plant biomass. I. glandulifera thus partially conforms to the endophyte-enemy release hypothesis, in that as an introduced species, it has an impoverished endophyte complement, acquired from the local environment. However, these endophytes represent a potential barrier to effective biological control and future weed control strategies need to find strains of rust that can overcome plant genetic resistance and the overlaying phenotypic resistance, conferred by endophytes. Future classical biological control programmes of weeds must therefore take into account the fungal bodyguards that invasive species may acquire in their introduced ranges.     

Mechanisms of resistance in Brassica carinata,B. napus and B. juncea to Pseudocercosporella capsellae

Studies were undertaken to compare susceptible and resistant host responses to Pseudocercosporella capsellae in cotyledons of Brassica carinata, B. juncea and B. napus in order to define the mechanisms of resistance in these three species. On both resistant and susceptible hosts, hyphal penetration was always through stomatal openings and without infection pegs or appressoria. On resistant B. carinata ATC94129P, up to 72% of spores disintegrated and, generally, germination (<22%) and germ tube lengths (<25 μm) were comparatively low. Resistant B. napus Hyola 42 had the lowest germination (8%) and susceptible B. carinata UWA#012 had the highest (51%). On resistant B. carinata ATC94129P, germ tube extension was impeded across 24–60 h post‐inoculation (hpi) and percentage stomatal penetration lower (4%) at 60 hpi compared with susceptible B. carinata UWA#012 (26%). Stomatal densities (stomata/14 757 μm²) on resistant B. juncea Dune (2·12) and B. napus Hyola 42 (1·62) were lower than for susceptible B. juncea Vardan (2·40) and B. napus Trilogy (2·03). Resistant B. carinata ATC94129P had greater stomatal density (1·89) than susceptible B. carinata UWA#012 (1·58). Overall, B. juncea had greater stomatal density (2·26) compared with B. napus (1·83) and B. carinata (1·74). In resistant B. carinata ATC94129P, P. capsellae induced 28% stomata to close, while in susceptible B. carinata UWA#012 no such closure was induced. Epicuticular wax crystalloids were present only on resistant B. carinata ATC94129P and probably also contribute towards resistance.     

Seed germination behavior of glyphosate‐resistant and susceptible Italian ryegrass (Lolium multiflorum Lam.)

Ryegrass (Lolium multiflorum Lam.) is one of the most difficult annual weeds to control in cultivation systems worldwide, especially in temperate regions. The widespread use of herbicides in the past two decades has selected resistant biotypes of ryegrass in crops in Southern Brazil. Ryegrass seeds are dormant when disseminated and germination can be staggered over time (crop‐growing season). Knowledge of the germination behavior of seeds from herbicide‐resistant plants has been little studied, but it would be very useful in integrated weed management. Thus, this study aimed to characterize the dynamics of the soil seed bank of two biotypes of L. multiflorum, one glyphosate‐resistant and the other glyphosate‐susceptible, under a no‐tillage system. The treatments were arranged in a bifactorial scheme, using seeds from biotypes (glyphosate‐resistant and glyphosate‐susceptible) with monthly periods of removal from field (one to 12 months). Seeds of each biotype were placed on the soil surface and covered with soil and straw to simulate no‐till conditions. The percentage of germinated, dormant, and dead seeds was evaluated every 30 days. The ryegrass seed bank of glyphosate‐susceptible and glyphosate‐resistant biotypes was reduced to 11 and 15% of dormant seeds, respectively, at the end of 12 months. However, there was no variation in germination, dormancy, and seed mortality between susceptible and glyphosate‐resistant ryegrass. Seeds of glyphosate‐resistant biotype and susceptible showed germination behavior with similar dynamics in the soil over a period of 12 months.     

Testing the performance of registered herbicides on the control of Campuloclinium macrocephalum (Asteraceae) in South African grasslands

We report on the performance of the registered herbicides picloram and metsulfuron‐methyl on the control of Campuloclinium macrocephalum (pompom weed) in grasslands. Herbicide trials in hydric and xeric grasslands were treated for three consecutive years in either summer (February) or autumn (April) and monitored for an additional 3 years after spraying ceased. Uncontrolled factors such as a host‐specific rust, fire and drought were observed from the second year of the study. Metsulfuron‐methyl and picloram did not differ in efficacy according to the timing of applications, but average mortality of marked plants was <80% expected of registered herbicides. Populations of C. macrocephalum in plots were reduced proportionately to the percentage mortality of marked plants. Picloram and metsulfuron‐methyl applied at 252 g a.i. ha^?1 and 45 g a.i. ha^?1, respectively, were not detectable by gas chromatography in the upper 25 cm of the soil profile during any of the sampling intervals from 0 to 56 days after treatment. Three annual applications of registered herbicide did not reduce C. macrocephalum successfully, and it is estimated that between five (summer) to seven (autumn) annual treatments are required to reduce weed density to <1 plant per plot (25 m²). Future research should focus on rust–herbicide interactions, the role of fire in seedbank management and fire as a treatment that could be integrated with chemical control.