Responses of non-primed or primed seeds of ‘Marketmore 76’ cucumber (Cucumis sativus L.) slurry coated with Trichoderma species to planting in growth media infested with Pythium aphanidermatum

Scientia Horticulturae 121 (2009) 54–62

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Responses of non-primed or primed seeds of ‘Marketmore 76’ cucumber (Cucumis sativus L.) slurry coated with Trichoderma species to planting in growth media infested with Pythium aphanidermatum W.G. Pill *, C.M. Collins, B. Goldberger, N. Gregory Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716-2170, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 October 2008 Received in revised form 29 December 2008 Accepted 7 January 2009

Aqueous slurries of commercial preparations of Trichoderma harzianum Rifai strain KRL-AG2 G41 (Th), T. virens Strain G-41 (Tv), or their combination (ThTv, at half rates each of the single application rate) were applied to ‘Marktetmore 76’ cucumber seeds (Cucumis sativus L.) that were non-primed or primed for 3 days at 25 8C either osmotically ( 2.5 MPa from 0.337 molal Ca(NO3)2) or osmomatrically ( 1.0 MPa from 0.135 molal Ca(NO3)2 plus 1.5 MPa from 50% water in exfoliated grade 5 vermiculite). Slurries were applied to seeds (1 mg per seed) either before or after priming. Seeds were sown in soilless, peatbased media with or without inoculation with Pythium aphanidermatum (Pa). Protection against damping-off caused by high pressures of Pa (16% emergence in non-coated, non-primed seeds) was increased by slurry coating Th on non-primed (76.4% emergence) or on osmotically primed seeds, with coating either before or after priming having no effect on efficacy (average 62.6% emergence). Slurry coating Th on osmomatrically primed seeds failed to increase final emergence percentage (FEP). Colony forming units per three seeds (CFU, all 103) was 2.8 for non-primed seeds, and 3.2 and 2.6, respectively, when osmotically and osmomatrically primed seeds were coated after priming. In a second study with lower disease pressure (58.1 FEP from non-coated, non-primed seeds), slurry coating of non-primed or osmotically primed seeds with Th, Tv or ThTv reduced percentage damping-off and increased FEP. The combination coating eliminated damping-off only in non-primed seeds, and tended to reduce percentage damping-off in osmotically or osmomatrically primed seeds compared to coating with Th or Tv alone. In a third study using only non-primed seeds, slurry coatings with mefenoxam fungicide, Th, Tv, or ThTv decreased total damping-off to 2.6%, 7.4%, 2.0%, and 0%, respectively, from the 30.1% occurring in non-coated seeds. Th, Tv or ThTv applied to growth media at the same rate as the seed coating (1 mg per seed) were generally as effective as the seed coatings, and only the ThTv growth medium application eliminated damping-off. A fourth experiment revealed that Th, Tv or ThTv remained viable on nonprimed seeds for up to 4 weeks (the longest storage duration) at 21 or 4 8C, but 21 8C storage resulted in faster seed germination by week 3 and higher CFU per three seeds by week 4. In summary, coating of non-primed seeds with Th, Tv or ThTv was more effective than coating primed seeds in reducing percentage damping-off. While priming treatments generally led to faster seedling emergence and greater seedling shoot fresh weight than was achieved with non-primed seeds, only for non-primed seeds was damping-off eliminated by the ThTv seed coating or growth medium application. Published by Elsevier B.V.

Keywords: Biological control Damping-off Seed priming Slurry coating Trichoderma spp. Pythium aphanidermatum

1. Introduction Several soil-borne pathogenic fungi including various species of Pythium, Phytophthora, Fusarium, Aphanomyces, and Rhizoctonia can cause pre- or post-emergence damping-off (Agrios, 1997). Damping-off is a common problem in many field and greenhouse crops, with little natural plant resistance to infection. Biological

* Corresponding author. E-mail address: [email protected] (W.G. Pill). 0304-4238/$ – see front matter . Published by Elsevier B.V. doi:10.1016/j.scienta.2009.01.004

control (biocontrol) provides a non-chemical means to control damping-off organisms that can reduce or eliminate the use of synthetic fungicides, and their use frequently is permitted within organic farming certification. Actinomycetes, bacteria and fungi have been used as both seed treatments and seed bed drenches to control damping-off (Fravel et al., 1998). While the Actinomycete Streptomyces griseoviridis and numerous strains of bacterial species including Bacillus subtilis, Enterobacter cloacae and Pseudomonas are available as commercial preparations, Trichoderma, one of several biocontrol fungal species, probably has received the most attention as a seed treatment

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

(Taylor et al., 1994; McQuilken et al., 1998). Seed treatment is more economical than drenching due to the smaller volume of inoculum needed (Bennett et al., 1992). Trichoderma, a mycoparasite that both attacks other fungi and stimulates growth of the host plant, is the most common saprophytic fungus in the rhizosphere. Modes of action of this fungus include mycoparasitism, antibiosis, competition for nutrients and space, tolerance to stress through enhanced root and plant development, solubilization and sequestration of inorganic nutrients, induced resistance, and inactivation of the pathogen’s enzymes (Scala et al., 2007). Yedida et al. (1999) found that Trichoderma harzianum (Th) entered primarily the epidermis and outer cortex of cucumber roots where strengthening of cell walls (increased callose and cellulose) occurred. They also noted increased activities of peroxidase and chitinase in leaves and roots providing evidence that Th may induce systemic resistance mechanisms. Many fungal biocontrol agents, including Trichoderma, are applied to seed as conidia or resting spores which must become active before interaction with the pathogen. The active fungal agent must persist in the spermoplane or spermosphere in sufficient quantity to protect the germinating seed (see reviews by Harman, 2006; Neumann and Laing, 2006). The slurry technique is a common method for applying bioprotectants to seeds (Taylor and Harman, 1990), and consists of mixing the bioprotectant with an aqueous binder and applying this mixture to seeds. Applying a Th suspension to cucumber seeds (without a binder) failed to provide protection against Pythium ultimum (Taylor et al., 1991). Various binders have been employed that have increased the ability of Trichoderma to protect against damping-off organisms. Included among these are gels such as hydroxyethyl cellulose (Hadar et al., 1984) and Pelgel (Nitragen; Inbar et al., 1996), an industrial film coating (vinyl acetate sticker; Cliquet and Scheffer, 1996), gelatin (Roberts et al., 2005), and ground oil palm mesocarp (Kanjanamaneesathian et al., 2003). These binder or coating materials may provide a food base for the Trichoderma. Nelson et al. (1988) found that polysaccharides and polyhydroxyl alcohols promoted Th growth. Stasz and Harman (1980) reported that Pythium species can infect seeds in less than 4 h after sowing, while Trichoderma spores can require about 12 h to germinate (Taylor et al., 1991). For Trichoderma to be effective, it must be active before attack by the pathogen. Taylor et al. (1991) found that when cucumber seeds were double coated, first with a Th slurry then a solid particulate (muck soil or Agro-Lig) plus binder (Pelgel or Polyox N-10), the resultant thin (<0.1 mm), uninterrupted layer (‘‘film-coated’’) over the seed surface was sufficient to slow infection by Pythium by about 6 h, which substantially improved biocontrol over that achieved with only the inner layer. In addition to improving biocontrol with Trichoderma by delaying attack by the pathogen with a physical barrier (Taylor et al., 1991), seed priming is another technique to promote Trichoderma colonization of the seed coat before planting. During priming, seeds are exposed to an environment of low water potential created osmotically or matrically (see reviews by Parera and Cantliffe, 1994; Pill, 1994; Welbaum et al., 1997), or by addition of a limited predetermined amount of water during drum priming (Bennett and Warren, 1997; Bennett and Whipps, 2008a,b). Primed seeds are held at the plateau phase of water concentration (phase II) following a rapid increase in water uptake (phase I of imbibition). During this plateau phase, pregerminative biochemical, physiological and anatomical activities occur that benefit subsequent germination, particularly under stressful seedbed conditions. Ideally, biological control agents added to seeds before or during priming (‘‘biopriming’’) would proliferate to provide greater protection against soil-borne pathogens than adding these agents after seed priming or to non-primed seed.

