DEVELOPMENT AND ASSESSMENT OF ORGANIC GROWING SUBSTRATES FOR TOMATO TRANSPLANT PRODUCTION AND DISEASE SUPPRESSION
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
CLAUDIA MIREILLE LAFRENIERE
In partial fulfillment of requirements
for the degree of
Master of Science
May, 2011
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1+1 Canada ABSTRACT DEVELOPMENT AND ASSESSMENT OF ORGANIC GROWING SUBSTRATES FOR TOMATO TRANSPLANT PRODUCTION AND DISEASE SUPPRESSION
Claudia Mireille Lafreniere Advisors: University of Guelph, 2011 Dr. Youbin Zheng Dr. Mike Dixon
Organic systems are rigidly regulated in Canada and specify that no synthetic materials be permitted in growing substrates. Research on organic growing substrates is generally not sufficient enough that practical and dependable use recommendations for organic growers can be made. This thesis is an investigation of organic growing substrates for the production of healthy organic tomato transplants and an investigation of the use of manure compost, alone or in combination with Clonostachys rosea, for the suppression of root disease caused by Pythium ultimum in organic tomato transplants. Several substrate formulations consisting of peat moss, coconut coir, fine perlite, fine vermiculite, manure compost, vermicompost, worm castings, yard waste compost, pine bark compost, and/ or aged pine bark were successful for growing healthy tomato transplants in 10-cm pots with organic fertilizer applications.
Transplant roots from sterilized substrates that were inoculated with P. ultimum were more fragile compared to other roots from other treatments, which suggested that some effect on roots was occurring. It was concluded that the establishment of P. ultimum in the root zone of transplants is more likely to occur when the transplants are grown in sterilized substrates that are not amended with the beneficial fungal endophyte C. rosea. ACKNOWLEDGMENTS
I would like to thank my advisory committee, Drs. Mike Dixon, Youbin
Zheng, and John Sutton for the opportunity to do this research and their time spent editing and providing me with helpful advice. I would also like to thank Dr.
Greg Boland for helpful edits, Dr. Paul Sibley for acting as chair at my defense,
Dr. Barry Micallef for accepting to be an external at my defense, and Ron Dutton,
Roger Shantz, and David Kerec for technical support and assistance in the greenhouse.
Sincere thanks to my Colleagues Celia Kennedy, Diane Cayannan, Donny
Cayannan, Jamie Lawson, Jamie Simpson, Linping Wang, Maria Dombrowski,
Matt Hannaberg, Dr. Micheal Stasiak, Olathe Maclntyre, Ping Zhang, and Tom
Graham for their advice and support.
A very special thanks goes out to Dr. Victoria Surrage for all the assistance, the late nights, the friendship, and advice throughout my research and to my family members for financial and emotional support.
I would also like to thank the Ontario Ministry of Agriculture, Food and
Rural Affairs and the Ontario Centre of Excellence for providing financial support,
De Ruiter Seeds Inc. (Lakewood, CO, United States), Dingo Farms (Bradford,
ON, Canada), Forterra Inc. (Puslinch, ON, Canada), Gro-Bark Organics Ltd.
(Milton, ON, Canada), Millenniumsoils Coir Inc. (St. Catharines, ON, Canada),
Peter van Straaten (University of Guelph, Guelph, ON, Canada) and Ed
Vermolen (Aldershot Greenhouses Ltd., Burlington, ON, Canada) for materials. TABLE OF CONTENTS
ACKNOWLEDGMENTS i
TABLE OF CONTENTS ii
LIST OF TABLES vi
LIST OF FIGURES viii
LIST OF ABBREVIATIONS x
CHAPTER 1: Introduction and Literature Review 1
1.1 Overview 1 1.2 Greenhouse Tomato Production in Canada 2 1.2.1 Types of Production Systems 2 1.2.2 Soilless Organic Growing Substrates 7 1.3 Compost 7 1.3.1 Compost Utilization: Waste Management, Enhanced Crop Production, and Climate Change Mitigation 9 1.3.2 The Composting Process 11 1.3.3 Strategies to Optimize the Use of Compost 19 1.4 Disease 25 1.4.1 Common Diseases of Tomato 26 1.4.2 Current Disease Management Strategies 26 1.4.3 Disease Management with Composts 28 1.4.4 Disease Management with Seeded/ Inoculated Microorganisms 31 1.4.5 Rhizosphere Competency of Microorganisms and the Carrying Capacity of Compost-Amended Growing Substrates 33 1.4.6 Mechanisms of Action of Disease Suppression 34 1.5 Thesis Objectives 37
CHAPTER 2: Developing Organic Growing Substrates for Tomato
Transplant Production 39
2.1 I ntroduction 39 2.2 Materials and Methods 42 2.2.1 Substrate Preparation 42 2.2.2 Chemical Properties of the Substrates 46 2.2.3 Growth Experiment 47 2.2.4 Growth Measurements 48 2.2.5 Statistical Analysis 49
n 2.3 Results and Discussion 49
2.4 Conclusion 57
CHAPTER 3: Zeolite as a Component in Organic Growing Substrates for
Tomato Transplant Production ". 58
3.1 I ntroduction 58 3.2 Materials and Methods 59 3.2.1 Substrate Preparation 59 3.2.2 Chemical Properties of the Substrates 59 3.2.3 Growth Experiment 60 3.2.4 Growth Measurements 62 3.2.5 Statistical Analysis 62 3.3 Results and Discussion 62
3.4 Conclusion 65
CHAPTER 4: Developing New Organic Growing Substrates for Tomato
Transplant Production 66
4.1 Introduction 66 4.2 Materials and Methods 67 4.2.1 Substrate Preparation 67 4.2.2 Growth Experiment 70 4.2.3 Chemical Properties of the Substrates 72 4.2.4 Growth Measurements 74 4.2.5 Leaf Chlorophyll Content and Gas Exchange Measurements 74 4.2.6 Statistical Analysis 74 4.3 Results and Discussion 75
4.4 Conclusion 88
CHAPTER 5: Using Processed Organic Waste in Growing Substrates for
Tomato Transplant Production 89
5.1 Introduction 89 5.2 Materials and Methods 91 5.2.1 Substrate Preparation 91 5.2.2 Growth Experiment 95 5.2.3 Chemical Properties of the Substrates 96 5.2.4 Growth Measurements 96 5.2.5 Statistical Analysis 97 5.3 Results and Discussion 98 5.4 Conclusion 110
iii CHAPTER 6: Rate of Inclusion of Manure Compost in Organic Growing
Substrates for Tomato Transplant Production 111
6.1 I ntroduction 111 6.2 Materials and Methods 113 6.2.1 Substrate Preparation 113 6.2.2 Growth Experiment 114 6.2.3 Chemical Properties of the Substrates 114 6.2.4 Growth Measurements and Leaf Chlorophyll Content Index 115 6.2.5 Physical Properties of the Substrates 115 6.2.6 Statistical Analysis 117 6.3 Results and Discussion 117
6.4 Conclusion 130
CHAPTER 7: Comparing the Physical Properties of Growing Substrates
Including Peat, Coir, and Aged Pine Bark to the Physical Properties of
Growing Substrates Including Peat, Coir, Perlite, and Vermiculite 131
7.1 Introduction 131 7.2 Materials and Methods 131 7.2.1 Substrate Preparation 131 7.2.2 Chemical Properties of the Substrates 133 7.2.3 Growth Experiment 134 7.2.4 Physical Properties of the Substrates 136 7.2.5 Growth measurements 137 7.2.6 Statistical Analysis 137 7.3 Results and Discussion 137
7.4 Conclusion 147
CHAPTER 8: Using Compost and Clonostachy rosea Inoculant to Suppress
Pythium ultimum in Tomato Transplants 148
8.1 Introduction 148 8.2 Materials and Methods 152 8.2.1 Substrate Preparation 152 8.2.2 Chemical Properties of the Substrates 152 8.2.3 Growth Experiment 155 8.2.4 Treatments 157 8.2.5 Pathogen 158 8.2.6 Growth Measurements 159 8.2.7 Microbiology 159 8.2.8 Statistical Analysis 163
IV 8.3 Results 164 8.4 Discussion 178 8.5 Conclusion 186
CHAPTER 9:Summary and Recommendations 187
9.1 Summary 186 9.2 Recommendations 195
REFERENCES 200
v LIST OF TABLES
Table 2.1 Abbreviations, and source information for materials 43
Table 2.2 Compacted bulk density, pH, electrical conductivity, and content of
