Final Report for GS09-085
The following is the resulting publication from the study “Evaluation of Simplicillium lanosoniveum as a Biological Control Agent.” It was submitted to the journal Biocontrol on 6-16-11.
Abstract: Simplicillium lanosoniveum (van Beyma) Zare & W. Gams (order Hypocreales, family Cordyciptaceae) is an antagonistic inhabitant of uredinial sori (pustules) of soybean rust (Phakopsora pachyrhizi). In previous laboratory studies, co-inoculations with S. lanosoniveum and P. pachyrhizi resulted in urediniospores that failed to germinate and fewer disease lesions. In the present field study, we used quantitative real time PCR to monitor colonization of S. lanosoniveum in diseased, field-grown soybean. Following inoculation with conidial inoculum of the antagonist, the fungus colonized leaf surfaces when plants were infected with P. pachyrhizi, either in a latent stage of infection or with disease symptoms. However, when plants were inoculated before infection, there was no increase of DNA of S. lanosoniveum suggesting that the pathogen must be present in order for the antagonist to establish itself on soybean leaf surfaces. Furthermore, we documented significantly lower amounts of DNA of P. pachyrhizi and lower disease severity when soybean leaves were colonized with S. lanosoniveum.
Soybean rust (SBR), caused by Phakopsora pachyrhizi authority, was first reported in Japan in 1904 and has since spread throughout Asia, Africa, and the Americas (Miles et al. 2008; Yorinori et al. 2005). Disease losses ranged from 10 to 90 %, though there are reports as high as 100% (Miles et al. 2008). SBR was first discovered in the US in 2004, where it quickly became established in the southeastern states (Schneider et al. 2005; Schneider et al. 2008; USDA). Yield losses have been reported between 35 and 40% in Louisiana and as high as 82% in Florida on susceptible varieties that were not sprayed with fungicides (Walker et al. 2011) (R. W. Schneider, D. R. Walker, personal communication).
Breeding efforts have yet to produce resistant cultivars (Hartman et al. 2005). Therefore, disease management studies have focused mainly on fungicide applications. These studies showed that preventative applications of protectant fungicides must be accurately timed and applied very early in the infection process for effective control of the disease (Schneider et al. 2008). This may lead to unnecessary fungicide applications, especially throughout the southern US, because growers fear rapidly escalating epidemics such as those seen in Africa and Asia.
An antagonistic fungus, Simplicillium lanosoniveum (van Beyma) Zare & W. Gams (order Hypocreales, family Cordyciptaceae), which colonized uredinial sori (pustules) of SBR and penetrated urediniospores was discovered in 2007 in soybean from Louisiana and Florida (Ward et al. 2011; Ward 2009) (Ward Phytopath, accepted for publication). Using detached leaf assays, we observed a reduction in production of sori in the presence of S. lanosoniveum as well as a significant reduction in viability of urediniospores. The objective of this study was to evaluate the effects of S. lanosoniveum under field conditions. Development of S. lanosoniveum and the rust pathogen using quantitative real-time PCR (qPCR), as well as visual disease ratings were monitored.
The objective of this study was to evaluate the effects of S. lanosoniveum under field conditions. Development of S. lanosoniveum and the rust pathogen using quantitative real-time PCR (qPCR), as well as visual disease ratings were monitored.
Primer and Probe Development
Simplicillium lanosoniveum. To develop highly specific primers and probe for detection and quantification of S. lanosoniveum, the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA gene repeat were sequenced from five isolates of S. lanosoniveum that had been recovered from soybean leaves collected in Louisiana and Florida, as well as two additional isolates (CBS101895 and CBS70486) obtained from Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) (Ward et al. 2011). Two other species of Simplicillium, (S. obclavatum (CBS 51082) and S. lamellicola (CBS 13837), and other phylloplane inhabitants (Fusarium spp. and Cladosporium spp.) were evaluated to eliminate overlapping sequences and to prevent false positives. Genomic DNA was extracted from 14-day old mycelia using Promega Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). Internal transcribed spacer primer pairs ITS1-F and ITS4 were used for DNA amplification (Gardes and Bruns 1993). PCR product was purified with Millipore Montage PCR Centrifugal Filters (Millipore, Billerica, MA) and adjusted to10 ng/µl. PCR products from each isolate were sequenced at the DNA Sequencing Core/ ICBR (University of Florida, Gainesville, FL) with the same primers used for amplification. All sequences were aligned with Clustal 2.0.11 (Conway Institute UCD, Dublin, Ireland). Primers and probe were designed to include all isolates of S. lanosoniveum and to exclude other species of Simplicillium and phylloplane inhabitants commonly isolated from soybean. Probe was labeled with 5’ 6-FAM (fluorescent reporter dye 6-carboxy-fluorescein) and with 3’ TAMRA (quencher dye 6-carboxytetramethyl-rhodamine). Primers and probe were purchased from Integrated DNA Technologies (Coralville, IA).
