Trichoderma harzianum ThzID1-M3 is a transformant strain of Thz (wild type) ThzID1. The parental isolate was obtained from field soil near Moscow, Idaho.ThzID1-M3 was derived by PEG-mediated cotransformation of ThzID1 with genes encoding GFP and hygromycin B resistance; it is mitotically stable, similar in morphology and biocontrol activity to the parent, and expression of GFP in soil has been demonstrated [9] . ThzID1-M3 was formulated with alginate as previously described [5] : the fungus was allowed to grow for 1 week on potato dextrose agar at 21C, and three 1-cm² pieces from sporulating culture were transferred to a 1-liter flask containing 500 ml potato dextrose broth. Flasks were placed on a rotary shaker (120 rpm) at room temperature and incubated for 1 week with 12 hours of light per day. Cultures were strained through cheesecloth and rinsed thoroughly with sterile water. Thirty-seven grams of hyphal biomass was blended briefly with 2 grams of wheat bran and 100 ml of 1% aqueous sodium alginate solution. Drops of the mixture were added to 0.25 M CaCl₂ solution, forming a pellet precipitate. Pellets were removed by straining, placed on waxed paper to air-dry at room temperature for 2 days then stored at 4C prior to use. The fungivorous nematode Aphelenchoides saprophilus was obtained from field soil near Moscow, ID, and populations were maintained on agar cultures of T. harzianum [6] . An isolate of S. sclerotiorum was grown on PDA for 7 days, then transferred to carrot discs and incubated 6 - 8 weeks at 25 8C. Sclerotia were harvested and airdried, then preconditioned for carpogenic germinationby incubating them on moist (saturated) sand at 10 C in the dark for approximately 24 months.

Latahco silt loam soil was obtained from the University of Idaho plant science farm near Moscow, ID. Soil analysis results (University of Idaho Analytical Services Laboratory) indicated that the soil contained 20% sand, 20% clay and 60% silt by weight, with 82.2 µg/g of plant-available iron per gram. Soil pH in soil/water (2:1) solution was approximately 5.9. Soil was sieved through a 2 mm mesh and allowed to air-dry at room temperature prior to use.

Trichoderma harzianum ThzID1, ThzID1-M3, and S. sclerotiorum were allowed to grow in 1-Liter flasks containing 500 ml of potato dextrose broth for 4 d at room temperature on a rotary shaker (120 rpm). Mycelium was harvested by filtering through cheesecloth and rinsing with sterile distilled water, then ground in a porcelain mortar with liquidnitrogen. Total genomic DNA was extracted using the DN easy plant DNA extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer0s instructions. Extracted DNA was eluted twice in 100 ml of Buffer AE (Qiagen) and the elutions were combined. Purified DNA was quantified spectrophotome- trically, and stored at −20 C prior to use. Several primer/probe sets specific for ThzID1-M3were designed based on a GFP gene sequence (Gen Bank Accessionno. U55761) using Beacon designer software versions 2 and 4 (Premier Biosoft International, Palo Alto, CA, USA). The Basic Local Alignment Search Tool (BLAST) was used to confirm the absence of significant homology with other known DNA sequences. Eight different primers and the probes exhibited low homology with sequences of soil microorganisms, including generic Trichoderma spp. For selected primer sets that did not give unintended signals (such as primer dimers and nonspecific bands), BLAST was repeated to find a probe with the lowest homology with other known fungal sequences. The best primer/probe set, designated TMeGFP2, contained a FAM reporter dye conjugated to the 50-terminaland a TAMRA quencher dye conjugated to the 30-terminal (Table 1).

To quantify S. sclerotiorum DNA, we used the TMSCL2 primer/probe set (Integrated DNA Technologies, Coralville, IA, USA) based on the S. sclerotiorum calmodulin gene sequence as described previously [10] . The TMSCL2 probe contained a HEX reporter dye andTMeGFP2 primer/probe set (Table 1). PCR conditions resulting in the lowest cycle threshold (Ct) value and the highest normalised fluorescence were selected, and PCR annealing and extension temperatures were optimised at 58 - 63 C. Standard curves obtained from each of the TMeGFP2 and TMSCL2 sets generated a linear fit with a slope of 3.4 to 3.6 with coefficient of determination (R²) values greater than 0.99, with a PCR efficiency higher than 90% over at least six orders of magnitude.