55

Harman and Taylor (1988) applied Th to cucumber seeds as an aqueous slurry then added the solid matrix and water and incubated this mixture for 4 days at 20 8C (solid matrix priming). The number of Trichoderma propagules increased 10-fold during priming to 103–104 colony forming units (CFUs) per seed, levels that were sustained after drying and a few days of storage. Following priming with a solid matrix of lignaceous shale (AgroLig), these seeds gave 96% emergence (compared to 64% with the aqueous slurry alone) in a P. ultimum-infested soil. Including Th during priming rather than Thiram fungicide gave greater seedling emergence. Solid matrices of bitumous coal or sphagnum peat resulted in fewer healthier plants, indicating the importance of choice of solid matrix by affecting pH or nutrient supply to the fungus. These workers found that priming using a liquid priming system with polyethylene glycol (all other conditions the same as in solid matrix priming) resulted in no Th recovery and poorer performance in P. ultimum-infested soil than was achieved with solid matrix priming. Wright et al. (2003a) noted during drum priming that Th and Tv survived but did not increase on the surface of carrot (Daucus carota L.) and parsnip (Pastinaca sativa L.) seed, and decreased on leek (Allium porrum L.) seed. They further noted that when applied as a post-priming treatment in a large priming system, recovery was greater than achieved when applied during priming. However, successful dual application of beneficial microorganisms on onion or carrot during drum priming was demonstrated recently (Bennett and Whipps, 2008a,b). We know of no reports of biopriming osmotically using inorganic salts. The primary objective of this study was to compare the efficacy of two commercially available Trichoderma species applied to cucumber seeds as a slurry coating before or after osmotic or osmomatric priming on seedling emergence and growth in a Pythium aphanidermatum-infested growth medium. A further objective was to examine the effects of storage (up to 4 weeks) of Trichoderma-coated non-primed seeds at 4 or 21 8C on seedling germination and on Trichoderma viability. 2. Materials and methods Seeds of ‘Marketmore 76’ cucumber (Cucumis sativus L.) were soaked in 0.625% (v/v) NaOCl for 2 min (50 g seeds per 300 ml with two drops of Tween 20 surfactant) then rinsed three times with sterile distilled water. 2.1. Experiment 1: seed priming and T. harzianum Initial work showed that when seeds were imbibed in water at 25 8C, germination started by 12 h and ended with 100% germination by 20 h. Seed moisture at germination was 41.7– 43.3%, oven dry weight (130 8C for 2 h). Further initial studies revealed that a minimum solute potential (cs) of 2.5 MPa at 25 8C was required to prevent seed germination for at least 3 days during seed priming. Osmotic priming of seeds was achieved using 0.337 molal Ca(NO3)2 ( 2.5 MPa at 25 8C according to the Van’t Hoff equation, cs = imRT, in which i = the dissociation constant, m = molality, R = universal gas constant, and T = 8K). Fifty seeds for each of four replications were placed on two layers of germination paper (Germination Blotter No. 385; Seedburo, Chicago, IL) moistened with 15 ml of the Ca(NO3)2 solution contained within 125 mm  80 mm  20 mm transparent polystyrene boxes. The boxes were placed in darkness in 25 8C incubators for 3 days. Matric priming using grade 5 exfoliated vermiculite (W.R. Grace, Columbia, MD) can provide a minimum of 1.5 MPa (50% water of the vermiculite dry weight) with any accuracy according to the moisture characteristic curve developed by Khan et al. (1992). To reach a water potential of 2.5 MPa, a 0.135 molal

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

56

Table 1 Final emergence percentage (FEP) and its angular transformation, days to 50% of FEP (E50), shoot fresh weight at 8 days after planting, and pre-emergence and total dampingoff (percentages and angular transformations) caused by Pythium aphanidermatum in response to Trichoderma harzianum (Th) or mefenoxam fungicide coatings on nonprimed, osmomatrically primed or osmotically primed seeds of ‘Marketmore 76’ cucumber. Seed treatment

Pythium control method

FEP (% (8))

Damping-off (% (8))

Minus

Plus

Pre-emergence Plus

Pythium aphanidermatum Non-primed

a

Total Plus

E50 (days)

Shoot fresh weight (mg/shoot)

Minusb

Mean

a

None Th Mefenoxam.

90.4 (72.0) abcc 90.3 (72.0) abc 94.2 (76.1) a

16.2 (23.7) j 76.4 (60.9) def 53.8 (47.2) gh

62.4 (52.2) a 9.7 (18.1) c 27.7 (31.8) bc

74.2 (59.5) a 13.9 (21.9) b 40.4 (39.5) ab

4.0 a 4.1 a 4.0 a

646 h 761 g 738 h

Primed-osmomatric

None Th before Th after Mefenoxam

91.2 86.2 91.4 91.1

(72.7) (68.2) (72.9) (72.6)

ab abcd ab ab

35.4 21.9 29.1 27.8

(36.5) (27.9) (32.6) (31.8)

hi ij hij hij

44.3 52.5 35.8 42.3

(41.7) (46.4) (36.8) (40.6)

ab ab ab ab

55.8 64.3 62.3 63.3

(48.3) (53.3) (52.1) (52.7)

a a a a

3.4 3.1 3.0 3.4

bc c c bc

815 821 783 919

d cd f ab

Primed-osmotic

None Th before Th after Mefenoxam

80.2 78.5 76.8 93.6

(63.6) (62.4) (61.2) (75.3)

bcde cde cde a

23.0 65.4 60.5 38.0

(28.7) (54.0) (51.1) (38.1)

ij efg fg hi

51.0 10.3 10.5 33.2

(45.6) (18.8) (18.9) (35.1)

ab c c bc

57.2 13.1 16.3 55.8

(49.1) (21.2) (23.8) (48.3)

a b b a

3.3 3.8 3.3 3.0

bc ab bc c

801 931 927 827

e a a bc

Significanced Pythium presence (Pa) Treatment (T) Pa  T a b c d

* *** **

NA **a NA

NA **a NA

Minus Pythium Plus Pythium

934 697

NA **a NA

** *** NS

Pre-emergence and total damping-off found only in plus Pythium aphanidermatum. No damping-off occurred in minus Pythium aphanidermatum media. E50 determined only in minus Pythium aphanidermatum media. Means for a variable within a main effect or interaction followed by the same letter(s) cannot be considered different according to LSD0.05. NS, , * **, ***, NANot significant or significant at P  0.05, 0.01, 0.001 or not applicable, respectively.