Upon alignment of ITS sequences of S. lanosoniveum, Simplicillium spp., and other soybean phylloplane inhabitants, the following sequences for primers and probe were selected (Fig 1). Forward primer SimpF-NW (5’-TTTATCCAACTCCCAACCC-3’) was specific to S. lanosoniveum. Reverse primer SimpR-NW (5’-ACGCGTAGTCCCGGGAG-3’) was specific to the Louisiana and Florida isolates but excluded the two CBS isolates of S. lanosoniveum by one or two bases. Probe SimpPR-NW (5’-FAM- CCGGGAGCCCCCTAG-TAMRA-3’) was specific to S. lanosoniveum, and the last two bases of the 3’ end were highly specific to S. lanosoniveum (Fig. 1). Optimization of primers and probe for the ABI 7000 yielded the following dilutions: 900 nM each forward and reverse primers and 200 nM probe. Primers and probe developed for S. lanosoniveum were very specific and had no cross reactivity when tested against S. lamilicolla, S. obclavatum, Fusarium spp. and Cladosporium spp. Sensitivity of the primers and probe was as low as 1.0 pg of DNA of S. lanosoniveum per 10 ng total genomic DNA as calculated with the standard curve at a mean Ct of 36.3.
Validity of the primers and probe was tested with both pure dilutions of S. lanosoniveum mycelial DNA spiked with soybean leaf DNA to identify possible inhibitors. A standard curve was generated by running six replications of 10-fold dilutions of 10 ng genomic DNA from S. lanosoniveum plus 10 ng of DNA from soybean. Next, we evaluated various primers and probe concentration combinations to test for sensitivity and to test the ability to detect lowest of S. lanosoniveum DNA. Specificity among S. lanosoniveum isolates was tested against pure dilutions of mycelial DNA of Fusarium and Cladosporium and soybean leaves infected with other pathogens such as Cercospora kikuchii and Septoria glycines. Assays were replicated twice.
Phakopsora pachyrhizi. Primers and probe for detection and quantification of P. pachyrhizi were selected from previous work (Frederick et al. 2002). Primers Ppm1 (5?-GCAGAATTCAGTGAATCATCAAG-3?) and Ppa2 (5?-GCAACACTCAAAATCCAACAAT-3?) were reported to be specific to P. pachyrhizi. Specific probe (5?-FAM-CCAAAAGGTACACCTGTTTGAGTGTCA-TAMRA-3?) was labeled at the 5’ end with the fluorescent reporter dye 6-carboxy-fluorescein (FAM) and at the 3’ end with the quencher dye 6-carboxytetramethyl-rhodamine (TAMRA). Primers and probe were purchased from Integrated DNA Technologies (Coralville, IA).
Field experiments were conducted in three soybean fields in Louisiana and one field in Florida in 2009 and 2010. Conidia and/or mycelial fragments were introduced to soybean leaves at various times as described below. SBR epidemics were initiated from naturally occurring inoculum; plants were not inoculated with the SBR pathogen, Leaf samples were collected weekly and DNA of both S. lanosoniveum and P. pachyrhizi was quantified by qPCR as described below.
Preparation of conidial inoculum. Conidial suspensions of S. lanosoniveum were produced by flooding petri dishes of 2-week old cultures with 20 ml sterile phosphate buffer (0.5 mM, pH 7.1) and rubbing colony surfaces with a glass rod (Ward et al. 2011). Two ml of this suspension were used to inoculate 100 ml of potato dextrose agar that had been poured in the bottoms of 2 L flasks. Flasks were shaken gently to spread inoculum across the agar surface. Cultures were incubated at 25?C in the dark for 7 days. At the time of inoculation in the field, flasks were filled with 2 L distilled water, shaken vigorously, and agar pieces were removed by straining the suspension through a wire mesh sieve with 500 µm openings. Conidial suspensions were adjusted to pH 7.0 with phosphate buffer (final concentration 0.5 mM) and amended with 100 µl Tween 20® per liter. The final inoculum concentration was approximately 106 conidia/ml. Plants were inoculated immediately after inoculum was prepared.