initial water-soluble nutrients in the individual materials 44
Table 2.3 Compositions of the growing substrates tested .46
Table 2.4 Substrates ranked by growth 53
Table 2.5 The pH, electrical conductivity, and content of water-soluble nutrients
in pourthru leachate of each substrate on days 29 and 30 55
Table 4.1 Compacted bulk densities and compositions of the substrates 68
Table 4.2 Compacted bulk density, pH, electrical conductivity, and content of water-soluble nutrients of each individual material 69
Table 4.3 Initial pH and electrical conductivity of the growing substrates 72
Table 4.4 Content of initial water-soluble nutrients in the substrates 73
Table 4.5 The pH and electrical conductivity of the substrates measured on days
24, 31, and 38 77
Table 4.6 Content of water-soluble nutrients in the substrates on day 38 80
Table 4.7 Leaf chlorophyll content index and photosynthetic rate on day 32....86
Table 5.1 Materials, abbreviations, and source information 92
Table 5.2 Initial pH, electrical conductivity, and compacted bulk density of the
individual materials 94
Table 5.3 The pH of each substrate, taken initially and subsequently 104
Table 5.4 Electrical conductivities measured on days 25, 32, and 36 104
vi Table 5.5 Content of water-soluble nutrients in the processed organic wastes used in substrates 106
Table 6.1 Initial pH, electrical conductivity, and compacted bulk density, of the individual materials 113
Table 6.2 Physical properties of substrates 127
Table 6.3 The pH of each substrate, on days 14, 25, 32, and 36 129
Table 6.4 Electrical conductivities measured on days 25, 32, and 36 130
Table 7.1 Substrate compositions 132
Table 7.2 The pH and electrical conductivity of substrates measured initially and subsequently 138
Table 7.3 The pH and electrical conductivity of the seedling substrate 139
Table 7.4 The pH and electrical conductivity of the transplant substrates 139
Table 8.1 Treatments 158
Table 8.2 Variance partition for fungal colony forming units 172
Table 8.3 Variance partition for bacterial colony forming units 173
Table 8.4 Multivariate analysis of variance of the microbial community profile. 176
Table 9.1 Substrates recommended for organic tomato transplant 196
vii LIST OF FIGURES
Figure 2.1 Percent seedling emergence at 24 days after sowing 50
Figure 2.2 Ease of removal of transplant roots from plug trays at 7 weeks after sowing 51
Figure 2.3 Growth responses at five weeks 52
Figure 2.4 Relationship between shoot dry weight and content of water-soluble nitrate on day 30 56
Figure 3.1 Demonstration of the pourthru method 60
Figure 3.2 Top view of selected transplants from second experiment 63
Figure 4.1 Growth responses measured at first and second harvest 78
Figure 5.1 Different composts used in fourth experiment 93
Figure 5.2 Growth responses at first, second, and third harvest 100
Figure 5.3 Leaf area at first, second, and third harvest 101
Figure 5.4 Root and shoot dry weight and root: shoot ratio at second and third harvest 102
Figure 5.5 Nutrient solubility as it is affected by pH change in a soilless growing substrate 105
Figure 5.6 Chlorophyll content index at first second and third harvest 109
Figure 6.1 Demonstration of the North Carolina State University porometer... 116
Figure 6.2 Growth responses at first, second, and third harvest 119
Figure 6.3 Leaf area at first, second, and third harvest 121
Figure 6.4 Chlorophyll content index at first, second and third harvest 122
Vlll Figure 6.5 Root and shoot dry weight and root: shoot ratio at second and third harvest 123
Figure 7.1 Porometer measurements before and after transplant growth 141
Figure 7.2 Top: leaf area versus substrate's initial air-filled porosity at first harvest; Bottom: relationship between leaf area and air-filled porosity 143
Figure 7.3 Stem diameter, stem height, leaf number, and leaf area of transplants 144
Figure 7.4 Fresh and dry weights of shoots of transplants 146
Figure 8.1 The substrate and fertilizer solution's pH and EC 154
Figure 8.2 Characteristic conidiophores and conidia of C. rosea 160
Figure 8.3 Characteristics used to detect Pythium sp 161
Figure 8.4 Unwashed and washed root systems of transplants 165
Figure 8.5 Leaf areas and shoot dry weights at first and second harvest 166
Figure 8.6 Incidence of recovery of C. rosea 168
Figure 8.7 Incidence of recovery of Pythium spp 169
Figure 8.8 Percentages for different outcomes recorded 170
Figure 8.9 Colony forming units counted from Trypticase soy agar and Potato dextrose agar on days 25 and 59 170
Figure 8.10 Microbial community profiles of treatment 175
Figure 8.11 Microbial community profiles taken from pourthru feachates on days
31 and 59 177
IX LIST OF ABBREVIATIONS
AAFC Agriculture and Agri-Food Canada
ADCM Anaerobically digested cattle manure
AFP Air-filled porosity
ANOVA Analysis of variance
APB '. Aged pine bark
BD Bulk density
BNQ Bureau de Normalisation du Quebec
°C Degrees Celsius
C Carbon
C:N Carbon to nitrogen ratio
Ca Calcium
CBD Compacted bulk density
CC Coconut coir
CCI Chlorophyll content index
CCME Canadian Council of Ministers of the Environment
CEC Cation exchange capacity
CEN Centre Europeen de Normalisation
CH4 Methane
CI" Chloride cm Centimeter
CO2 Carbon dioxide
EC Electrical conductivity
x EnvC Environment Canada
EuC European Commission
Fe Iron
FP Fine perlite
FV Fine vermiculite g Gram
GB Growblock
H20 Water
H30+ Hydronium ion
HNO2" Nitrous acid
HNO3 Nitric acid
IFOAM International Federation for Organic Agriculture Movements
ISHS International Society of Horticultural Science
K Potassium
L Liter m Meter
MANOVA Multivariate analysis of variance
MC Manure compost
MC+E Manure compost with eggshells
Mg Magnesium mg Milligram mL Milliliter mS Millisiemens
xi MU University of Missouri
N Nitrogen
N2 Atmospheric nitrogen
N20 r Nitrous oxide
Na Sodium
NCSU North Carolina State University
NDI Nitrogen drawdown index
NH3 Ammonia
NH4+ Ammonium
NH4HCO3 Ammonium bicarbonate
N02" Nitrite
N03" Nitrate
NOSB National Organic Standards Board
OCO Organic Council of Ontario
OH" Hydroxide ion
OMAFRA Ontario Ministry of Agriculture Food and Rural Affairs
OMRI Organic Materials Review Institute
OORA Ontario Organic Research Advisory
P Phosphorous
PBC Pine bark compost pH Approximate p[H]; the negative common logarithm
+ (base 10) of the molar concentration of dissolved hydronium ions (H30 )
PM Peat moss
xii 3 P04 " Phosphate umol Micromole uS Microsiemens v/v By volume
RCBD Randomized complete block design
S Sulfur
SAS Statistical analysis software
SD Standard deviation
S04 Sulphate
SRC Spanish River Carbonatite™
SV Superfine vermiculite
TP Total porosity
VC Vermicompost
WC Worm castings
WHC Water-holding capacity
YWC Yard waste compost CHAPTER 1 introduction and Literature Review
1.1 Overview
Research on growing substrates needed for organic crops is generally not yet sufficiently advanced that practical and dependable use recommendations for organic growers can be made (Kuepper 2004). Organic crop production in
Canada is rigidly regulated with specifications that no synthetic materials, such as wetting agents, fertilizers, and pesticides, be permitted in the growing substrate (Government of Canada 2008a and b). Besides physical support of root systems, substrates for organic crops should provide adequate water, root zone aeration and nutrients to support good plant growth and productivity
(Handreck and Black 2002). The substrates may be any permissible material or combination of materials that meet the requirements for organic crop production.