Ca(NO3)2 solution ( 1.0 MPa) was added at 50% of vermiculite dry weight. We term this novel treatment that relied on additive matric and solute potentials, osmomatric priming. Osmomatric priming was conducted in 28.4 ml souffle´ containers. Fifty seeds (1.22 g) were added to 6.10 g of vermiculite, a 1:5 seed:vermiculite weight ratio that was selected to minimize a decline in matric potential during seed imbibition. After addition of 3.05 g of Ca(NO3)2 solution to the vermiculite, the seed-vermiculitesolution was stirred thoroughly. The lidded containers then were placed in darkness in 25 8C incubators for 3 days. Some seeds were slurry coated with T. harzianum Rifai strain KRL-AG2 G41 (Th, PlantShield; BioWorks, Victor, NY) before priming. The slurry was stirred into the 50 seeds for each treatment at 0.2 ml per 50 seeds (1 mg Th per seed), a volume sufficient to cover the seed surfaces thoroughly. When dried, the seeds were primed. Following osmomatric priming, seeds were sieved from the vermiculite, rinsed for 10 s under running water, then allowed to dry for 3 days at 21 8C and 40% relative humidity before planting. Osmotically primed seeds were treated identically except that the seeds were washed from the box into the sieve. Non-primed seeds also were coated with Th. A fungicide seed treatment consisted of nonprimed or primed seeds coated with an aqueous suspension of Mefenoxam (Syngenta, Research Triangle Park, NC) at 6 mg of active ingredient per g of seed (0.2 ml stirred into 50 seeds and allowed to dry). Seeds of the 11 treatments (Table 1) were sown into a peat-lite medium (Sunshine Mix No. 1, Bellevue, WA) that had been inoculated or not inoculated with P. aphanidermatum. The contents of three, 10-cm dia. Petri dishes containing this fungus growing on acidified potato dextrose agar (aPDA) were blended into 300 ml of reverse osmosis water, and this stirred into 4 l of grade 3 horticultural vermiculite. The inoculated vermiculite was mixed thoroughly into the peat-lite at 42 ml l 1. The inoculated and noninoculated peat-lite contained within 17 cm  12 cm  6 cm plastic flats (1 l capacity of peat-lite) was kept well-watered and

warm (30 8C) for 4 days before planting. Flats of the 11 (seed treatments and Pythium control method)  2 (minus or plus Pythium) factorial were arranged in a split block design (main plot = Pythium; subplots = seed treatments) with four replications. Seeds were sown into five 1 cm deep  12 cm long furrows within each flat. The furrows then were filled with a fine peat-lite (RediEarth, SunGro Horticulture, Bellevue, WA). The flats were watered twice daily (08:00 and 15:00 h) in a greenhouse set at 25/22 8C (day/night) under natural light (March–April, 2006). The numbers of seedlings emerged (hypocotyl arch visible) were counted daily until there was no further increase over 2 days. The number of shoots that lodged due to post-emergence damping-off also was counted. Percentage total damping-off was the sum of pre- and post-emergence damping-off. From these data, the angular transformation of the final emergence percentage (FEP) and percentages of pre-emergence and total damping-off, and days to 50% of FEP (E50, an inverse measure of emergence rate) were calculated. Percentage pre-emergence damping-off was estimated as: (FEP in minus-Pythium media) (FEP + post-emergence damping-off in plus-Pythium media). At 8 days after planting, 15 representative shoots without obvious disease symptoms were cut from the central area within the tray at the peat-lite surface and the fresh weight per shoot was determined. Emergence and shoot weight data were subjected to analysis of variance. 2.2. Experiment 2: seed priming and T. harzianum and/or T. virens Details of this experiment are the same as for Experiment 1 except that T. virens strain G-41 (Tv, Root Mate; BioWorks, Victor, NY) was included alone or combined with Th (ThTv, each at halfrate) as a seed coating before or after osmotic or osmomatric priming. Non-primed seeds also were treated with the single or combined Trichoderma species. Fungicidal coating of seeds was excluded. This experiment was conducted under the same

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

greenhouse temperature settings as in Experiment 1 (25/22 8C; day/night) but with the natural light of July, 2007. Data collection and analysis was identical to that of Experiment 1.

57

rate) were calculated. FGP and G50 data and CFU values for the 10 3 dilution were subjected to analysis of variance. 3. Results

2.3. Experiment 3: T. harzianum and/or T. virens as a seed coating or growth medium drench Non-primed seeds were coated with Th and/or Tv using the method described in Experiment 2. Seed coatings were compared to growth medium applications of Th and/or Tv. The Trichoderma suspensions were applied to growth media at the highest commercially recommended concentration of 370 mg l 1 water (185 mg l 1 of each for the combined species). Suspensions were mixed into non-Pythium-inoculated peat-lite at 143 ml l 1 (the volume of growth medium in the plastic tray). We hereafter refer to this Trichoderma application method as growth medium drench. The weight of Trichoderma inoculum per seed was equal as a seed coating or as a growth medium drench (1 mg per seed). The Trichoderma-drenched peat-lite was placed into the flats in the greenhouse where they were watered daily for 2 days before onehalf of these flats was inoculated with P. aphanidermatum. After 4 days, non-primed seeds either non-coated or coated with Th, Tv, ThTv or mefenoxam, were sown into flats with or without the Trichoderma-treated media and with or without Pythium. The experiment was arranged in split block design (Pythium = main plots) but the flats (subplots; the 11 treatments listed in Table 3) were spaced at least 0.5 m apart to reduce the risk of Trichoderma cross-contamination. This experiment was conducted under the same greenhouse temperature settings as in Experiment 2, but with the natural light of August, 2007. Data collection and analysis were identical to those of Experiment 2. 2.4. Experiment 4: short-term storage of Trichoderma-coated non-primed seed Non-primed seeds were coated with Th, Tv or ThTv as described in Experiment 3. Treated seeds were stored at 4 or 21 8C for 1, 2, 3 or 4 weeks in 14.8 ml plastic souffle´ cups with the lids secured (50 seeds per cup). Storage times were scheduled so that on the same day seeds from the 3 (Trichoderma coating)  2 (storage temperature)  5 (storage weeks, including 0 weeks) factorial were subjected to both Trichoderma viability and seed germination assays. Treatments (tubes) were replicated three times. To determine colony forming units (CFUs), three seeds were placed in 10 ml of sterilized distilled water within a 15 ml centrifuge tube. The tube was vortexed for 3 min, then 1 ml transferred to another centrifuge tube containing 9 ml of sterilized distilled water. Following 1 min of vortexing, 1 ml of this tube’s solution was transferred to another tube containing 9 ml of sterilized distilled water. For each treatment, the dilutions proceeded to 10 4. A 100 ml aliquot from each tube was transferred to aPDA in 10-cm diameter Petri dishes. The plates were incubated for 3 days at 21 8C under natural light. CFUs were counted over 5 days during which colonies at first were white then turned green as sporulation occurred. The remaining 47 seeds from each treatment were placed on two layers of germination paper (Germination Blotter No. 385; Seedburo, Chicago, IL) moistened with 15 ml of deionized water within 125 mm  80 mm  20 mm transparent polystyrene boxes. Boxes were placed in darkness in 21 8C incubators. The three replications (boxes) of each treatment were arranged in a completely randomized design. The numbers of seeds germinated (having a visible radicle) were recorded and removed daily until no further germination occurred. From these data, the angular transformation of the final germination percentage (FGP) and days to 50% FGP (G50, an inverse measure of germination