Preparation of mycelial inoculum. Cultures of S. lanosoniveum were flooded as described above. Two ml of conidial suspension were added to 250 ml potato dextrose broth and shaken at 200 rpm on an orbital shaker for 7 days at 25?C in the dark. Cultures were strained through a mesh sieve (500 µm openings), rinsed twice with deionized water, and blended in 500 ml sterile phosphate buffer (0.5 mM, pH 7.1). Two hundred fifty ml of this suspension were added to each liter of conidial suspension to produce inoculum of 104 colony forming units (CFU) per ml of mycelial fragments.
To monitor colonization of S. lanosoniveum and P. pachyrhizi on soybean leaves, 10 trifoliolate leaves were sampled weekly from each plot beginning in the late vegetative stages or early reproductive stages of growth. Leaves were stored in freezer bags at -20?C until they were processed for DNA extraction. The soybean crops were maintained according to recommended protocols with regard to insect and weed control and fertilization (Levy et al. 2011).
Field 1: The purpose of this experiment was to compare isolates of S. lanosoniveum and to determine whether they colonized sori of SBR under field conditions. Soybean cultivars Asgrow 6202 (Monsanto Corp.), Deltagrow 4770 (Deltagrow Corp.), Deltagrow 4771 (Deltagrow Corp.), and Delta King GP-533 (Armor Seed Co.) were grown at the Louisiana State University Agricultural Center’s Ben Hur Research Farm near Baton Rouge, LA. Plants were sampled in October and November 2009 while they were at the R5 to R6 stage of reproductive growth (Fehr et al. 1971). Plots were four rows wide by 9 m long and arranged in a randomized complete block with four replicates per cultivar. When disease severity in all plots reached at least 15% (85 DAP), conidial suspensions of 106 spores ml of isolates BH081707-1A (GenBank accession number HQ270477) or D082307-2A (GenBank accession number HQ270476) were applied to each of the center two rows with a hand-held sprayer at approximately 18 to 20 ml/m2 (Ward et al. 2011). The nontreated control received no treatment. Seven days after inoculation, 10 trifoliolate leaves were sampled from each plot for disease assessment and quantification of DNA by qPCR. Plots were inoculated again immediately after sampling (92 DAP) and sampled again on day 14 (99 DAP).
Field 2: SBR was detected within 44 km of the research farm on June 5, 2009, 46 days before soybeans were planted. To determine when S. lanosoniveum affected SBR infection, we inoculated soybeans with S. lanosoniveum before and after disease symptoms occurred. Soybean cultivar Asgrow 6202 was planted in July 2009, at the Ben Hur Research Farm, near Baton Rouge, LA. Plots were 8 rows wide by 9 m long arranged in a randomized complete block with 4 replicates per treatment. The following four treatments were included: 1) inoculation with S. lanosoniveum at first flower (R1/R2); 2) inoculation with S. lanosoniveum at first occurrence of rust (<2.5% severity) (R5); 3) application of pyraclostrobin fungicide (Headline, BASF Corp.) (876 ml product in 187 liters of water per ha) at R1/R2; and 4) nontreated control. There were four replications per treatment, and the experiment was arranged in a randomized complete block design. The center 4 rows of each plot were sprayed until leaves were wet (approximately 18 to 20 ml/m2) with conidial suspensions (106 spores/ml) of S. lanosoniveum isolate D082307-2A with a hand-held garden sprayer. Ten trifoliolate leaves were collected each week beginning during mid-vegetative stages (V4) and continuing through senescence (R7). One hour after each inoculation, three trifoliolate leaves were sampled from each plot to quantify initial inoculum.
Corn was included as a nonhost control in this experiment in order to examine colonization of S. lanosoniveum in the absence of SBR. Corn leaves were inoculated with conidial suspensions as described above. Three rows, each 9 m long, were inoculated with 106 spores/ml of S. lanosoniveum. Three leaves from each plot were sampled 1, 7, 14, and 21 days after inoculation. The experiment was conducted twice.