Presently, the Canadian greenhouse industry is booming (Purdy 2005) and the tomato is the most popular greenhouse crop in North America. Valued at
~$177 million in the year 2000 in Ontario alone (AAFC 2008), Canada is the largest producer of greenhouse tomatoes in North America (Cook and Calvin
2005). For these reasons, the research for this thesis was focused on greenhouse tomatoes. As most greenhouse tomatoes start off as. transplants
(Russo 2005) growing substrates for organic tomato transplants were developed and tested. Environment and waste management issues were also addressed, as these topics are directly relevant to organic production systems. Plant pathogens and disease management strategies relevant to the use of organic growing substrates were also major issues discussed here.
1 1.2 Greenhouse Tomato Production in Canada
Greenhouse crop production in Canada is a fast growing industry; the total area dedicated to greenhouse production increased by >2 million m2 from 2007 to 2009 (Mailvaganam 2010). According to Agriculture and Agri-Food Canada
(AAFC), in 2007, 20.6% of greenhouse production in Canada was for tomato
(AAFC 2008), which is the most popular greenhouse crop in Canada, but cucumber, lettuce, and pepper crops are also produced in large quantities.
Greenhouse tomato production (>50 kgm"2) is up to ten times greater than that in a field (Dorais et al. 2010). Altogether, greenhouse vegetables, in 2003, were valued at nearly $606 million in Ontario, which was more than triple that of
Ontario field production (Purdy 2005; Cook and Calvin 2005). In 2007, greenhouse vegetables accounted for >$789 million in farm cash receipts; and
533 000 tons of fresh vegetables (excluding potatoes) were exported that year, with a large percentage from greenhouse vegetables (AAFC 2007). By comparison, >182 000 tons of greenhouse-grown tomatoes in Canada were valued at $288 million in the year 2000 (AAFC 2008).
1.2.1 Types of Production Systems
High-Energy Efficiency Production Systems
High-energy efficiency production systems are conventional and they are characterized by reduced labor and recycling and increased inputs (e.g. chemical fertilizers and pesticides, fuels, and water). These systems cause over- consumption of environmental resources, contribute to global warming, and result in environmental contamination (Pearson 2007).
2 Low-Energy Efficiency Production Systems
Reduction in dependence of the production system on external inputs while maintaining or increasing yields is an ideal goal that would reduce input costs as well as negative impacts on the environment. Low-energy efficiency production systems are becoming more popular. These systems are known for recycling inputs and are considered regenerative systems because they minimize inputs and minimize impacts on the outside environment (Pearson 2007). Other terms that are often used to describe low-energy efficient systems are
'sustainable' and 'organic'.
A sustainable system is one that can maintain itself biologically, economically, and/or socially; it is often defined in relation to the total amount of inputs needed to maintain biodiversity and yields (Pearson 2007). Certified organic production systems, for example, are considered sustainable when compared to conventional production systems, mostly in terms of environmental sustainability (Andersson et al. 2005). There is increasing consumer interest to purchase organic produce (Martin 2007; Parsons 2002; Bulluck III er al. 2002).
However, the proportion of consumers buying organic produce remains small and health concerns appears to be the main motive over environmental concerns
(Homes and Macey 2008; Andersson et al. 2005). A definition of an organic system [International Federation of Organic Agriculture Movements (IFOAM)] is:
"a production system that sustains the health of soils,
ecosystems and people. It relies on ecological
processes, biodiversity and cycles adapted to local
3 conditions [...]. Organic agriculture combines
tradition, innovation and science to benefit the shared
environment and promote fair relationships and a
good quality of life for all involved" (IFOAM 2009).
Organic technologies have been used for many years. Benefits of organic practices in the field, which include increased soil organic matter, moisture retention, and total carbon (C) and nitrogen (N) content, lead to the conservation of water resources, increased soil cation exchange capacity (CEC), decreased soil bulk density, and increased soil microbial diversity. Microbial diversity can suppress pathogens and thus has the potential to reduce pesticide use; therefore, soils managed under organic production are often considered healthier and more productive than soils managed under conventional production
(Pimental et al. 2005; Bulluck III et al. 2002; Workneh et al. 1993). Adoption of organic practices in the field can make conventional agriculture more sustainable, affordable, and environmentally sound. Reduced external inputs and improved waste management are results of organic practices (Monteny er al.
2006; Follett 2001). As ecological processes that foster plant nutrition, conserve soil, and save water are improved, the financial situation of the growing system is optimized, and the environment is conserved (Pearson 2007; Pimental et al
2005). The same is true for organic practices in the greenhouse.
Greenhouse production is necessary in Canada as cold temperatures preclude field production during the months of October through April. In 2007, there were 10.7 million m2 of greenhouses for vegetable production in Canada
4 (Statistics Canada 2008) whereas in 2005, only 310 000 m2 of greenhouses in
Canada were dedicated to organic vegetables (Macey 2006). Approximately 80% of all organic products consumed in Canada are imported (Homes and Macey
2008). However, as Canada is the largest producer of greenhouse tomatoes
(Calvin and Cook 2005) there is much potential for greenhouse tomato growers in Ontario to adopt organic practices because there is a market for organic products driven by rising demands from consumers concerned with food safety and the environment (Martin 2007; Bulluck III et al. 2002; Parsons 2002). The organic council of Ontario (OCO) claims that only 1% of all agriculture in Ontario conforms to organic production standards, capturing only 15% of the organic market (OCO 2008). Canada (primarily the area of Leamington, ON) has advanced greenhouse technology, which can be adapted to organic techniques to increase supplies of organic produce in Canada (Zheng et al. 2011). However, implementation of organic agriculture in Canada is slow because organic standards are restrictive and growers feel they have insufficient knowledge and information to grow crops successfully (Martin 2008; Martin 2007; Diaz-Perez et al. 2006; Peet et al. 2004; Rippy et al. 2004; Martin 2001).
Worldwide regulation for organic production is nonexistent; several standards are used to define organic production. For instance, the European
Commission (EuC) states that hydroponic production, which is the production of plants in aqueous solution, is prohibited in European organic greenhouse systems (EuC 2008), but the National Organic Standards Board (NOSB) of the
United States allows hydroponics in organic greenhouse systems (NOSB 2008).
5 Canadian regulation, on the other hand, initially did not prohibit hydroponic production (in 2006); however, revisions made in 2008 stated that hydroponic production is prohibited (Government of Canada 2008a and b).
Discrepancies between national regulations of organic production systems create both opportunities and obstacles for Canadian greenhouse growers. Most
Canadian greenhouse produce is exported to the United States, and since the
United States has a more relaxed regulatory system in place for organic production, Canadian growers can easily export organic produce to the United
States. However, as a vast majority of Canadian greenhouse growers are descendants of European nations, they obtain most of their growing techniques from Europe. Canadians and Europeans are at the forefront of the greenhouse technology, primarily because of climate (AAFC 2008). Canadian organic greenhouse growers depend on European technology. However, European organic regulation prohibits the use of soilless growing substrates so there is little to no research available on soilless organic substrates in Europe. Canadians growing organically must therefore conduct their own research on soilless growing substrates. Other obstacles to widespread adoption of organic production systems include unpredictable yields, and risks associated with shifting to new fertility and pest management techniques (Martin 2008; Peet et al.
2004). In addition, funds to support organic research are limited, and growers tend to keep their findings to themselves. This lack of research and communication among growers of organic crops in Canada is a major obstacle that needs to be overcome (Zheng et al. 2011).
6 1.2.2. Soilless Organic Growing Substrates
Soilless growing substrates, for container grown plants, are most commonly used in Canadian greenhouses to avoid soil-borne pathogens and avoid nutrient deficiencies associated with the repeated use of the same soil.
Currently, there is a very limited supply of reasonably priced and certified organic growing soilless substrates available in Canada. Growers tend to mix their own substrates through trial and error. Spagnum peat moss, coconut coir, perlite, vermiculite, and zeolite are all examples of components in organic soilless growing substrates (see section 2.1 Introduction for more information on these materials). Compost is produced from organic waste and is also often included in soilless growing substrates for organic production. Compost is a major topic in this research.