3.1. Experiment 1: seed priming and T. harzianum In general, Pythium presence reduced FEP irrespective of Th or fungicide seed coating, although Th was more effective in promoting seedling emergence than fungicide coating in nonprimed or osmotically primed seeds than in osmomatrically primed seeds (Table 1). In media not inoculated with Pythium, no damping-off was apparent and seed treatments had little effect on FEP except for generally lower values for osmotically primed seeds coated with Th before or after priming (Table 1). In Pythiuminoculated media, seedling emergence was 16.2% from non-primed seeds without Th or fungicide coating; seeds of this treatments having 74.2% total damping-off, most of which was pre-emergent. Applying Th or fungicide to the non-primed seeds increased FEP to 76.4% and 53.8%, respectively. Th or fungicide coating of osmomatrically primed seeds failed to increase FEP above that of non-treated seeds (35.4%). The FEP of osmotically primed seeds, however, was greater when Th was applied before or after priming (average 63.0%) than in non-treated seeds (23.0%) or fungicidetreated seeds (38.0%). Coating osmotically primed seeds with Th either before or after priming had no effect on pre- or total damping-off percentage; but, in both non-primed and osmotically primed seeds, Th coating reduced damping-off more than the fungicide. Seed coating with Th reduced total damping-off from 74.2% to 13.9% in non-primed seeds, and from 57.2% to an average 14.7% in osmotically primed seeds. Non-primed seeds had slightly slower seedling emergence (higher E50) than primed seeds in minus-Pythium media, but the 1.1 day range in E50 of all seed treatments would be of little practical significance (Table 1). Pythium presence, compared to its absence, reduced shoot fresh weights by an average 25%. Shoot fresh weights were lower from non-primed seeds than from primed seeds. Th coating of non-primed seeds gave greater shoot fresh weights than fungicide-coated or non-coated seeds. For osmotically primed seeds, shoot fresh rates were in the order Thcoated > fungicide-coated > no coating. 3.2. Experiment 2: seed priming and T. harzianum and/or T. virens Since timing of coating seeds with Th, Tv, or ThTv (either before or after priming) had no effect on variables, responses to the combined timing data are presented in Table 2. In media either without or with Pythium, seeds of both priming methods had lower FEP than those of non-primed seeds, irrespective of Pythium control method. In media without Pythium, lack of Pythium control method effect on FEP was associated with the absence of dampingoff. Although FEP was greater as a result of coating seeds with Th, Tv or ThTv (85.8% average) than in seeds provided no coating (76.7%) in Pythium-inoculated media, Pythium control method interacted with seed treatment in influencing the percentages of both preemergence and total damping-off. For non-primed or osmotically primed seeds, coating with Th, Tv, or ThTv reduced percentages of both pre- and total damping-off below that of non-coated seeds. For osmomatrically primed seeds, only ThTv reduced percentages of pre- and total damping-off below that of non-coated seeds. Most of the damping-off in osmomatrically primed seeds was preemergent, while in osmotically primed seeds most of the dampingoff was post-emergent. With non-primed seeds, however, the biocontrol treatments were more effective than with the primed seeds, in that Th and Tv reduced total damping-off to 9.5% and 1.3%, respectively, compared to the 28.7% of non-treated seeds. Only in

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

58

Table 2 Final emergence percentage (FEP) and its angular transformation, days to 50% of FEP (E50), shoot fresh weight at 8 days after planting, and pre-emergence and total dampingoff (percentages and angular transformations) caused by Pythium aphanidermatum in response to Trichoderma harzianum (Th), T. virens (Tv), or their combination (ThTv) coatings of non-primed, osmomatrically primed or osmotically primed seeds of ‘Marketmore 76’ cucumber. Treatment

E50 (days)

FEP (% (8))

Shoot fresh weight (mg/shoot)

Minus

Plus

Minus

Plus

Minus

Plus

Pythium aphanidermatum Seed treatment Non-primed Primed-osmomatric Primed-osmotic

99.1 (86.9) aa 96.8 (82.4) b 91.3 (74.2) c

89.6 (75.2) c 82.1 (66.4) d 83.4 (67.3) d

1.33 c 0.94 e 1.21 d

1.56 a 0.99 e 1.43 b

602 c 685 a 651 b

555 d 703 a 634 b

Pythium control method None Th Tv ThTv

96.0 96.5 94.4 93.9

76.7 84.5 86.1 86.8

1.18 1.12 1.15 1.10

1.27 1.31 1.29 1.28

668 671 633 647

630 649 643 646

(82.7) (81.7) (79.0) (78.2)

a a a a

(61.4) (67.8) (71.1) (71.5)

c b b b

Plusb

b b b b

a a a a

a a b ab

b a ab ab

Plusb

Pre-emergence damping-off (% (8)) Non-primed Pythium aphanidermatum Pythium control method None 11.2 (19.4) a Th 3.0 (7.0) b Tv 0.7 (2.3) c ThTv 0 (0) c

Total damping-off (%, (8))

Primed-osmomatric

Primed-osmotic

Non-primed

Primed-osmomatric

Primed-osmotic

12.1 15.9 12.7 3.4

8.1 0.4 4.0 0.7

28.7 (32.0) a 9.5 (17.2) cde 1.3 (3.3) f 0 (0) f

19.6 19.8 14.3 6.3

16.2 8.4 12.5 5.8

(19.4) a (21.9) a (17.3) a (6.6) b

(16.3) a (1.2) c (6.6) b (2.5) c

(26.2) (24.8) (18.3) (12.8)

ab ab bcde e

(32.0) (15.3) (20.1) (12.1)

a de bcde e

Significancec

FEP (8)

E50

Shoot fresh weight

Pre-emergence damping- off (8)

Total damping-off (8)

Seed treatment (S) Pythium control (PC) S  PC Pythium presence (Pa) S  Pa PC  Pa S  PC  Pa

*** NS ** * ** ** NS

*** NS NS *** NS NS NS

*** NS NS NS * NS NS

*** ** NS *** *** *** NS

* *** ** *** * *** **

a b c

Means followed by the same letter(s) within an interaction cannot be considered different according to LSD0.05. No damping-off occurred in minus Pythium aphanidermatum media. NS, , * **, ***Not significant, or significant at P  0.05, 0.01, or 0.001, respectively.

non-primed seeds was damping-off eliminated by coating them with ThTv, while in primed seeds the ThTv coating resulted in an average 6.1% total damping-off. Irrespective of Pythium presence or absence in media, E50 was in the order non-primed seeds > osmotically primed seeds > osmomatrically primed seeds (Table 2). Values of E50 and shoot fresh weight were inversely related. Coating seeds with biocontrol agents had no effect on E50 and had very little effect on shoot fresh weight. Pythium presence in media, compared to its absence, increased E50 for all Pythium control methods and for non-primed seeds and osmotically primed seeds. Only for non-primed seeds was E50 higher and shoot fresh weight lower in the presence of Pythium compared to its absence. 3.3. Experiment 3: T. harzianum and/or T. virens as a seed coating or growth medium drench Using only non-primed seeds, we compared responses to Th, Tv or Th + Tv when used as a seed coating or as a growth medium drench. In media without Pythium, no damping-off occurred, and Pythium control method had no effect on FEP (Table 3). In media inoculated with Pythium, however, FEP decreased to 69.3% from 99.4% in non-coated seeds, a response associated with 11.2% and 30.1% pre- and total damping-off, respectively. Only seed coating with Tv or ThTv resulted in no reduction in FEP in Pythium inoculated media (99.3% average) compared to that in media without Pythium (98.1 average). Coating seeds with fungicide or Th raised FEP to 92.6% and 91.2%, respectively, above the 69.3%