Field 3: In July 2010, soybean cultivar Pioneer 95Y20 was planted at the University of Florida North Florida Research and Education Center in Quincy, Florida. There were four treatments in this experiment: 1) inoculation with S. lanosoniveum at first flower (R1); 2) inoculation with S. lanosoniveum at beginning seed development (R3); 3) application of pyraclostrobin fungicide as described above at V6/R1; and 4) nontreated control. Plots were four rows wide by 9 m long, and six replications per treatment were arranged in a randomized complete block design. The center two rows were treated with suspensions of 106 spores/ ml plus 104 CFU/ ml of S. lanosoniveum isolate D082307-2A. Plants were sprayed until visibly wet (approximately 18-20 ml/m2). Ten trifoliolate leaves were sampled weekly from each plot beginning during vegetative stages (V6) of growth and ending at the onset of senescence (R7) (80 DAP). SBR was detected in kudzu approximately 180 m from the plots at 8 DAP. However, SBR was not detected in this field until 68 DAP when plants were in the R5 growth stage.
Numbers of Sori
Immediately after sampling, sori were counted on each apical leaflet from each of the 10 trifolioliates collected from each plot. Numbers of sori were counted within three fields of vision (5 cm2) per leaf with a dissecting microscope at 25x magnification, and sori per/cm2 were calculated for each leaflet.
After determing numbers of sori, leaves from each plot were stored at -20?C in Ziploc® plastic bags. For processing, leaves were ground in liquid nitrogen with mortar and pestle. Subsamples of 50 mg of ground leaf material were transferred to 1.5 ml microcentrifuge tubes and ground again with plastic pestle grinders for 30 sec in extraction buffer (Qiagen, DNeasy Plant Mini Kit). Samples were incubated on a heat block at 55?C for 30 minutes during which time they were vortexed twice. Genomic DNA was extracted from the ground leaf samples using Qiagen’s DNeasy Plant Mini Kit (Qiagen; Hilden, Germany) according to the manufacturer’s protocol. Final DNA concentrations were determined as described above and diluted to10 ng/?l for use in qPCR assays.
qPCR was used to quantify the amount of DNA of S. lanosoniveum and P. pachyrhizi from extractions above. Triplicate samples were tested in a total volume of 25 ?l per reaction. Each test for S. lanosoniveum included 10 ng template DNA, 12.5 µl TaqMan® Universal PCR Master Mix (Applied Biosystems; Carlsbad, CA), 900 nM each forward and reverse primer and 200 nM probe. Reactions for P. pachyrhizi included 10 ng template DNA, 12.5 µl TaqMan Universal PCR Master Mix, 15 mM primers Ppm1 and Ppa2, and 10mM FAM probe (Frederick 2002; Z.Y. Chen and S. Park, unpublished). The qPCR instrument (ABI 7000; Applied Biosystems; Carlsbad, CA) was run on the following protocol: initial denaturation at 95?C for 10 min followed by 40 cycles of 15?C for 15 sec and 60?C for 1 min. The instrument software package (Applied Biosystems, Carlsbad, CA) automatically analyzed the critical threshold values (Ct) for each reaction (Lees et al. 2002). Using absolute quantification, Ct values were converted to picograms (pg) DNA based on standard curves.
Data was analyzed using JMP software version 9 (SAS Institute; Cary NC). Data from experiments were first tested for normality and homogeneity of variances then subjected to analysis of variance (ANOVA) in order to compare means of treatments. To evaluate sori counts, numbers of sori per field of vision were averaged for each leaflet, and leaflet means were analyzed accordingly for each plot. Plot data for each treatment was analyzed using ANOVA. DNA concentrations were analyzed similarly by averaging triplicate subsamples for each plot. Plot means were analyzed for each treatment using ANOVA. Statistical significance was established at P < 0.05.
Field 1: In the late-planted field, rust severity was at least 25% at the time of inoculation with S. lanosoniveum. One hour after inoculation with isolate D082307-2A, there were 4.0 ± 0.82 pg DNA of S. lanosoniveum per 10 ng soybean DNA. At 14 days after inoculation, this value increased to 7.15 ± 2.96 pg, while the nontreated control yielded 0.24 ± 0.04 pg (P = 0.02; F1, 8 = 3.22). DNA concentration of S. lanosoniveum isolate BH081707-1A was detected at 1.26 ± 0.43 pg by day 14 compared to the nontreated control (P > 0.01; F1, 8 = 2.41).