1.3 Compost
Composts are decayed organic matter often used as partial fertilizer for plants. A definition commonly used to describe compost is "aerobically stabilized or matured organic matter"; however, anaerobically processed organic matter may also be stable and mature (Komilis and Tziouvaras 2009). Composts are used increasingly in Canada as soil or growing substrate amendments, or as substitutes for peat moss, bark, and animal manures (Favoino and Hogg 2008;
Chong 2005). However, disadvantages of compost use exist. Obnoxious odors, possible presence of contaminants (heavy metals, organic chemicals, non biodegradable materials), potential phytotoxicities, variability in quality between batches, and non-optimal pH leading to nutrient deficiencies prevent more
7 widespread use of compost. Waste generated from greenhouse production systems first needs to be segregated (i.e. non-organic materials such as plastics must be removed); increased labor resulting from tedious sorting is a laborious burden to some. Nonetheless, composting organic waste from greenhouse use is an effective way to dispose of organic waste hygienically, and to reduce landfill.
Compost must be produced following product quality standards and regulations for compost and composting, which are provided by Canadian organizations: (1) The Standards Council of Canada [through the Bureau de normalization du Quebec (BNQ)]; (2) Agriculture and Agri-Food Canada (AAFC);
(3) The Canadian Council of Ministers of the Environment (CCME); (4) the
Ministry of the Environment (MOE); (5) The Canadian Food Inspection Agency
(CFIA); and (6) the Environmental Protection Act (EPA 2008; CFIA 2007; CCME
2005; MOE 2004; Lefebvre et al. 1994). Furthermore, organic standards for composting must be followed by organic growers; including: the 'Organic
Production Systems General principles and Management Standards'
(Government of Canada 2008a); and The 'Organic Production Systems
Permitted Substances Lists' (Government of Canada 2008b). Regulations are important to ensure compost quality for both conventional and organic systems.
For successful marketing, compost composition must be consistent with appropriate physical and chemical properties (Boulter et al. 2002c; Forste 1997).
Growers interested in on-site composting may construct their own composting bins rather than buying custom ones. A study at the University of
British Columbia (Cheuk et al. 2003) found that an on-site-in-vessel composting
8 container system, made from a used refrigerated shipping container, could produce high-quality compost from greenhouse wastes. A 10% yield increase was observed with the use of the compost to grow tomatoes (Cheuk et al. 2003).
In this case (over a five year period), the net savings due to composting rather than disposing of the waste were $8 000 annually (Cheuk et al. 2003). On-site composting eliminates hauling and tipping fees, which can accumulate to more than $100 000 per year for a 100 000 m2 greenhouse (Cheuk et al. 2003).
Therefore, on-site composting is likely to be a more sustainable practice than landfill as it may generate added revenue rather then added expenditure; growers and home owners alike can benefit from such on-site composting
(Cheuk er al. 2003). Compared to large scale (industrial) composting, ammonia
(NH3), methane (CH4), and nitrous oxide (N20) emissions may be five times greater in small-scale composting whereas large-scale composting may consume or generate up to 53 times more energy, water use, transportation, infrastructure, electricity, waste, and/or volatile organic compounds. Overall on- site composting of organic waste can be a good alternative or complement to industrial composting [especially in low-density areas of population], especially if the compost is used to grow other crops or as an added source of income
(Martinez-Bianco et al. 2010).
1.3.1 Compost Utilization: Waste Management, Enhanced Crop Production, and Climate Change Mitigation
Waste management is a problem of global proportion. As food security issues are on the rise, and concerns with waste management cannot be
9 overlooked, there is a trend towards compost utilization to enhance crop production and reduce environmental degradation and landfill (Martinez-Bianco et al. 2010; Cheuk et al. 2003). Properly prepared compost can be used as a soil or growing substrate conditioner that not only contributes to fertility, but also structure, porosity, organic matter, water-holding capacity, CEC, pH buffering, and disease suppression (Boulter et al. 2000; Hsu and Lo 1999). Compost can also be used to fight soil erosion, and to mitigate greenhouse gas emissions
(Favoino and Hogg 2008).
The most important and significant terrestrial pool of C storage is in our soils (Follett 2001). Compost applied to soils helps build up organic C and improves physical and chemical properties to ultimately mitigate soil erosion and greenhouse gas emissions, which are currently caused by land misuse and soil mismanagement (Martinez-Bianco et al. 2010, Favoino and Hogg 2008; Insam and Wett 2008; Lai 2004). Declines in soil organic C is an issue throughout the world (Follett 2001), and although losses of soil organic C to the atmosphere may be offset by C sinks such as peat moss bogs, oceans, and soils, future global warming adds uncertainty to future trends in C sequestration, which add more significance to tackling this issue now (Monteny et al. 2006; Bellamy et al. 2005;
Lai 2004; Follett 2001). The application of quality compost (free of contaminants) to soils promotes the build-up of soil organic matter, which restores soil quality, helps prevent flooding and desertification, enhances biomass production, and purifies surface and ground water. In addition, compost improves soil structure
(Cogger 2005; Lynch et al. 2005), thereby reducing consumption of fuel and
10 associated emissions that would be released to till and work the soil (Favoino and Hogg 2008). Compost can also reduce the use of chemical fertilizers synthetic pesticides, which also helps mitigate greenhouse gas emissions by moderating fossil fuel consumption needed to produce and apply these chemicals (Favoino and Hogg 2008; Chen era/. 1992). As an alternative to peat moss in growing substrates, compost helps to reduce C02 emissions associated with the drainage and degradation of peat moss bogs, which are major C sinks when left undisturbed (Favoino and Hogg 2008; Ozores-Hampton et al. 1999;
Lamanna et al. 1991). Compost also helps to increase plant growth, thus increasing C sequestration in plants and temporarily reducing carbon dioxide
(C02) from the atmosphere (Lynch et al. 2005; Gonzalez and Cooperband 2002).
1.3.2 The Composting Process
Composting is an environmentally friendly, biological, and aerobic process that breaks down organic matter and converts it to a more stable, useful, and affordable product (Cheuk et al. 2003; Boulter et al. 2000). Composting is a dynamic process involving continuous changes in chemical, physical, and biological properties. Variations in the composting process will have an impact on the quality, the physicochemical properties, and the microbial composition of the compost such as: differences in length of time to process the compost, which depends on the size of the composting operation; whether or not the pile is covered or turned; the location and size of the compost pile; the amount and quality of raw organic material used; the nutrient content and mineralization rates; the temperature reached; the level of aeration in the pile; the moisture
11 level; the C to N ratio (C:N) of the feedstock; the salt level; the pH; the abundance and composition of microorganisms present throughout the process; the time of year; the physical practices; the degree of contamination with soil; and any other additional amendments (Insam and Wett 2008; Larney and Hao
2007; Curtis et al. 2005; Dianez et al. 2005; Raviv et al. 2004b; Boulter et al.
2000; Larney era/. 2000; Hoitink et al. 1997; Boehm et al. 1993). Re-colonization of composts by beneficial microbes can be augmented by inoculation or it can be natural assuming all basic requirements for successful composting are present
(i.e. temperature and adequate mixing, good moisture, and oxygen; see below).
Compost Temperature
Many factors affect the microbial community during the composting process but temperature is a major factor. The microbial community present in the compost follows a predictive successional pattern with distinct temperature stages (Boulter et al. 2002c; Boulter et al. 2000). True composting occurs in three temperature stages. In the first stage (days 1 to 3), temperatures reach 40 to 50°C and sugars are decomposed, C02 production is increased, and mesophilic, zymogenous (i.e. pertaining to the excretion of zymogens, which are enzyme precursors) fungi and bacteria are present. In the second stage, temperatures reach 55 to 70 and sometimes 80°C, and cellulose, hemicellulose, and lignins are degraded. Methane production increases, and actinomycetes and thermophilic bacteria are present. The heat is generated by microbial metabolism and kills many plant pathogens and seeds; however, turning the pile regularly ensures proper heat penetration of the whole pile. Many microorganisms
12 metabolize efficiently at a certain optimum temperature and, generally, optimum decomposition occurs near 55°C. In the final phase known as the curing stage, decomposition continues, temperatures decline, and re-colonization by mesophilic autochthonous microorganisms (some of which are beneficial for disease suppression) occurs. Biomass decreases up to 95% from initial state,
CH4 emission also decreases, and N20 production increases (Boulter et al. 2000;
Hermann and Shann 1997). However, not all composting procedures occur in definite temperature stages. Backyard compost bins rarely reach the high temperatures of true composting. Instead, small-scale composting is often just rotting vegetation without much heat (Boulter er al. 2000).
The C:N of Compost
Not all organic wastes can be used as a single base to produce high- quality compost. Proper composting often requires more than one input to optimize the physical and nutrient components of the end product (Forste 1997).