FEP of non-coated seeds in media inoculated with Pythium. Combining Th and Tv as a seed coating eliminated damping-off, with Th alone giving higher percentage of total damping-off than Tv or ThTv. In media inoculated with Pythium, percentages of pre-and postemergence damping-off were reduced greatly by Trichoderma growth medium drenches to an average 1.5% and 3.2%, respectively, compared to those in media not drenched (11.2% and 30.1%, respectively; Table 3). Whereas seed treatment with ThTv eliminated damping-off, growth medium drench with ThTv reduced total damping-off to 1.9%. E50 was little affected by Pythium control method or Pythium presence or absence, with a range of only 0.44 days among all seed coating or growth medium drench treatments (Table 3). Drenches of Th, Tv or ThTv compared to seed coating consistently resulted in a small (average 0.28 day) increase in E50. Shoot fresh weight at 8 days after planting was unaffected by Pythium presence or absence. Apart from higher shoot fresh weights with Th or Tv as a seed coating than as a growth medium drench, shoot fresh weights were little affected by Pythium control method (11.7% range of the mean 364.5 mg per shoot). 3.4. Experiment 4. short-term storage of Trichoderma-coated non-primed seed Final germination percentage was unaffected by storage of nonprimed Trichoderma-treated seeds for up to 4 weeks at 4 or 21 8C (average 97.0%; data not shown). Except for an increase in G50 with

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

59

Table 3 Final emergence percentage (FEP) and its angular transformation, days to 50% of FEP (E50), shoot fresh weight at 8 days after planting, and pre-emergence and total dampingoff (percentages and angular transformations) caused by Pythium aphanidermatum in response to Trichoderma harzianum, T. virens, or their combination as either a coating of non-primed seeds of ‘Marketmore 76’ cucumber or as a growth medium drench. Mefenoxam fungicide coating of non-primed seeds is included. FEP (% (8)) Minus

Pythium aphanidermatum Pythium control method Seed coating None Mefenoxam T. harzianum T. virens T. harzianum plus T. virens

99.4 96.9 98.8 99.2 99.4

(87.7) (79.9) (86.8) (85.5) (87.7)

Plus

ab abc a a a

Significancec Pythium control (PC) Pythium presence (Pa) PC  Pa b c

Pre-emergence

Total

Plusa

Plusa

d bc bc a a

11.2 (19.4) a 1.3 (4.6) bc 3.0 (7.0) b 0.7 (2.3) bc 0 (0) c

30.1 (33.3) a 2.6 (9.3) c 7.4 (15.9) b 2.0 (9.8) c 0 (0) d

93.8 (76.1) bc 98.1 (83.2) ab 96.3 (80.5) ab

2.0 (5.7) b 1.3 (4.5) bc 1.3 (4.6) bc

4.5 (9.9) c 3.1 (8.5) c 1.9 (5.6) cd

69.3 92.6 91.2 97.1 99.0

Growth medium drench (seeds not coated) T. harzianum 97.5 (82.2) ab T. virens 98.8 (86.8) a T. harzianum plus T. virens 96.9 (81.5) ab

a

E50 (days)

Damping-off (% (8))

(56.4) (74.2) (72.7) (80.2) (85.9)

*** *** ***

*** *** ***

*** *** ***

Shoot fresh weight (mg/shoot)

Minus

Plus

1.40 1.53 1.30 1.38 1.24

1.51 1.58 1.59 1.60 1.56

bc ab c c c

1.54 ab 1.67 a 1.56 ab

Mean

ab ab ab a ab

581ab 550 b 603 a 563 ab 566 ab

1.68 a 1.61 a 1.56 ab

553 b 537 c 563 ab

*** ** *

** NS NS

Pre-emergence and total damping-off found only in plus Pythium aphanidermatum media (none in minus Pythium aphanidermatum media). Means for a variable within a main effect or interaction followed by the same letter(s) cannot be considered different according to LSD0.05. NS, , * **, ***Not significant, or significant at P  0.05, 0.01, or 0.001, respectively.

Although CFU  10 3 per three seeds generally remained constant at 4 8C, it tended to increase between weeks 3 and 4 in 21 8C storage irrespective of Pythium control method (Table 4). Storage at 21 8C compared to 4 8C resulted in a 3.4-fold increase in CFU  10 3 per three seeds by 4 weeks of storage.

ThTv from 0.88 days after 3 weeks to 1.00 days at 4 weeks of storage, G50 varied little (Table 4). While temperature had no effect on G50 (average of 0.92 days) with up to 2 weeks of storage, by 4 weeks of storage G50 had increased to 1.10 days at 4 8C and decreased to 0.77 days at 21 8C.

Table 4 Time to 50% of final germination percentage (G50) and colony forming units (CFUs) per three seeds after storage of non-primed ‘Marketmore 76’ cucumber seeds coated with Trichoderma harzianum, T. virens, or their combination following 0–4 weeks storage at 4 or 21 8C storage. Treatment

Storage (weeks)

G50 (days) Pythium control method T. harzianum T. virens T. harzianum plus T. virens Storage temperature (8C) 4 21 CFU X 10 4 21

3

0

1

2

3

4

0.99 aba 0.91 c 0.90 c

0.90 c 0.93 bc 0.92 c

0.91 c 0.98 ab 0.90 c

0.89 c 0.98 ab 0.88 c

0.89 c 0.94 abc 1.00 a

0.93 bc 0.93 bc

0.92 bc 0.91 bc

0.94 bc 0.92 bc

0.96 b 0.87 c

1.10 a 0.77 d

1.09 d 1.09 d

1.48 c 2.16 bc

1.92 bc 1.71 bc

1.64 bc 2.65 b

1.16 cd 3.93 a

per three seeds

Pythium control method

4 21

T. harzianum

T. virens

T. harzianum plus T. virens

2.15 a 2.17 a

1.14 b 2.64 a

1.15 b 2.12 a

Significanceb

G50

CFU  10

Pythium control method (PC) Weeks of storage (W) PC  W Storage temperature (T) PC  T WT PC  W  T

** NS *** *** NS *** NS

NS ** NS *** * ** NS

NS,

*, **, ***Not significant, or significant at P  0.05, 0.01, or 0.001, respectively. Means for a variable within an interaction followed by the same letter(s) cannot be considered different according to LSD0.05. b The angular transformation of final germination percentage was unaffected by any factor or interaction (mean FEP value = 97). a