Concentration of DNA of P. pachyrhizi was determined as a measure of disease potential. On day 14, plots inoculated with isolate D082307-2A had a mean of 11.03 ± 0.87 ng DNA of P. pachyrhizi, which was significantly lower than 16.54 ± 1.64 ng detected in the nontreated control (P = > 0.01; F1, 7 = 2.62). In plots inoculated with isolate BH081707-1A, a mean of 21.71 ± 4.43 ng DNA of P. pachyrhizi was detected, which was not significantly different from the nontreated control (P = 0.03; F1, 7 = 5.5). DNA of P. pachyrhizi increased between days 7 and 14 in all treatments, but isolate D082307-2A resulted in significantly less DNA of P. pachyrhizi than isolate BH081707 or the nontreated control by the end of the study (Fig. 2).
Numbers of sori were detected at 129.39 ± 22.08 sori/cm2 for isolate D082307-2A and 164.22 ± 13.64 sori/cm2, compared to the nontreated control at 138.39 ± 22.08 sori/cm2. Numbers of sori/cm2 did not differ between inoculated treatments and the nontreated control (P = 0.51; F3, 27 = .82).
Field 2: SBR symptoms were observed on soybeans at 68 DAP when plants reached the R5 growth stage. DNA of P. pachyrhizi was detected at 30 days after planting, which resulted in a latent infection period of 38 days. By physiological maturity (R7, 79 DAP), early inoculations (R1) resulted in 0.75 ± 0.15 ng of DNA of P. pachyrhizi, which was not statistically different from 0.85 ± 0.17 ng detected in fungicide treated leaves (P = 0.14; F1, 18 = 1.01). Inoculations that were made after rust symptom development resulted in 1.26 ± 0.07 ng of rust DNA, which was not significantly different from 1.17 ± 0.05 ng detected in the nontreated control (P = 0.21; F1, 18 = 0.86; df = 1, 18) (Fig. 3).
By the time soybean plants reached physiological maturity (79 DAP), the nontreated control had a mean of 196.8 ± 8.60 sori per cm2, while the mean number of sori in the R1 treatment was 143.4 ± 16.0 sori per cm2 (P = 0.04; F1, 38 = 5.74). The fungicide treatment also had a significantly lower number of sori by the end of the study, 137.8 ± 18.05, as compared to the nontreated control (P = 0.03; F1, 38 = 6.32) (Fig. 4).
Simplicillium lanosoniveum increased in all inoculated treatments, with sharp increases in DNA 7 days after application. At 59 DAP (R5), rust severity increased to 25%, and DNA of S. lanosoniveum reached 0.31 ± 0.02 pg in the R1 treatment compared to the nontreated control (0.0 ± 0.0 pg) (P < 0.01; F1, 6 = 0.25). When disease severity was 50% (73 DAP), the early inoculated treatment (R1) had 0.25 ± 0.05 pg DNA of S. lanosoniveum and the treatment that was inoculated at >2.5% disease severity contained 0.10 ± 0.01 pg DNA of S. lanosoniveum compared to the nontreated control 0.0 ± 0.2 pg (P < 0.01; F1, 6 = 0.32 and P < 0.01; F1, 6 = 0.68, respectively). By R7, DNA of S. lanosoniveum decreased sharply in all treatments.
In order to determine initial amounts of inoculum, S. lanosoniveum was quantified in each treatment one hour after inoculation. In the three inoculation treatments, 0.04 ± 0.05 pg and 0.06 ± 0.0 pg were detected in the R1 and <2.5% rust treatments, respectively. Nontreated control contained no DNA of S. lanosoniveum.
The corn treatments yielded a mean of 0.06 ± 0.10 pg of S. lanosoniveum one hour after inoculation, which was similar to the amount detected in soybean 1-hour after inoculation. In the first repetition, corn samples yielded 0.50 ± 0.0 pg of S. lanosoniveum after 7 days, but on day 14 DNA of S. lanosoniveum was not detected. There was no rainfall during this period. In the second replication, 2.5 cm of rain was recorded 3 days after inoculation. DNA of S. lanosoniveum was not detected upon sampling at 7 days after inoculation.