To produce a quality compost a good balance is needed between materials with high C:N, materials with low C:N, and bulk materials with rich structure (Muller
2009). The optimum initial C:N of the feedstock is between 20:1 and 25:1. During proper composting, C is released. Carbohydrates are broken down into simple sugars and organic acids, incorporated into bacterial protoplasm, and finally released as CO2, which lowers the C:N but, in some cases, the C:N may increase due to losses of N being greater than losses of C. Proteins (source of N)
+ are generally broken down into peptides, amino acids, and ammonium (NH4 )
13 compounds, incorporated into bacterial protoplasm or released as atmospheric N
(N2) or leached out of the compost as NH3 (Boulter et al. 2000).
The C:N of compost can be used to determine compost maturity and stability. An ideal compost C:N is 10:1 (Larney and Hao 2007) although adequately composted material may have a C:N anywhere between 5:1 and 20:1
(Boulter et al. 2000). Because of this variability, C:Ns are not precise indicators of compost maturity and stability, but they can still be useful for application of compost. When an immature compost with high C:N is applied to soil, the high C levels cause the N to be immobilized in microbial biomass and results in impaired plant growth due to N deficiency. If an immature compost with a low C:N is applied, the potential to incorporate pollutants, or materials with phytotoxic properties becomes high (Boulter et al. 2000).
Compost Aeration
Aeration is an important, often limiting factor, for decomposition. Aerobic decomposition products are NH3, C02, water, heat, and humus, whereas anaerobic decomposition products are CH4, C02, and intermediates (i.e. organic acids and alcohols). Ways of maintaining aeration in the compost pile include natural, passive, and forced aeration methods. An example of natural aeration is a windrow compost system where compost piles are turned periodically with machines. Passive aeration involves the use of aerating pipes at the base of the pile to increase the rate of composting; however, with passive aeration there is a risk of uneven aeration, which could result in uneven composting of the pile.
Forced aeration is often performed in a closed- or in-vessel system and
14 designated by a high degree of process control. Although more costly than natural and passive aeration, forced aeration is suited to hazardous wastes
(Boulter et al. 2000). The pH of compost is a good indicator of aeration where high pH is characteristic of well-aerated compost and low pH indicates the likelihood of anaerobic conditions (Boulter et al. 2000; Hoitink et al. 1997).
Compost pH
Compost pH is generally initially acidic (pH <6) due to the presence of organic acids that are formed during the early stages of decomposition (Day and
Shaw 2001). Organic acids commonly found in immature composts are: acetic, propionic, isobutyric, butyric, isovaleric, valeric (Wiles et al. 2001), lactic, formic
(Veeken et al. 2000), humic, and fulvic acids (Hsu and Lo 1999). As composting proceeds the pH reaches near neutral as these acids are converted to CH4 or
CO2 and released to the atmosphere (Day and Shaw 2001; Boulter et al. 2000).
Wiles et al. (2001) found that acetic and butyric acids were emitted in the greatest amounts from swine waste and sawdust compost, followed by propionic, valeric, isovalyric, and isobutyric acids. The mineral decomposition of organic C also affects compost pH. Organic C in immature compost consists mainly of sugars, hemicellulose, phenolic substances, organic and amino acids, peptides, and other easily biodegradable substances, whereas most of the organic C in mature compost is in the form of humic substances. Humic substances are resistant to further degradation and have a high pH buffering capacity, which helps stabilize the pH of the finished compost (Hsu and Lo 1999).
15 During decomposition, compost pH generally rises because of the decomposition of organic N (i.e. protein, amino acid, etc.), which releases NH3.
When NH3 dissolves in water it generates hydroxide ions (OH") by accepting protons from water and the resulting pH of the mixture is alkaline; however, NH3 is a weak base because it reaches an equilibrium state before all NH3 molecules
+ have accepted protons from water. The protonated form of NH3 is NH4 , which is
+ the conjugate acid of NH3. When NH4 dissolves in water, protons are transferred
+ to water and generate hydronium ions (H30 ) that acidify the solution (Bertran et al. 2004; Olmstead and Williams 2001). The pH of compost is therefore dependent on many factors.
+ The mineralization of organic N produces NH3 and NH4 via
+ ammonification; however, NH3 and NH4 production is highly dependent on many variables during the composting process. Maintaining a lower pH (<9) in a high
C:N substrate reduces NH3 production and maintaining a high pH (>9) in a low
C:N substrate at high temperature and aeration promotes the production of NH3
(Pagans et al. 2006; Raviv et al. 2004b; Wiles et al. 2001). Conversely, NH3 will also have an affect on the pH of the substrate depending on the amount of aeration, the temperature, and the pH. NH3 is neutralized by dissolved CO2, which produces ammonium bicarbonate (NH4HCO3) that maintains the pH near neutral. But, if aeration is high, and the C02 content of the compost is diluted by this action, this causes the pH to rise furthermore because NH4HC03will convert back to NH3, water (H20), and C02 (Wiles et al. 2001). NH3 is basic in aqueous form (1.0 M = pH 11.6) but if a strong acid is added lowering the pH to neutral
16 + (7), 99% of the NH3 is protonated to become NH4 , which is the mildly acidic
+ conjugate acid of NH3; however, at very high pH (>9) NH4 are unstable and end up being converted back to NH3 and under aerobic conditions, nitrification
+ converts NH4 into nitrites (NO2"), which are salts (or conjugate bases) of nitrous acids (HNO2; weak acid), and nitrates (NO3"), which are salts (or conjugate bases) of nitric acids (HNO3; strong acid) ultimately resulting in a decline in the
+ mildly acidic NH4 , an increase in the conjugate bases NO2" and NO3", and an increase in pH (Larney and Hao, 2007). Most finished composts are therefore slightly alkaline (pH 7.5 to 8.5) and stable (Bertran et al. 2004; Day and Shaw
2001; Hsu and Lo 1999).
Compost pH affects microbial colonization; low pH (<5.0) precludes colonization by bacteria, which require a pH between 6 and 7.5, whereas fungi can tolerate pH between 5.5 and 8.0. For fungi, pH tolerance depends more on nutrient supply than pH itself. Adequate composting can be achieved within a pH range of 5.5 and 5.9 (Wiles et al. 2001), but the optimum pH for decomposition is considered anywhere between 6.5 and 8.5 (Day and Shaw 2001).
Microorganisms in Compost
Quality composts are teaming with a diverse array of microorganisms.
Thermophilic and aerobic composting involves mesophilic, thermotolerant and thermophilic aerobic microorganisms (e.g. actinomycetes, bacteria, and fungi).
Actinomycetes are strict aerobic saprophytes. They thrive on a wide range of C- sources and are mostly found in well-aerated portions of the compost pile (within
-15 cm of the surface). Actinomycetes grow optimally between 25 and 30°C and
17 a pH between 5 and 9. Actinomycetes grow more slowly than bacteria and fungi, and therefore are poor competitors in high-nutrient substrates but excellent competitors in low-nutrient substrates (Boulter et al. 2000).
Eubacteria (true bacteria), unlike fungi and actinomycetes, are present throughout the whole composting process. Bacteria present in the initial stages of composting are mesophiles (e.g. the genera: Pseudomonas, Achromobacter,
Flavobacterium, Micrococcus, and Bacillus). Bacteria present in the thermophilic stage are thermophiles (e.g. genera: Bacillus, Thermomonospora, Thermus,
Micropolyspora, and Hydrogenobacter), and are mostly gram-positive. However, as the compost cures, cellulose-degrading bacteria are present and gram- negative bacteria (e.g. genera: Pseudomonas, Serratia, Klebsiella, and
Enterobacter) replace gram-positive bacteria. Actinomycetes and bacteria (e.g. genera: Xanthomonas, Janthinobacterium, Flavobacterium, and Bacillus), which can be antagonists of pathogens, are often present in the finished compost
(Boulter etal. 2002c; Boulter era/. 2000; Hermann and Shann 1997).
Fungi are extremely important decomposers. Fungi decompose the complex polymeric substrate known as lignocellulose with hydrolytic and oxidative enzymes. Genera that are commonly found in compost are mesophilic fungi, such as Thermomyces, Penicillium, Aspergillus, Geotrichum, Mucor, and thermophilic fungi, such as Cladosporium (Boulter et al. 2000).