3

per three seeds

60

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

4. Discussion Seed priming is used to improve emergence of crops, particularly under adverse seedbed conditions (Parera and Cantliffe, 1994; Pill, 1994; Welbaum et al., 1997). In a preliminary study we discovered that 2.5 MPa was necessary to prevent germination of seeds over a 3-day priming period, with 2.33 MPa giving 7% on day 3. In a preliminary imbibition study, we noted that 100% germination occurred at between 12 and 16 h of exposure to water at 25 8C, when seed moisture was 41.7–43.3% (oven dry weight) compared to 5.6% in non-imbibed seeds. Three days of priming in 2.5 MPa Ca(NO3)2 resulted in 31.1% seed moisture. The water potential we selected to avoid germination within 3 days of priming is considerably lower than the 1.7 MPa mannitol solution used by Passam and Kakouriotis (1994) to prime cucumber seeds for 3 days at 25 8C. Harman and Taylor (1988) primed cucumber seeds for 4 days at 20 8C in a lignaceous shale matrix at 60% moisture, which resulted in a matric potential > 0.1 MPa (Khan et al., 1992). Presumably, the osmotic components of Agro-Lig lowered the total water potential so that priming without germination occurred. Preliminary work showed that a seed:vermiculite ratio of 1:5 was needed to maintain 1.5 MPa matric potential since lower ratios caused vermiculite water content (and matric potential) to decrease markedly over the 3-day priming period. Previous work has shown that combining priming with the application of beneficial microorganisms can improve crop establishment and several methods have been used to achieve such ‘‘biopriming’’ (Harman and Taylor, 1988; Callan et al., 1991; Jensen et al., 2004). These have generally involved coating the seeds with a microorganism suspension, and then priming the seed using a variety of methods, including incubation under moist conditions in a plastic bag (Callan et al., 1991), or incubation in a moistened organic carrier such as finely ground shale or coal (solid matrix priming) (Harman and Taylor, 1988). While survivability of beneficial fungal isolates may be decreased by competition with indigenous microorganisms as they proliferate during priming (Wright et al., 2003b), this should be reduced or eliminated by disinfesting seeds before priming. Coating seeds with Th (Table 1) or with Th, Tv or ThTv (Table 2) either before or after osmotic priming compared to equivalent applications to osmomatrically primed seeds resulted in greater reductions in percentage damping-off. At least for Th we determined that CFU per three seeds (all 10 3) was greater when the Th was applied after (3.2  0.8 3) than before (1.4  0.7) osmotic priming. For osmomatric seeds, however, there was little difference in CFU per three seeds whether the Th was applied before (2.8  0.3) or after (2.6  0.5) priming, values similar to those where Th slurry was applied to non-primed seeds (2.8  0.7). Osmomatrically primed seeds (and non-primed seed) had higher CFU values than those of osmotically primed seeds (when the Th was applied after priming), and yet the osmotically primed seeds (and non-primed seed) had lower total damping-off (16.3%) than the osmomatrically primed seeds (63.3% average). Thus, we can conclude either that the higher CFU values were not necessary for damping-off prevention or that osmomatrically primed seeds were more susceptible to damping-off than osmotically primed seeds. The inverse relationship between E50 values due to seed treatment (non-primed > osmotically primed > osmomatrically primed) (Tables 1 and 2) and percentage damping-off suggests that susceptibility to the pathogen was directly related to speed of seedling emergence or germination. Earlier penetration of the Pythium-inoculated medium by the radicle of osmomatrically primed than of osmotically primed seeds with presumably less (time for) movement of the biocontrol agent to the radicle from the seed coat may have increased the susceptibility of the osmomatrically primed seeds to damping-off. Likewise, in media

treated with Pythium, E50 (non-primed > osmotic > osmomatric) was inversely related to the percentage of damping-off, lending credence to the idea that the more slowly seeds germinate or emerge, the greater the probability that the biocontrol agent can contact the emerging radicle and become established on the root and in the rhizospere, thus providing greater protection against the Pythium. Taylor et al. (1991) noted that infection of cucumber seeds with P. ultimum can occur very quickly after seed sowing. These authors first coated the seeds with a slurry of Th, then a slurried mixture of solid particulate (Agro-Lig or muck soil) plus binder (Pelgel or Polyox N10), a process they termed double coating. The resultant thin (<0.1 mm), uninterrupted layer over the seed surface was considered to be sufficient to slow infection by Pythium by about 6 h, and biocontrol was substantially improved over that obtained by a simple Th slurry. Sporangia of P. ultimum began to germinate within 3–4 h in response to exudates from bean seeds (Bennett et al., 1992) and yet conidiospores of Trichoderma require 12–14 h to germinate (McQuilken et al., 1998). In the case of non-primed seeds, the Trichoderma conidiospores must germinate and colonize the seed surface during seed imbibition after planting. Based on our data, colonization was hypothesized to be substantial in these non-primed seeds since FEP was high and damping-off was low. Since the effect of coating seeds with Th either before or after priming (Table 1) had no effect on FEP or percentage damping-off, presumed colonization of the seed during priming was of no advantage. While we found no increase in CFU during either osmotic or osmomatric priming, Harman and Taylor (1988) observed a 10fold increase in Th during solid matrix priming of cucumbers seeds. Obtaining high numbers of biocontrol agents is critical since a linear increase in cucumber stand in Pythium-infested soil occurred in response to a logarithmic increase in Th from 0.1 to 10 mg per seed (Taylor et al., 1991). It is possible in the present study that conditions during priming were not conducive to Trichoderma growth. During priming, food support for the Trichoderma in the spermosphere would result only from the clay-based Trichoderma carrier and seed exudates that include sugars, amino acids, organic acids and phenolic compounds (Nelson, 2004). It is possible during osmotic, and less so osmomatric priming, that such seed exudates diffuse away from the seed following concentration gradients, and thus away from the Trichoderma which had been slurry coated onto the seed surface. With drum priming, however, there would be no diffusion of seed exudates away from the seeds. Recently, Bennett and Whipps (2008a,b) noted that biocontrol microorganism proliferation during drum priming was strongly seed-species dependent. Although proliferation occurred on carrot, declining numbers on onion seeds (due to inhibitory seed exudates) had to be compensated for by applying a higher inoculum rate (Bennett and Whipps, 2008b). Nelson et al. (1988) applied Trichoderma conidia to seed in a suspension of methyl cellulose, together with various substrates such as polysaccharides and polyhydoxyl acids which promoted biological control activity. Cliquet and Scheffer (1996) found that coating cucumber and radish seeds using an industrial film coating technique with a 0.5% vinyl acetate sticker reduced damping-off caused by P. ultimum and Rhizoctonia solani. Our preliminary research revealed that 1.5% (w/v) hydroxyethyl cellulose as a binding agent for Th compared to a water slurry had no effect on seedling emergence or damping-off in media infested with Pythium. Harman and Taylor (1988) found that solid matrix priming in Agro-Lig, a lignaceous Leonardite shale with pH 4.1 was more effective than bitumous coal with pH 6.6 or slurry coatings on seeds in promoting Th growth on cucumber seeds and in reducing damping-off due to P. ultimum. Trichoderma grows well in acidic environments as demonstrated by the increased ability of Th to control seed rots when HCl was added to a cellulosic seed coating