PCR standards were developed as follows: first, purified DNA of T. harzianum ThzID1-M3 or S. sclerotiorum was serially diluted (1:10). Standard curves were constructed by plotting threshold cycles (Ct) for ten-fold dilution series of the extracted pure DNA vs. logarithmically transformed DNA amounts of the dilution series. Ct value specified as the cycle number at which the fluorescent signal exceeded background fluorescence, a linear regression equation of a standard curve, and PCR efficiency were determined by the Bio-Rad iCycler IQ software program version 3.1 (Bio-Rad, Hercules, CA). ThzID1-M3 DNA standards ranged from 170 fg to 170 ng and S. sclerotiorum DNA standards ranged from 1 pg to 100 ng. Real-time PCR amplifications of total DNA extracted from sclerotia were performed using the iCycler IQ (Bio-Rad). The reactions were carried out in a real- time PCR plate (VWR International, West Chester, PA) with a total volume of 25 ml per single reaction.

The TMeGFP2 set was tested for potential false positive reactions with DNA from soil by adjusting nonsterile soil to a moisture content of either −50 kPa or −500 kPa, then added 20 g of soil to plastic containers (8 cm × 6.3 cm × 6.3 cm) and incubating at 22 C. After 0, 3, 5, 7, 14, and 21 d, replicate containers were destructively sampled. Soil was homogenized by suspending in 80 ml of 50 mM sodium phosphate buffer [pH 7.0], and placing on a rotary shaker (200 rpm) for 1 h. Total DNA was extracted from a 0.25 ml subsample from each suspension, using the PowerSoil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA). qRT-PCR amplification of extracted DNA was performed using the TMeGFP2 primer/probe set, with conditions as described above. No false positive results were obtained from soil that wasn’t inoculated with T. harzianum ThzID1M3.

To correlate fungal biomass with qRT-PCR results, known weights (1, 2, 3, 4, 8 or 9 mg, dry weight) of mycelium were placed in 1.5 ml microtubes and ground with liquid nitrogen. Total genomic DNA was extracted using the DNEasy plant DNA extraction kit (Quiagen Inc., Valencia, CA, USA). DNA was twice eluted in100 ml of AE buffer, and the elutions were combined and stored at 20 C prior to use. The relationship between fungal biomass and DNA estimates, for all three species, using the qRT-PCR assay was quantified using linear regression. All standard curves obtained from qRT-PCR runs with the above primer/probe sets generated a linear fit with R2 ≥ 0.98, with ≥90% PCR efficiencies.

Ability of A. saprophilus to reduce both colony radial growth and recoverable populations of T. harzianum in

^aForward primers. ^bReverse primers. ^cTaqMan probes: T. harzianum ThzID1-M3 probe is labelled with the reporter dye FAM (6-carboxy-fluorescein) on the 5’ end and the quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine); S. sclerotiorum probe is labelled with the reporter dye HEX (hexachlorofluorescein) and the quencher dye IBFQ (Iowa Black FQ by Integrated DNA Technologies). ^dAntisense probe.

soil was previously shown [6] . Nematodes (10 nematodes/g of soil, males and females, mixed life stages) were added to soil with pelletized T. harzianum; controls consisted of sterile water. After 5, 10, 20, or 30 days, recoverable populations of ThzID1-M3 and total recoverable Trichoderma spp. were determined by plating samples on selective media, and nematodes were extracted with a Baermann funnel and counted.