Vermicompost
Traditional thermophilic composting is generally more time-consuming than non-thermophilic (i.e. mesophilic) vermicomposting, which uses
18 earthworms. Earthworms can be used to fragment organic matter into fine particles by passing it through their grinding gizzard. Mesophilic vermicomposting results in a product low in soluble salts with high CEC (Arancon et al. 2004); vermicomposts also provide nutrients for plants and contain plant growth regulators such as indoles (i.e. indole acetic acid-like substances), humic acids, cytokinins, and auxins, which can improve plant growth (Paul and Metzger 2005;
Edwards 2004; Canellas et al. 2002). In terms of bacterial populations, one study found that composts were mainly composed of the phyla Actinobacteria and
Firmicutes, whereas vermicomposts were dominated by other bacterial phyla:
Chloroflexi, Acidobacteria, Bacteroidetes, and Gemmatimonadetes (Fracchia et al. 2006). Anastasi et al. (2004) found that fungal populations are variable in terms of abundance and diversity in vermicomposts compared to composts.
However, these results should not be regarded as universal as the final microbial community present in any compost is largely dependent on the level of maturity and the quality of the material being composted (Hermann and Shann 1997).
1.3.3 Strategies to Optimize the Use of Compost
Nutrient content
In addition to compost, organic fertilizers are often required in organic production (Larrea 2005). Composts provide low amounts of nutrients and release these nutrients at a lower rate compared to synthetic forms of fertilizer
(Diaz-Perez et al. 2006). An imbalance of plant nutrients often exists in compost; when rates of compost applications are based on N content, excessive amounts of phosphorous and insufficient amounts of potassium are likely to be applied
19 (Muchovej and Pacovsky 1997). However, research (discussed below) suggests there is an opportunity to optimize the supply of most plant nutrients available in composts, enabling growers to limit the need for supplementary fertilization
(Raviv et al. 2005; Nielson and Thorup-Kristensen 2004; Raviv et al. 2004b).
Ammonification gives rise to NH3, which ultimately volatilizes or converts
+ to NH4 depending on pH and air movement. Later in the composting process,
+ nitrification converts NH4 to NO2' and NO3", ultimately resulting in a decline in
+ NH4 and an increase in NO2" and NO3", which leads to higher pH (Larney and
Hao 2007). N losses to volatilization of NH3 are driven primarily by low C:N, high pH, and high aeration during the composting process (Raviv et al. 2004b). True composting, with continuous turning of the pile, results in greater losses of N than composting without turning (stockpiling); however, N losses during the composting process can be minimized using high C:N or slightly acidic additives
(Raviv er al. 2005). According to Raviv et al. (2004b) acidic orange peels help retain more N in composted manure than additives with high C:N (i.e. grape marc and wheat straw). Adding phosphogypsum (a by-product of phosphorous fertilizer manufacturing) to the compost feedstock also reduces N losses and increases sulfur content of the compost, which can then be used on crops with high sulfur demands growing on sulfur-deficient soils (Larney and Hao 2007).
Predicting nutrient losses and availability in composts helps tailor compost application rate to plant needs (Larney and Hao 2007). Standardized testing prior to using compost, to reveal nutrient mineralization rates, which can be predicted from chemical properties of composts, can reveal the nutrient content of compost
20 for optimum application rates (Larney and Hao 2007). For example, total C
+ evolved from composts can be predicted from the NH4 content and the
NH4+:N03" ratio, and phosphorous availability can be predicted from total water- extractable phosphorous and total phosphorous content.
Compost Quality
Improving the quality and management of composts is a major priority
(Anton et al. 2005). The most important requirement of compost before it is used is maturity and stability. Maturity refers to the effect of compost on plant growth by presence or absence of phytotoxins; immature compost can be phytotoxic and may contain NH3 salts, heavy metals, and/or organic acids, whereas stability refers to the degree of decomposition by microorganisms (Komilis and
Tziouvaras 2009). A cheap and simple maturity and stability test for compost has been sought for many years (Boulter et al. 2000), and still there is no universally accepted standard for assessing compost (Komilis and Tziouvaras 2009).
Compost manufacturers would benefit from determining economic viability of composts in terms of maturation, stability, and presence of beneficial microbial agents (Trillas et al. 2006). Carbohydrate, lipid, and other organic compound fractions have been evaluated as potential indicators of compost maturity
(Boulter et al. 2000). In addition, compost temperature, NH3 volatilization, CO2 evolution (respiration), and N2O production, which are all indicators of microbial activity, can also be used in compost maturity tests (de Guardia er al. 2010a and b; Scheuerell et al. 2005; Boulter et al. 2000). Compost maturity is considered to
+ have been reached if the NH4 :N03" ratio is less than 1:1 (by weight) (Larney and
21 + Hao 2007; Pare et al. 1998); however, maturity may be reached at NH4 :N03" ratio of 4:1 (Brewer and Sullivan 2003). Temperature is proportional to the total amount of organic matter that biodegrades during composting; this provides insight into optimizing feedstock formulations to increase temperature during composting (de Guardia etal. 2010a). The seed germination index (Zucconi et al.
1981) is still widely used for testing compost maturity (Komilis and Tziouvaras
2009), and the C:N may be used as an index of compost stability where a C:N of
10:1 (by weight) is considered very stable (Larney and Hao 2007). However, because of variability, C:Ns are not precise indicators of compost stability.
While maturity and stability tests based on indirect quantitative microbial assessments are useful it is even more important to assess microbial activity in a more qualitative way as distinct microbial features, shifts, and changes in microbial function may provide information on the maturity and stability of compost (Boulter et al. 2002c; Boulter et al. 2000). Such microbial assessments for compost may include microbial ratio indices, C-source utilization assays, and identification of bacterial isolates and metabolic traits (Boulter et al. 2000).
Phospholipid fatty acid analysis allows for a real-time evaluation of compost maturity, provided that microbial communities are proven to change over the composting process in a consistent and predictable manner (Boulter et al. 2002b;
Boulter et al. 2002c; Boulter et al. 2000; Hermann and Shann 1997).
Phospholipid fatty acid analysis can be used to estimate microbial diversity, to fingerprint the community structure, and measure microbial biomass; it is a more robust method of quantifying microbes than culture plating techniques. Moreover,
22 phospholipid fatty acids degrade quickly upon cell death, which is important for estimation of the active microbial community (Boulter et al. 2002c). However, this type of analysis is too complicated and expensive for routine analysis of compost, and thus should only be used in studies to optimize the composting process and to evaluate the compostability of new materials and the distinct microbial features of new composts (Boulter et al. 2002c; Hermann and Shann
1997). Furthermore, microbial community structure and shifts are influenced by environmental properties; therefore, studying these interactions can reveal information on microbial function in compost (Boulter et al. 2000). A number of tests have been proposed based on physical and chemical indices, enzymatics, microbial and plant bioassays, and physicochemical parameters. Yet maturity and stability remain difficult to define. More importantly, many technologies are just too sophisticated and expensive to serve as routine monitoring tools (Boulter et al., 2000). However, the assessment of microbial involvement in compost is critical to further understand their role in disease suppression.
Genetics
Parasites of fungal pathogens, such as Trichoderma spp., produce enzymes (proteases, chitinases, and glucanases) to extract nutrients from their fungal host, and some of these parasites also produce antibiotics to initially weaken the host. When mixed, these enzymes and antibiotics can be synergistic
(Woo et al. 2002; Lorito 1998). Advances in technology have allowed genetic manipulations of microorganisms for the benefit of growers. Protoplast fusion was successfully used >20 years ago to combine two rhizosphere competent
23 fungal strains to produce progeny that are more effective in disease suppression and that are more rhizosphere competent than either parental strain (Harman
1992). Experimentation with transgenic plants expressing beneficial genes involved in disease suppression, or transgenic microorganisms co-expressing antibiotic and enzymatic genes in synergistic combinations, substantially improves our understanding and application of genetic manipulations for use in disease suppression. However, environmental concerns and regulations are likely to delay adoption of these new technologies. The use of transgenic organisms for disease control has been discussed (Lorito 1998), but transgenic organisms are definitely not allowed in organic production systems (Government of Canada 2008a). Only extensive testing will reveal any commercial value and practicality of transgenic plants and microorganisms to reduce chemical inputs for more sustainable plant production. Nonetheless, genetic advances including targeted or non-targeted gene isolation, sequencing, transformation, expression, and regulation may be used to further understand the mechanisms of microorganisms involved in disease suppression.
