Evaluation of Simplicillium lanosoniveum as a Biological Control Agent

Evaluation of Simplicillium lanosoniveum as a Biological Control Agent

Summary

Documentation of a mycoparasitic fungus determined that Simplicillium lanosoniveum, a naturally occurring fungus, colonized and parasitized Phakopsora pachyrhizi, causal agent of Asian soybean rust. We performed field trials to monitor natural infection and establishment of ASR. Also, S. lanosoniveum was applied as inoculum to determine whether the antagonist colonized and parasitized sori under field conditions. When used as an inoculum before rust symptoms occurred, S. lanosoniveum provided disease control similar to that of the fungicide Headline.

Objectives/Performance Targets

We hypothesized that S. lanosoniveum meets the criteria for a successful biological control agent. However, in order to effectively evaluate this antagonist, we examined its life cycle on the soybean leaf surface as it related to the soybean rust disease cycle under field conditions.

The objectives of this study were to:

1. Develop real time quantitative PCR (q-PCR) diagnostic test for S. lanosoniveum. Quantitatively monitor naturally occurring S. lanosoniveum in the field to determine whether it meets the criteria as a biological control agent as it interacts with P. pachyrhizi.
2. Determine the native host range for S. lanosoniveum in Louisiana. Sample and assay other rusts in Louisiana for the presence of S. lanosoniveum to determine the native host range.
3. Evaluate S. lanosoniveum in field plots. Apply S. lanosoniveum to field plots and use q-PCR to evaluate establishment and colonization and compare it to disease incidence and severity.

The evaluation of an organism for potential as a biological control agent appears frequently in recent scientific literature. Our experimental design is based on the criteria discussed by Andrews [1]. However, our approaches are unique in that P. pachyrhizi is an obligate parasite.

In Phase I of the evaluation process, antagonists are selected according to preliminary studies. Our previous research used leaf inoculations to determine the effects of S. lanosoniveum on rust sori development. Additionally, scanning electron microscope studies confirmed the trophic response of the fungus to sori.

Phase II includes the putative mycoparasite in in vivo studies under controlled laboratory conditions. Our previous work included detached leaf assays in which co-inoculations of disease-free leaves with S. lanosoniveum and P. pachyrhizi clearly demonstrated a significant decrease in development of rust sori. Additionally, inoculations of rust-infected soybean leaves revealed that S. lanosoniveum not only decreased additional lesion development, but it also encouraged a significantly higher percentage of red-brown lesions, which have been associated with resistance reactions. Moreover, the antagonist produced a significantly higher number of brown urediniospores that failed to germinate.

Phase III proposes testing of the organism under field conditions. We examined the habitation and epidemiology of S. lanosoniveum by using the q-PCR assay above to monitor the organism in the presence of soybean rust to determine whether it meets the criteria for a biological control agent, i.e. environmental tolerances and rate of establishment. Additionally, we collected rusts from other plant species to evaluate host specificity of S. lanosoniveum. Objectives 1 and 2 addressed these issues.

Accomplishments/Milestones

We used q-PCR to develop an assay to monitor naturally occurring S. lanosoniveum as well as to monitor the introduced mycoparasitic fungus under field conditions. This technique is more sensitive than serological assays or conventional PCR because it quantifies fungal DNA, and it is more reproducible than other methods.

1. A q-PCR assay was developed to examine and quantify the emergence, colonization, and establishment of S. lanosoniveum as it relates to the Asian soybean rust disease cycle. Isolates collected regionally along with additional isolates from Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands) were sequenced with primers from the internal transcribed spacer region (ITS) of the nuclear DNA repeat to identify potential probe regions. Sister taxa, as well as other phylloplane inhabitants, were evaluated to prevent false detections. Next, we designed primers and probe from these sequences and tested them for sensitivity (ability to detect small amounts of DNA) and accuracy (specificity among S. lanosoniveum isolates).

Field samples were collected weekly beginning in the early vegetative stages, August 10, 2009. Samples consisted of pooled subsamples from 30 leaflets collected from mid-canopy at the LSU AgCenter Research Farm near Baton Rouge, Louisiana. DNA was extracted from each sample to quantify the amount of P. pachyrhizi and S. lanosoniveum DNA by q-PCR (Figure 1). Results document the survival of S. lanosoniveum for 8 weeks after the first inoculation (flowering). DNA of the antagonist was detected at as much as one pictogram and as little as 200 femtograms (0.21 picograms) in all three treatments, however, there was a steep decline as soybeans began to senesce. Similarly, DNA of the rust pathogen continued to increase with time, but the earliest treatment resulted in a significantly lower amount of rust DNA by senescence (Figure 1).

2. Throughout Year 1, we sampled rusts on other plant species and assayed for the presence of S. lanosoniveum to determine its native host range. The mycoparasite was not found on other rusts.

3. Following laboratory screenings, one Louisiana isolate of S. lanosoniveum was selected for field plot inoculations for evaluation as a biological control agent. Rust establish naturally, with the first symptoms occurring on September 27, 2009.