Field 3: In fields that were planted before soybean rust was detected in the area, SBR symptoms developed at R6 (66 DAP). By the end of the experiment (R6), one sorus was detected in each of two plots. A qPCR assay value of 0.07 ± 0.01 pg DNA of P. pachyrhizi indicated that there was latent infection in some plots as early as 48 DAP (R3). Amounts of DNA of P. pachyrhizi remained low throughout this study, and there were no statistical differences in the amounts of DNA of P. pachyrhizi among treatments (Fig. 5).
Initial inoculum levels of S. lanosoniveum (one hour after inoculation) were 4.0 ± 0.08 pg and 4.50 ± 0.08 pg per 10 ng of total DNA in the R1 and R3 treatments, respectively. By the end of the study (R7), DNA of S. lanosoniveum decreased to 2.36 ± 1.40 pg in early inoculations (V6 treatment). Late inoculations (R3 treatment) resulted in a significant increase to 75.92 ± 24.12 pg compared to 0.12 ± 0.07 pg in the noninoculated control (P = 0.023; F1, 10 = 10.34).
- Fig 3 Population dynamics of (A) Phakopsora pachyrhizi and (B) Simplicillium lanosoniveum as assessed by monitoring DNA concentrations of these organisms in soybean leaves using quantitative PCR. Assays were conducted on soybean leaves collected from field plots that had been subjected to the following four treatments: Plants were inoculated with S. lanosoniveum either at R1 when there were no symptoms of soybean rust (R1) or when rust severity was no more than 2.5% (Rust <2.5%). Plants were sampled one hour after inoculation and once per week thereafter. Other treatments included the fungicide pyraclostrobin (Headline) applied at R1 or a nontreated control (No treatment). Bars represent standard error of the mean.
- Fig 5 Population dynamics of (A) Simplicillium lanosoniveum and (B) Phakopsora pachyrhizi as assessed by monitoring DNA concentrations of these organisms in soybean leaves using quantitative PCR. Assays were conducted following inoculation of field-grown soybean plants with the antagonist, S. lanosoniveum. Samples were collected from field plots that had been subjected to the following four treatments: Plants were inoculated with S. lanosoniveum at either the V6 or R3 growth stages when there were no symptoms of soybean rust. Sampling began one hour after inoculations and continued every 7 to 10 days thereafter. Other treatments included the fungicide pyraclostrobin at R3 (Headline R3) and a nontreated control (Control). Bars represent standard error of the mean.
- Fig 2 Population dynamics of (A) two isolates of Simplicillium lanosoniveum and (B) Phakopsora pachyrhizi as assessed by monitoring DNA concentrations of these organisms in soybean leaves using quantitative PCR. Assays were conducted following inoculation of field-grown soybean plants with the antagonist, S. lanosoniveum. Inoculum was applied 85 and 92 days after planting (DAP), and leaves were sampled 7 days after each inoculation (92 and 99 DAP). Bars represent standard error of the mean.
- Fig 4 Effects of inoculation of soybean leaves with Simplicillium lanosoniveum on soybean rust as assessed by (A) numbers of sori per cm2 leaf area and (B) percent diseased leaf area as a function of days after planting. Plants were inoculated with S. lanosoniveum either at R1 when there were no symptoms of soybean rust (R1) or when rust severity was no more than 2.5% (Rust <2.5%). Plants also were sprayed with the fungicide pyraclostrobin (Headline) at R1, or they were not treated (No treatment). Bars represent standard error of the mean.
- Fig1 Sequence alignment of the internal transcribed spacer (ITS) region from Simplicillium spp., Fusarium sp., and Cladosporium sp. Nucleotide differences that occur between S. lanosoniveum and other species are highlighted with open boxes. Primer and probe sequences are shown by arrows.
Educational & Outreach Activities
Peer Reviewed Publications:
Ward, N. A., Schneider, R. W., and Aime, M. C. 2010. Colonization of soybean rust sori by Simplicillium lanosoniveum. Fungal Ecology (4)303-308
Ward, N. A., Schneider. 2010. Suppression of Phakopsora pachyrhizi, causal agent of SBR, by the mycophilic fungus, Simplicillium lanosoniveum. Phytopathology. (accepted for publication)
Submitted, Peer-reviewed Publications:
2011. Mycoparasitism of Phakopsora pachyrhizi by Simplicillium lanosoniveum and effects on soybean rust. (Phytopathology)
2011. Field evaluation of Simplicillium lanosoniveum as a biological control agent of soybean rust. (BioControl)*Note: this is the primary work resulting from this grant. The above report is the text reported in this report.