The use of molecular techniques to relate gene sequence to function is promising. Phylogenetic relationships between non-culturable microorganisms and known culturable species may one day be used to devise new culturing techniques (Hill et al. 2000). These process-level assessments provide limited information describing community-level changes that are involved in disease suppression. Tests to trace shifts in metabolic activities by microorganisms and shifts in microbial communities exist, but have not been perfected as they
24 exclude certain non-culturable organisms (van Bruggen and Semenov 2000).
Studies of community shifts over time may provide additional information on whether the presence of active and potentially beneficial microorganisms correlates to disease suppression. The role of the pathogen, as it induces microbial shifts and factors that stimulate the growth of specific antagonists, needs to be further examined (Hagn et al. 2008; de Brito Alvarez et al. 1995).
Scheuerell et al. (2005) found no significant relationship between pathogen suppression and individual microbial populations found in different composts. Despite advances in microbial identification and enumeration, knowledge of structure and function, and interactions and synergisms among these microbes, is still in its infancy and the extent to which the host and the pathogen influence the microbial community is still in question.
1.4 Disease
A major drawback of organic crops, which leads to the reluctance of growers to switch to organic production, is the risk associated with new pest management techniques. This is because organic regulations prohibit the use of synthetic pesticides (Martin 2008; Government of Canada 2008a; Peet et al.
2004). Although chemical treatments against plant pathogens are still common in conventional systems, rising concerns with health and the environment, the emergence of pesticide resistant pests, and the deregistration of several pesticides may limit the future use of pesticides in conventional production systems (Massart and Jijakli 2007; Boulter et al. 2000). Growers, in general, are therefore searching for alternatives to synthetic pesticides. Strategies to further
25 improve compost utilization, which depend on compost quality, consistency, and efficiency, will be continuously needed (Kuo et al. 2004).
1.4.1 Common Diseases of Tomato
Major fungal diseases of greenhouse tomatoes include: early blight
{Alternaha solan i); fu sari urn crown and root rot (Fusarium oxysporum f. sp. radicis-lycopersici); fusarium wilt (Fusarium oxysporum f. sp. lycopersici); corky root rot (Pyrenochaeta lycopersici); gray leaf spot (Stemphylium solani); botrytis blight or gray mold (Botrytis cinerea); leaf mold (Fulvia fulva or Cladosporium fulvum); powdery mildew (Oidium lycopersicum, Erysiphe sp.); septoria leaf spot
(Septoria lycopersici); pythium root rot and wilt (Pythium spp.); and phytophthora root rot (Phytophthora spp.). Major bacterial diseases include: bacterial canker
(Clavibacter michiganensis subsp. michiganensis); bacterial speck
(Pseudomonas syringae pv. tomato); and pith necrosis (Pseudomonas corrugata) (Zhai er al. 2009; Barkley 2004; Hasna et al. 2007; Dodson et al.
2002; Workneh et al. 1993). Nematodes (Meloidogyne incognita) also parasitize greenhouse tomato roots (Siddiqui 2004).
1.4.2 Current Disease Management Strategies
Pest control for organic systems aims at enhancing crop health and reducing yield losses. Current practices to control pests in organic greenhouse production systems include: integrated pest management practices; the use of disease free seed and resistant varieties; root grafting; sanitation measures; biological organisms; soil steaming or solarization; crop monitoring and rotation; establishment of a balanced ecosystem; cultivation; traps; mulches; and
26 greenhouse moisture control (Government of Canada 2008a; Barkley 2004;
Hasna et al. 2007; van Loenen et al. 2003). When the aforementioned practices cannot prevent pests, substances in the 'Organic Production Systems Permitted
Substances Lists', such as copper products, and insecticidal soaps, may be applied with proper documentation (Government of Canada 2008b).
Over 50 years ago, nursery growers in North America were starting to explore composted barks as alternatives to peat moss to lower the cost of growing substrates. Since then, composted organic matter has been linked to the improvement of plant growth and the suppression of disease, which lead to the use of composts as replacements or reducers of fungicides, or as tools to slow the development of fungicide resistance (Boulter et al. 2002a; Boulter et al.
2002b; Boulter et al. 2000; Hoitink et al. 1997). Today, compost is becoming more and more popular as a means to suppress disease (Zhai et al. 2009; Hasna et al. 2007; Dianez et al. 2005; Scheuerell er al. 2005; Abbasi et al. 2002; Chaoui et al. 2002). Several literature reviews on suppression of plant diseases with compost have been published (Noble and Coventry 2005; Litterick et al. 2004;
Scheuerell and Mahaffee 2002; Hoitink and Boehm 1999; Hoitink et al. 1997;
Hoitink and Fahy 1986). Suppression of plant pathogens with compost or other organic amendments is in line with sustainable plant production and the needs of the organic sector; it is a realistic alternative to chemical treatment of crops.
However, as composts differ in their ability to suppress disease, they generally have a positive or no effect on disease suppression and rarely stimulate infection by pathogens (Hasna et al. 2007; Termorshuizen et al. 2006;
27 Tuitert et al. 1998). Variations in the composting process may influence disease suppression, so the composting process must be standardized (CCREF 2004;
Forste 1997; Lefebvre er al. 1994). Standardizing the composting process, however, does not necessarily ensure consistent disease suppression. To substantiate the claims that composts can be effective tools for disease suppression, it is necessary to first appreciate and understand the complex nature of compost and disease suppression (Hermann and Shann 1997).
1.4.3 Disease Management with Composts
Suppression of plant diseases with composts is attributed to increased microbial activity, soil health, and fertility (Craft and Nelson 1996; Boehm et al.
1993; Chen et al. 1988). Composts have been recognized as suppressive agents against the development of disease caused by many plant pests (i.e. Fusarium,
Pythium, Phytophthora, Pyrenochaetai, Rhizoctonia, Xanthomonas, Verticillium,
Cylindrocladium, Streptomyces, and Sclerotium spp. and nematodes).
Efficacy of disease suppressive composts in agronomic and horticultural crops vary depending on many factors such as microbial activity (Zhai et al.
2009; Boehm er al. 1993), fluctuations in feedstock materials or in batches from the same feedstock (Escuadra and Amemiya 2008; Craft and Nelson 1996), number and timing of application (Boulter et al. 2002a and b) maturity level
(Tuitert et al. 1998), and environment (Hoitink et al. 1997). It has been reported that increased microbial diversity from mixing several composts (chicken manure, coffee, sawdust and wheatbran) promoted suppression of Fusarium wilt of spinach in the field (Escuadra and Amemiya 2008). However, numerous plant
28 diseases caused by Fusarium spp. (i.e. F. oxysporum, F. melonis, F. basilici, F. radicis-lycopersici, and F. radicis-cucumerinum) have been suppressed by compost on its own (Yogev et al. 2006).
Although increased microbial activity or diversity is an important characteristic of compost that is often associated with disease suppression, high microbial activity is not the only requisite for disease suppression. Some soil- compost mixtures may not suppress disease even though microbial activity is
+ high (Zhai et al. 2009). Composts having lower microbial, NH4 , and C contents, and higher calcium content were more associated with suppression of corky root rot disease of tomato (caused by Pyrenochaeta lycopersici) than composts with
+ higher microbial activity and NH4 levels. Suppression in the former composts was likely by means of microbial competition for nutrients (low C content) and increased protection from cell wall degrading enzymes (high calcium content).
The latter compost exhibited increased microbial activity and diverse bacterial activity, including copiotrophic bacteria and actinomycetes, as a result of high
+ NH4 levels, but was not suppressive to corky root rot (Hasna er al. 2007).
However, suppression of corky root rot has also been attributed to elevated microbial activity (Workneh and van Bruggen 1994; Workneh et al. 1993).