We investigated the possibility that colonized sclerotia may serve as refuges that enhance survival of T. harzianum, compared to survival in the surrounding soil, as follows. T. harzianum ThzID1-M3 was formulated into pellets as described above. Sclerotia of S. sclerotiorum (surface-sterilized in 10% bleach solution) were attached to glass microscope slides using cyanoacrylate glue, either withThzID1-M3 pellets attached approximately 15 mm away from sclerotia, or without ThzID1-M3 pellets. The slides then were placed in Petri dishes which were filled with nonsterile soil, with nematodes present, adjusted to −100 kPa matric potential. The dishes were sealed with parafilm to maintain a relatively constant moisture content, and were incubated at 22 C. After 20, 30, or 40 d, sclerotia were (destructively) sampled and either sectioned for microscopic observation or held at −20 C for subsequent DNA extraction and qRT-PCR analysis. Sclerotia sampled for microscopic observation were embeddedin paraffin, and 20 mm thick sections were cut with a microtome. Sections were viewed at 400× magnification using a Nikon Eclipse E1000 epifluorescence microscope (Nikon, Melville, NY, USA), to observe GFP fluorescence of ThzID1-M3. From captured images, fluorescing ThzID1-M3 hyphal segments were quantified using Image Pro Plusimaging software (Media Cybernetics, Bethesda, MD, USA). Hyphal segments were quantified based on pixel numbers on the images, and values then were converted to area (mm²). A haemocytometer was used as a measuring grid to calibrate an appropriate conversion factor.

Amounts of T. harzianum ThzID1-M3 DNA as well as S. sclerotiorum DNA inside sclerotia were quantified using the real-time PCR assay with the TMeGFP2 and TMSCL2 primer/probe sets. Sclerotia were ground with liquid nitrogen, and DNA was extracted using the DN easy plant DNA kit. Amounts of S. sclerotiorum DNA and ThzID1-M3 DNA contained in each sclerotium were determined as described above.

Also, in order to quantify ability of T. harzianum ThzID1-M3 to grow and persist outside of sclerotia, at each sample time the surrounding soil was sampled and the amount of T. harzianum ThzID1-M3 DNA there was quantified. Total DNA was extracted from 0.25 ml (containing 0.05 g soil) of each homogenized soil suspension using the Power Soil DNA Isolation kit.

We also used epifluorescence microscopy of the GFP-expressing ThzID1-M3 to quantify hyphal proliferation (biomass) in the same treatments, using the methods of Orr and Knudsen [11] . Subsamples (0.01 g of soil) from each homogenized soil suspension were filtered through 0.8-µm membrane filters and viewed at 100× with a Nikon Eclipse E1000 epifluorescence microscope (Nikon, Melville, NY) to observe hyphal fragments with GFP activity. Membrane filters were prepared and examined in their entirety for any fungal segments expressing GFP. Autofluorescence within the range of GFP from substances such as minerals, organic debris, plant tissue, and other microbes was alleviated with a triple filter cube with DAPI/FITC/TRITC (excitation wavelengths of 380 - 400, 470 - 490 and 545 - 565 nm; dichroic mirror wavelengths of 425 - 470, 500 - 540 and 570 nm; and barrier wavelengths of 450 - 465, 510 - 540 and 580 - 620 nm). From each object, two images were captured under both FITC and TRITC filters with a Hamamatsu digital camera (model C4742-95), and color-combined using MetaMorph computer software. A conversion factor from pixels (of captured images) to micrometers was determined as a length of 0.99 µm using ImagePro Plus imaging software. For each captured image, radii and lengths of visible hyphae were measured using ImagePro Plus imaging software. Calculated biomass values were expressed as µg/g of soil. Recoverable populations expressed as colony-forming units (CFU) per gram of soil were also determined by plating soil serial dilutions on selective medium containing hygromycin B.

Experimental data were analysed using SAS package 9.0 (SAS Institute, Cary, NC, USA). Linear regression was performed for Ct values vs. log-transformed fungal DNA concentrations and for Trichoderma harzianum ThzID1-M3 mycelial weights vs. DNA estimates from the real-time PCR assay. Estimated amounts of ThzID1-M3 DNA and Sclerotinia sclerotiorum DNA in sclerotia were analyzed over time using analysis of variance with significant differences occurring at the level of P < 0.05. Estimated amounts of ThzID1-M3 DNA and Sclerotinia sclerotiorum DNA in sclerotia were analyzed over time using analysis of variance.