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

of cucumber (pH 3.1–3.7; Harman and Taylor, 1988). In the present study, the 2.5 MPa Ca(NO3)2 osmotic priming solution had a pH of 5.8 and the vermiculite plus 1.0 MPa Ca(NO3)2 had a pH of 6.1. Apparently the lower pH during osmotic relative to osmomatric priming failed to promote Trichoderma growth during priming (CFU values of 1.4  103 and 2.8  103, respectively, by the end of priming). Jackson et al. (1991) noted that optimal growth of various Trichoderma isolates occurred between pH 4.6 and 6.8. They also noted that hyphal extension decreased with decreasing water potential but that Trichoderma was more tolerant of osmotic (NaCl) than of matric (polyethylene glycol) potential. Harman and Taylor (1988) found no Th on tomato or cucumber seeds after priming in polyethylene glycol and seeds performed poorly compared to those with solid matrix priming plus Trichoderma. Trichoderma proliferation on seeds in our study may have been promoted had we provided additional nutrients, as noted by Harman and Taylor (1988). The Agro-Lig these researchers used to matrically prime cucumber seeds, besides having a pH favourable to Trichoderma proliferation, contained considerable quantities of organic and inorganic nutrients as well as growth promoting substances. Agro-Lig also provides iron which favours the ability of Trichoderma to compete with other soil microflora (Hubbard et al., 1983). While the combination of Th and Tv applied to osmomatrically primed seeds tended to reduce the incidence of pre- and total damping-off (Table 2), it eliminated damping-off in non-primed seeds (Tables 2 and 3). Disease pressure was lower in Experiments 2 and 3 than in Experiment 1, as evidenced by lower incidences of damping-off, even though the Pythium inoculation protocol was identical. All three experiments were conducted in the same greenhouse with identical temperature settings (25/22 8C; day/ night). However, it is possible, for instance, that growth medium temperatures were affected differentially by the timing of the experiments (1 = March–May; 2 = July; 3 = August) which may have influenced the level of Pythium pathogenicity or the effectiveness of Th and/or Tv. Combinations of microorganisms, such as the ThTv used in the present study, may be better able to colonize the rhizosphere than single isolates by inhabiting different niches and providing a more stable rhizosphere community (Bennett and Whipps, 2008b). A combination may also permit toleration of a broader range of environmental conditions (Pierson and Weller, 1994) and, as multiple modes of action may be exhibited by combinations of microorganisms, mixtures of microorganisms may target plant pathogens more effectively. Recently, combinations of a bacterium and a fungus were established on carrot and onion seeds using drum priming and these microorganism were shown to transfer onto the roots and into the rhizosphere after the seeds were planted (Bennett and Whipps, 2008a,b). Only for non-primed seeds, coating seeds with an aqueous slurry of mefenoxam fungicide resulted in greater FEP (53.8%) than non-coated seeds (16.2%), but this control method was less effective than Th which gave greater protection against P. aphanidermatum (76.4%) (Table 1). In Experiment 3 (Table 3), however, non-primed seeds coated with fungicide gave a lower level of total damping-off (2.6%) than seeds coated with Th (7.4%). Coating the seeds with Tv gave similarly low levels of total damping-off as coating them with fungicide (2.3% average), but not as low as coating seeds with ThTv which eliminated dampingoff. It is possible that if the mefenoxam had been coated on the seeds before priming, greater protection against damping-off would have resulted since the fungicide is systemic and would have been absorbed by the embryo during priming. Taylor et al. (1994) showed that fungicidal seed coatings were of variable effectiveness against disease depending on the crop planted and year of planting. Kanjanamaneesathian et al. (2003) noted that Th

61

cultured on ground mesocarp fiber of oil palm as a seed coating of Chinese kale was not as effective as a seed coating with metalaxyl against damping-off caused by P. aphanidermatum. Taylor et al. (1994) showed that Captan and Apron fungicides were compatible with Th when applied as a slurry seed treatment. However, seed coatings of fungicides with biological control agents or fungicides alone would not be permitted by organic growers. Likewise, growers of vegetable sprouts or microgreens (leafy crops harvest at the 2 through 4 true-leaf stage; see Lee et al., 2004) could not use fungicide coated seeds because of the lengthy preharvest interval (often 28 days) applied to most commercial fungicides. In addition to the ability of some Trichoderma isolates to control plant diseases either by inducing resistance or by direct attack, they can promote plant growth (Harman, 2006; Neumann and Laing, 2006). These authors have attributed many factors to the growth promoting properties of some Trichoderma isolates, including increased plant growth hormone synthesis (indoleacetic and gibberellic acids), enhanced nutrient uptake, enhanced dissolution of soil nutrients, enhanced root development, increased root hair production, and deeper rooting. In the present study, Trichoderma-induced growth promotion was variable. In Experiment 1, Th promoted shoot fresh weight in non-primed or osmotically primed seeds irrespective of the presence or absence of Pythium, while in Experiments 2 and 3 there was little or no growth promotion. These differential responses would most likely be attributable to differences in experimental conditions, primarily temperature, during the sequence of the experiments or to differential levels of Trichoderma propagule viability on the seeds. Cucumber growth promotion in Trichoderma-treated plants has been reported (Kleifield and Chet, 1992; Yedida et al., 2001). Inbar et al. (1996) observed greater seedling height (24%), leaf area (96%), and plant dry weight (25%) at 18 days after planting Th-treated cucumber seeds than in non-treated seeds. Harman and Taylor (1988) noted a marked increase in seedling growth rate and in earliness of emergence from seeds treated with solid matrix priming and Trichoderma, however, this was observed only in Pythium-infested soil. In the present study, the effect of Pythium on shoot fresh weight of symptomless plants was variable. Pythium reduced shoot fresh weight irrespective of seed treatment or seed coating by an average 25% in Experiment 1 (Table 1). In Experiment 2, Pythium reduced shoot fresh weights of seedling only from non-primed seeds or seeds given no Pythium control method (Table 2). Pythium had no effect on shoot fresh weight in Experiment 3 (Table 3). Taylor et al. (1991) noted that the presence of P. ultimum slowed emergences and growth of seedling without obvious disease symptoms, but that the reduction in growth was overcome by solid matrix priming and Trichoderma. In the present study, E50 was increased by about 0.2 days in Pythium-infested media but this generally had no effect on shoot fresh weights at 8 days after planting. While sufficient numbers of the Trichoderma conidiospores must survive seed application and be able to grow in the rhizosphere of the germinating seeds, such seeds must have an acceptable shelf-life. Seeds must be stored at low moisture levels, so the inoculants must be able to survive a period of low water potential. We noted that germination percentages of non-primed seeds coated with Th, Tv or ThTv was unaffected with up to 4 weeks storage at 4 or 21 8C, however, G50 increased slightly between 3 and 4 weeks for ThTv and with storage at 4 8C rather than at 21 8C (Table 4). CFU per three seeds remained constant at 4 8C but increased between week 3 and 4 at 21 8C. Thus, Trichodermacoated seeds can be stored successfully for up to 4 weeks, with some proliferation occurring between weeks 3 and 4 at 21 8C. Cliquet and Scheffer (1996) showed that conidia of some