Experimental layout was a randomized complete block design with three replications. Simplicillium lanosoniveum was introduced to 10×30 ft. field plots at the rate of one liter of a 106 spores/ml suspension per plot with a hand held garden sprayer. Three treatment times included: flowering, the first susceptible growth stage; both flowering and <5% rust incidence; and <5% rust incidence only. An unsprayed and a fungicide (Headline) treatment were included as controls.

Field plots were sampled weekly as described above. Before DNA extraction, we documented the number of rust sori per cm2. By comparing these counts to both nontreated control samples and fungicide treated samples, we determined the impact of S. lanosoniveum on disease development (Figure 2). Results indicate that the earliest inoculation with S. lanosoniveum yielded in a reduction in disease lesions equivalent to that of the fungicide. Conversely, the treatment imposed at the first incidence of disease yielded no control.

Finally, we used a visual disease severity rating scale based on percentage of affected area to evaluate field plots (Figure 3). These visual ratings were compared to quantitative measurements of S. lanosoniveum detected in q-PCR assays. Results indicate that the visual disease severity rating mimicked the quantitative sori rating. Disease severity of the early inoculation was lower than the fungicide treatment from the first disease symptoms through senescence. At the R7 stage, physiological maturity of the plant, they were equal. Unlike the quantitative treatment, however, the late inoculation also resulted in less disease than the nontreated control. It was not as effective, however, as the early inoculation or the fungicide treatment.

By conducting field trials, we assessed the effects of S. lanosoniveum on soybean rust in field-grown plants. R1 lowered disease and amount of rust DNA. Although we anticipated colonization on the phylloplane, there was no consistent visual colonization by S. lanosoniveum. In 2010, we will evaluate various inoculum formulations containing mycelial fragments and/or conidial spore suspensions.

• Figure 1. Quantitative measurements of DNA of (A) Simplicillium lanosoniveum and (B) Phakopsora pachyrhizi as determined by realtime PCR (q-PCR). Figure reflects measurements taken between pod formation (R4) and physiological maturity.
• Figure 3. Effects of Simplicillium lanosoniveum on soybean rust in 2009 field trials. Lesions per sq cm2 were used as a quantitative severity rating, and S. lanosoniveum proved effective as a biological control agent against the rust pathogen. First symptoms of rust appeared on 9/27/2009, R5 stage of growth.
• Figure 2. Effects of Simplicillium lanosoniveum on soybean rust in 2009 field trials. Lesions per sq cm2 were used as a quantitative severity rating, and S. lanosoniveum proved effective as a biological control agent against the rust pathogen. First symptoms of rust appeared on 9/27/2009, R5 stage of growth.

Impacts and Contributions/Outcomes

Phakopsora pachyrhizi was first discovered in North America in Louisiana in 2004. Since then, it has been shown to overwinter in the Gulf South, spreading to 10 to 20 states each year, including the largest producers of soybean, the Midwestern states. Because of preventative fungicide applications, potential for disease spread and destruction has been minimized. Efficacy of fungicides varies with application time, which may lead to unnecessary or ineffective preventive spray applications. However, this continual overuse of pesticides decreases populations of natural enemies, which suppress populations of harmful organisms. There is also the threat of fungicide resistance and secondary pest outbreaks as well as safety and environmental considerations. Nevertheless, an estimated 21,000 tons of fungicides are applied to soybeans in the US each year. Less than 1% are biologically based.

Of the 76 million acres of soybeans planted in 2009 in the US, over 13.5 million acres are grown in SARE’s Southern Region. Current research has determined that preventative applications of fungicides are necessary to protect crops against infection by P. pachyrhizi. Typically, chemical fungicides are applied at approximately 13.5 oz per acre for the control of soybean rust, which is equivalent to approximately 11.5 million pounds per application in the Southern Region alone. In high risk areas, two treatments are applied. Furthermore, there are no curative fungicides effective beyond 5% infection. The dilemma of excessive pesticide applications is detrimental to both environmental quality and human health.

The development of S. lanosoniveum as a biological control agent has the potential to directly reduce these excessive pesticide applications. Elucidating the in planta relationships between the mycophilic fungus and soybean rust is crucial in determining whether the antagonist constitutes an effective biological control agent. Our research objectives include the development of an assay to monitor the establishment of this antagonist on the phylloplane and an exploratory field trial to monitor its colonization. Thus, our ultimate goal is to develop a biological control agent to be used as an alternative to preventative fungicides. In an effort to provide alternatives to inefficient and unsustainable practices, we aim to provide sustainable solutions to not only small niche markets, but also to large agribusinesses, which will have substantial environmental impacts on soil and water quality; wildlife, including endangered species; human health and safety; grower income resulting from fewer sprays and reduction of secondary pests, as well as new opportunities like production of specialty and organic produce.

Key findings indicate that the early inoculation (flowering stage) with S. lanosoniveum delayed disease and resulted in reduced sori (lesion) count by physiological maturity of the soybean. Simplicillium lanosoniveum survived 8 weeks in the early inoculation, and ultimately resulted in the lowest disease severity, lowest amount of P. pachyrhizi DNA, and the highest amount of DNA of S. lanosoniveum.

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