Ward, N. A., Schneider, R. W., and Robertson, C. L. 2010. Field evaluations of Simplicillium lanosoniveum as a biological control agent for Phakopsora pachyrhizi. Invited speaker, University of Florida Soybean Rust Shortcourse, August, 2010.
Ward, N. A., Schneider, R. W., Aime, M. C., Robertson, C. L. 2010. Leaf surface interactions between Phakopsora pachyrhizi, the soybean rust pathogen, and the mycoparasite Simplicillium lanosoniveum. The 9th International Symposium of the Microbial Ecology of Aerial Plant Surfaces, December 2009, New Orleans, LA
Ward, N. A., Schneider, R. W., Aime, M. C., and Robertson, C. L. 2010. Characterization of Simplicillium lanosoniveum as a possible mycoparasitic interaction in sori of Asian soybean rust. Sigma Xi Annual Spring Banquet, March 2010, Baton Rouge, LA
Ward, N. A., Schneider, R. W., and Robertson, C. L. 2010. Field evaluations of Simplicillium lanosoniveum as a biological control agent for Phakopsora pachyrhizi. Phytopathology 100:S134
Ward, N. A., Schneider, R. W., and Robertson, C. L. 2010. Field evaluations of Simplicillium lanosoniveum as a biological control agent for Phakopsora pachyrhizi. Southern Soybean Disease Workers, March 2010, Pensacola Beach, FL
Ward, N. A., Schneider, R. W., Giles, C. G., and Robertson, C. L. 2010. Development of a screening protocol for assessing baseline sensitivity to fungicides for Phakopsora pachyrhizi, the soybean rust pathogen. Phytopathology 100:S203
In previous studies, we documented through scanning electron microscopy that hyphae of S. lanosoniveum wrapped around urediniospores of P. pachyrhizi and colonized sori (Ward et al. 2011). We also used detached leaf assays to evaluate the effects of S. lanosoniveum on SBR. When inoculated with S. lanosoniveum, soybean leaves contained significantly fewer sori and viability of urediniospores was significantly reduced (Ward and Schneider 2011). To evaluate the effects of S. lanosoniveum under field conditions, we conducted field trials in Louisiana and Florida in 2009 and 2010. qPCR was used to quantify the establishment of S. lanosoniveum and its effects on SBR on both diseased soybeans and disease-free plants under field conditions.
In Field 1, we tested two isolates of S. lanosoniveum used in previous experiments (Ward et al. 2011). This field study utilized several commercial soybean cultivars, all with similar degrees of disease severity (25%). Isolate D082307-2A colonized and established in sori in all cultivars. Mycelia were often visible under low magnification with a dissecting microscope. Isolate BH081707-1A, on the other hand, did not readily colonize sori. Mycelia were visible in less than 1% of sori, and amounts of DNA were not significantly different from that of the nontreated control. In Field 2 and Field 3, we inoculated only with isolate D082307-2A.
Weather appeared to have an effect on colonization of S. lanosoniveum in soybean fields. The Field 1study was conducted in October and November 2009. We suspect that there was more S. lanosoniveum in sori, as assessed with qPCR and visible in sori, in this late-planted field than in the other studies because conditions were favorable for SBR development. Average high temperature was 23?C, and there was 9.2 cm rainfall during the month of the study. In Field 2 in 2009, we occasionally observed mycelia in sori. This field study was conducted from August through October during which time temperatures were lower than normal because of frequent rainstorms and cloudy skies. Maximum daytime temperatures averaged 30? to 33?C, and precipitation ranged from 5.5 to 9.5 cm per month (www.lsuagcenter.com). Field 3 study was conducted in August and September 2010. Rainfall at this site was similar to Field 2 (7 to 11 cm per month), however, maximum daytime temperatures averaged 36?C (www.fawn.ifas.ulf.edu).