62

W.G. Pill et al. / Scientia Horticulturae 121 (2009) 54–62

Trichoderma strains applied to cucumber seeds as a carboxymethyl cellulose coating or an industrial film (0.05% vinyl acetate) coating were able to survive storage in sealed pouches for 5 months at 4 8C or 3 months at 15 8C. We conclude that slurry coating of osmotically primed or nonprimed seeds with a combination of Th and Tv is at least as effective as mefenoxam coating or as the ThTv combination growth medium drench in reducing damping-off in a P. aphanidermatum-infested seedbed. Elimination of damping-off, however, occurred only with non-primed seeds coated with the ThTv combination, a response that occurred without Trichoderma-induced increased seedling growth. Seeds coated with Th, Tv or ThTv can be stored for 4 weeks with the Trichoderma viability remaining fairly stable at 4 8C, and increasing from week 3 to 4 at 21 8C. Acknowledgment The authors are grateful to BioWorks, Victor, NY for providing samples of Plant Shield and Root Mate. References Agrios, G.N., 1997. Plant Pathology, 4th ed. Academic Press, London. Bennett, A.J., Whipps, J.M., 2008a. Beneficial microorganism survival on seed, roots and in rhizosphere soil following application to seed during drum priming. Biol. Control 44, 349–361. Bennett, A.J., Whipps, J.M., 2008b. Dual application of beneficial microorganisms to seed during drum priming. Appl. Soil Ecol. 38, 83–89. Bennett, M.A., Warren, J.E., 1997. Seed hydration using the drum priming system. HortScience 32, 1220–1221. Bennett, M.A., Fritz, V.A., Callan, N.W., 1992. Impact of seed treatments on crop stand establishment. HortTechnology 2, 345–349. Callan, N.W., Mathre, D.E., Miller, J.B., 1991. Field performance of sweet corn seed bio-primed and coated with Pseudomonas fluorescens AB254. HortScience 26, 1163–1165. Cliquet, S., Scheffer, R.J., 1996. Biological control of damping-off caused by Pythium ultimum and Rhizoctonia solani using Trichoderma spp. applied as industrial film coatings on seeds. Eur. J. Plant Pathol. 102, 247–255. Fravel, D.R., Connick Jr., D.R., Lewis, J.A., 1998. Formulation of microorganisms to control plant diseases. In: Burgess, H.D. (Ed.), Formulation of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments. Kluwer Academic Publishers, Dordrecht, pp. 187–202. Hadar, Y., Harman, G.E., Taylor, A.G., 1984. Evaluation of Trichoderma koningii and T. hartzianum from New York soil for biological control of seed rot caused by Pythium spp. Phytopathology 74, 106–110. Harman, G.E., Taylor, A.G., 1988. Improved seedling performance by integration of biological control agents at favorable pH levels with solid matrix priming. Phytopathology 78, 520–525. Harman, G.E., 2006. Overview of mechanism and uses of Trichoderma spp. Phytopathology 96, 190–194. Hubbard, J.P., Harman, G.E., Baker, R., 1983. Effect of soilborne Pseudomonas spp. on the biological control agent, Trichoderma harmatum, on pea seeds. Phytopathology 73, 655–659. Inbar, J.A., Menendez, A., Chet, I., 1996. Hyphal interaction between Trichoderma harzianum and Sclerotinia sclerotiorum and its role in biological control. Soil Biol. Biochem. 28, 757–763. Jackson, A.M., Whipps, J.M., Lynch, J.M., 1991. Effects of temperature, pH and water potential on growth of four fungi with disease biocontrol potential. World J. Microbiol. Biotechnol. 7, 494–501.

Jensen, B., Knudsen, I.M.B., Madsen, M., Jensen, M., 2004. Biopriming of infected carrot seed with an antagonist, Clonostachys rosea, selected for control of seedborne Alternaria spp. Phytopathology 94, 551–560. Khan, A.A., Maguire, J.D., Abawi, G.S., Ilyas, S., 1992. Matriconditioning of vegetable seeds to improve stand establishment in early field plantings. J. Am. Soc. Hortic. Sci. 117, 41–47. Kanjanamaneesathian, M., Phetcharat, V., Pengnoo, A., Upawan, S., 2003. Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum, a causal agent of damping-off of Chinese kale seedling. World J. Microbiol. Biotechnol. 19, 825–829. Kleifield, O., Chet, I., 1992. Trichoderma harzianum-interaction with plants and effect on growth response. Plant Soil 144, 267–272. Lee, J.S., Pill, W.G., Cobb, B.B., Olszewski, M., 2004. Seed treatments to advance greenhouse establishment of beet and chard microgreens. J. Hortic. Sci. Biotechnol. 79, 565–570. McQuilken, M.P., Halmer, P., Rhodes, D.J., 1998. Application of microorganisms to seeds. In: Burgess, H.D. (Ed.), Formulation of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments. Kluwer Academic Publishers, Dordrecht, pp. 255–285. Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Trichoderma-induced biological control of Pythium seed rot and pre-emergence damping-off of peas. Soil Biol. Biochem. 20, 145–150. Nelson, E.B., 2004. Microbial dynamics and interactions in the spermosphere. Ann. Rev. Phytopathol. 42, 271–309. Neumann, B., Laing, M., 2006. Trichoderma: an ally in the quest for soil system sustainability. In: Uphoff, N.T. (Ed.), Biological Approaches to Sustainable Soil Systems. CRC, Taylor and Frances, New York, NY, pp. 491–500. Parera, C.A., Cantliffe, D.J., 1994. Presowing seed priming. Hortic. Rev. 16, 109–141. Passam, H.C., Kakouriotis, D., 1994. The effects of osmoconditioning on the germination, emergence and early plant growth of cucumber under saline conditions. Sci. Hortic. 57, 233–240. Pierson, E.A., Weller, D.M., 1994. Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology 84, 940– 947. Pill, W.G., 1994. Low water potential and presowing germination treatments to improve seed quality. In: Basra, A.S. (Ed.), Seed Quality: Basic Mechanisms and Agricultural Implications. Haworth Press, Binghampton, NY, pp. 319–359. Roberts, D.P., Lohrke, M.M., Meyer, S.L.F., Buyer, J.S., Bowers, J.H., Baker, C.J., Li, W., de Souza, J.T., Lewis, J.A., Chung, S., 2005. Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber. Crop Protect. 24, 141–155. Scala, F., Raio, A., Zoina, A., Lorito, M., 2007. Biological control of fruit and vegetable diseases with fungal and bacterial antagonists: Trichoderma and Agrobacterium. In: Chincholkar, S.B., Mukerji, K.G. (Eds.), Biological Control of Plant Diseases. Haworth Press, Binghampton, NY, pp. 151–190. Stasz, T.T., Harman, G.E., 1980. Interactions of Pythium ultimum with germinating resistant or susceptible pea seeds. Phytopathology 70, 27–31. Taylor, A.G., Harman, G.E., 1990. Concepts and technologies of selected seed treatments. Ann. Rev. Phytopathol. 28, 321–340. Taylor, A.G., Min, T.G., Harman, G.E., Jin, X., 1991. Liquid coating formulation for the application of biological seed treatments of Trichoderma harzianum. Biol. Control 1, 16–22. Taylor, A.G., Harman, G.E., Nielsen, P.A., 1994. Biological seed treatments using Trichoderma harzianum for horticultural crops. HortTechnology 4, 105–109. Welbaum, G.E., Shen, Z., Oluouch, M.A., Jett, L.W., 1997. The evolution and effects of priming vegetable seeds. Seed Technol. 19, 209–235. Wright, B., Rowse, H.R., Whipps, J.M., 2003a. Application of beneficial microorganisms to seeds during drum priming. Biocontrol Sci. Technol. 13, 599–614. Wright, B., Rowse, H.R., Whipps, J.M., 2003b. Microbial population dynamics on seeds during drum and steeping priming. Plant Soil 255, 631–640. Yedida, I., Benhamou, N., Chet, I., 1999. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl. Environ. Microbiol. 65, 1061–1070. Yedida, I., Srivastva, A.K., Kapulnik, Y., Chet, I., 2001. Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants. Plant Soil 235, 235–242.