Despite temperature extremes, S. lanosoniveum failed to colonize field-grown leaves unless SBR was present in some form. The fungus was an aggressive colonist when sori were present. Additionally, S. lanosoniveum colonized leaves when SBR was in a latent stage, even in the absence of sori. It is likely that spores of P. pachyrhizi on leaf surfaces may have provided a sufficient nutrient source to sustain S. lanosoniveum until sori developed. This phenomenon was observed in Field 2 in the earliest (R1) treatment. In this instance, latent infection was detected, but disease symptoms did not occur until 30 days later. Simplicillium lanosoniveum colonized soybean leaves early in the disease cycle. In Field 3, on the other hand, S. lanosoniveum did not readily colonize leaves following the early (R1) inoculation. There was no latent infection at this stage, and we suspect that this was because rust inoculum was extremely low or nonexistent. The later inoculation (R3) was applied 2 weeks before disease symptoms developed. Latent infection was detected and we suspect that rust inoculum was present in large enough quantities to sustain the antagonist. At this point, S. lanosoniveum began to colonize soybean leaves.
In early inoculations in Field 2, amounts of S. lanosoniveum increased significantly for the first 4 weeks after inoculation, although it fluctuated from week to week. However, at 73 DAP (R7), there were sharp decreases in amounts of S. lanosoniveum in the two inoculated treatments. This decrease was not associated with weather extremes such as high temperatures or dry weather. Sori were heavily sporulating at this point, but we observed many phylloplane inhabitants as leaves senesced. Soybean leaves became increasingly chlorotic and sori developed necrotic margins. We suspect that competition for nutrients or exudates from uredinial inhabitants may have affected growth of S. lanosoniveum. This phenomenon was not observed in Field 3 because SBR did not develop until late in the study, disease severity was extremely low, and amounts of SBR DNA were too small to distinguish differences.
Initial amounts of S. lanosoniveum inoculum applied to soybeans in these field studies were quantified. Approximately 0.06 pg of DNA per 10 ng soybean DNA was detected when conidia alone were used as inoculum in Field 2. This was similar to results obtained with corn, which served as a nonhost control. In corn, there was no increase in amounts of DNA 7 days after inoculation, and then there was a sharp decrease in amounts of S. lanosoniveum. When rain occurred after inoculation of corn leaves, S. lanosoniveum was not detected on the day 7 sampling. In Field 3, 4.0 to 4.5 pg DNA of S. lanosoniveum was detected one hour after inoculation. In this field study, both conidia and mycelia were used as inoculum. In both Field 2 and Field 3, S. lanosoniveum was not detected 14 days after application unless rust (latent infection or visible sori) was present. In all field studies, S. lanosoniveum colonized diseased soybean leaves and failed to colonize disease-free leaves.
In Field 2, there were significant differences in numbers of sori per cm2 between the treated and the nontreated control. Additionally, this field contained more red-brown sori, which resulted in fewer secondary sori and more necrotic tissue surrounding primary sori (McLean 1983; Ward and Schneider 2011) (Ward unpublished pustule age study). Overall, disease was less severe in treatments inoculated with S. lanosoniveum. In Field 1, on the other hand, numbers of sori were extremely high when we began the experiment, and by day 14, disease severity reached a plateau, and defoliation had already begun, which is a reasonable explanation for the lack of significant differences in sorus production. However, we observed that there were differences between treatments in sorus size and apparent age as observed in Field 2. In Field 3, only two plots showed disease symptoms, but only one sorus was detected in each plot.
Simplicillium lanosoniveum was effective in slowing the rate of disease development, and this was reflected in the DNA assays in two of the three field experiments. In the third experiment conducted in 2010, following hard freezes and significant reductions in alternative hosts such as kudzu, SBR was not detected on soybean in Florida until July. Moreover, SBR was not detected within 22 km of this field until 11 days before symptoms were discovered. Once rust inoculum was sufficient or sori developed, S. lanosoniveum colonized sori.
We conclude that S. lanosoniveum is an ideal candidate for biological control because it readily colonized soybean leaves and was effective in reducing disease severity. Results from these and other studies warrant the use of this antagonistic fungus as part of an IPM program in combination with other cultural practices or as a biological control agent in organic soybean production systems (Ward et al. 2011; Ward and Schneider 2011). Additional research should include the effects of sunlight on survival and colonization of leaf surfaces by S. lanosoniveum and the development of formulations with extended shelf life.
Areas needing additional study
This study should be repeated in grower fields.