Craft and Nelson (1996) found a strong correlation between microbial activity and suppression of Pythium graminicola on creeping bent grass, which was observed for a wide variety of composts including different feedstock. Turkey litter compost, however, did not suppress P. graminicola and even inhibited seed germination in the laboratory, but it suppressed P. graminicola in the field despite
29 having a low initial microbial activity reading. A high level of NH3, which is often associated with inhibition of seed germination, was likely involved in the low microbial activity of the turkey litter compost and consequently precluded P. graminicola suppression in laboratory assays. However, in the field, volatilization of NH3 likely occurred allowing natural re-colonization of the compost by microorganisms present in the field and thereby re-establishing disease suppression (Craft and Nelson 1996). Similarly, in a study comparing 36 composts for suppression of P. ultimum, composts exhibiting NH3 volatilization were associated with greater survival of cucumber seedlings compared to composts without NH3 volatilization, but there was no difference in survival of healthy seedlings when compost with and without NH3 volatilization was used against Rhizoctonia solani (Scheuerell et al. 2005).
Vegetable, fruit, and garden waste compost from two composting facilities in the Netherlands were compared for suppression of Rhizoctonia solani under different maturation ages (one to seven months) with and without the addition of cellulose. Freshly collected compost and long-matured compost (five to seven months) conferred suppression to R. solani, but short-matured compost (three months) stimulated infection. The addition of cellulose conferred suppression in short-matured compost from one facility but not the other (Tuitert et al. 1998).
However, in another study, storage of composts for up to one year did not reduce its ability to suppress turfgrass disease (Boulter et al. 2002b).
In turfgrass experiments, incidence and severity of dollar spot (Sclerotina homoeocarpa), fusarium patch (Microdochium nivale), and typhula blight
30 (Typhula ishikariensis) were further suppressed with increasing numbers of compost applications but not totally suppressed (Boulter et al. 2002a; Boulter et al. 2002b). Although compost may not totally prevent turfgrass disease, it may increase the rate of recovery of turfgrass from disease by increasing nutrient levels and radiant heat absorption, which stimulate growth (Boulter et al. 2002a).
Similar to the use of composts, the use of vermicomposts for disease suppression is also associated with biological activity. However, improvements in growth and yield in absence of disease are linked to hormones or humates present in the vermicompost, which act like plant-growth promoters (Arancon er al. 2004; Arancon et al. 2003). Suppression of disease with vermicomposts due to biological elements has been recognized (Edwards er al. 2004; Rivera et al.
2004; Szczech and Smolinska 2001; Szczech 1999), but claims that vermicomposts are superior disease suppressants than composts, under the notion that vermicompost have higher microbial activity and higher nutrient contents (Subler et al. 1998; Edwards 1995), should not be made because the quality of the finished product is highly dependent on the initial inputs and the process (Tognetti et al. 2005). Vermicomposts do, however, differ in terms of microbial composition when compared to composts (see section 1.3.2 The
Composting Process - Microorganisms in Compost).
1.4.4 Disease Management with Seeded/ Inoculated Microorganisms
Intentional microbial inoculation of compost may be used to achieve a desired microbial community in the compost. The most widely used fungi for successful disease suppression in plant production are Trichoderma spp.
31 (Harman et al. 2010); they are often used successfully as inoculum for compost to provide more consistent disease suppression (Abbasi et al. 2007; Hoitink et al.
2006; Huang et al. 2006; Trillas et al. 2006; Hjeljord and Tronsmo 1998).
Trichoderma asperellum strain T-34 established well in grape marc composts aged 1.5 to 3.0 years and suppressed disease caused by Rhizoctonia solani in cucumber seedlings; however, the ability of 7". asperellum to suppress this disease was compromised when inoculated into younger compost (0.5 to 1.0 year). While establishment of R. solani in the younger compost occurred, the cellulose-rich compost repressed chitin-degrading enzymes of Trichoderma spp., which reduced the suppressive capacity of the fungus (Trillas et al. 2006).
Easily cultured microorganisms may be applied to seeds, or added directly to the soil, substrate, or leaf surface. Several bacteria applied to seeds or roots control fungal plant pathogens (Whipps 2001). Harman et al. (2010) lists several beneficial microorganisms for disease suppression and/or promotion of plant growth. Fungi like Trichoderma and Gliocladium spp. have been used to suppress several plant pathogens (i.e. Pythium, Fusarium, Botrytis, Rhizoctonia,
Phytophthora, and Sclerotinia spp.) in many crops (i.e. tomatoes, cabbage, radish, beans, cotton, grapes, corn, lettuce, onions, peas plums, apples, and carrots) (Abbasi et al. 2007; Scheuerell et al. 2005; Hjeljord and Tronsmo 1998;
Elad and Chet 1980), and, more recently, yeasts have been explored as potential inoculum to control fungal plant pathogens and promote plant growth (El-Tarabily and Sivasithamparam 2006). Trichoderma harzianum seemingly exhibited improved antagonism under conditions that were unfavourable for the pathogen
32 such as high doses of pentachloronitrobenzene, high pH, or low temperature.
Thus, there is much potential to integrate different means to improve microbial activity and optimize disease suppression (Elad and Chet 1980).
Benefits of adding compost to soils include pH stabilization and rapid water infiltration due to greater soil aggregation (Stamatiadis et al. 1999); benefits are also linked to increased microbial activity, which increases nutrient mineralization, soil health, and pathogen suppression, and reduces the need for chemicals (Cheuk et al. 2003; Drinkwater et al. 1995; Workneh and van Bruggen
1994; Workneh et al. 1993). However, it is still very important to achieve a better understanding of the microbial community dynamics involved in producing the many benefits mentioned above. Most importantly, understanding microbial dynamics to further our understanding of biological elements as tools to suppress disease, can eventually lead to more successful replacement of the many chemicals on which conventional growers are largely dependent, and of which organic growers are forbidden to use (Hermann and Shann 1997).
1.4.5 Rhizosphere Competency of Microorganisms and the Carrying
Capacity of Compost-Amended Growing Substrates
Nutrients (i.e. carbohydrates, lipids, chitin, etc.) are essential for sustaining biological activity. Root exudates originating mainly but not exclusively from root tips, and other nutrients (i.e. root mucilages, secretions, lysates, and sloughed off cells) are constantly being released into the substrate directly surrounding roots
(Andrews and Harris 2000). These nutrient sources provide microorganisms with the opportunity to thrive in the root zone environment or, in other words, achieve
33 rhizosphere competence. Rhizosphere competence is the physiological and genetic ability of microorganisms to colonize roots and substrate surrounding roots (Harman 1992). While the root is a main source of nutrients, compost also provides nutrients for microorganisms (Welbaum et al. 2009).
Nutrients in compost support microbial activity, but the carrying capacity of compost (i.e. the length of time of this support), is difficult to predict because of variability with compost application rate, the soil or growing substrate composition, fluctuations in temperature and moisture, the cultural practices used, and the nutrient content of the compost feedstock. Nutrients in compost, however, play a critical role in the decomposition of organic matter (Huang et al.
2006), and the microbial composition of compost is continuously changing as organic matter decomposes and the compost matures (Hermann and Shann
1997). Eventually, however, nutrient supply may be exhausted and naturally present or introduced rhizosphere competent bacteria and fungi, such as
Azospirillum, Enterobacter, Pseudomonas, Trichoderma, and Penicillium spp.
(Bashan 1998; Harman 1992), may not survive to suppress disease.
1.4.6 Mechanisms of Action of Disease Suppression
General (indirect) suppression typically involves contact biological control agents and in some cases they must be applied directly to the site of infection. It also involves manipulation of the microbial community of the growing substrate by means of crop practices, cultural practices, and added amendments (e.g. compost) (Boulter et al. 2000). General suppression is associated with high microbial activity, which generally suppresses various plant pathogens or induces
34 microbiostasis (Benito et al. 2006; Mandeibaum and Hadar 1990; Chen et al.
1988). Microbiostasis involves several mechanisms such as: 1) antibiosis or inhibition with antimicrobial compounds induced by interspecific competition for nutrients (Dianez er al. 2005; Mandeibaum and Hadar 1990); 2) competition for colonization sites and nutrients on seeds and roots (Pantelides et al. 2009; Chen and Nelson 2008); 3) competition for iron through production of siderophores
(Dianez et al. 2006; de Brito Alvarez et al. 1995); 4) elicitation of plant resistance mechanisms (Ekengren 2008; Panina et al. 2007 Hoitink et al. 2006); 5) interruption of germination of pathogen propagules through antagonistic colonization of the pathogen propagule and other abiotic affects (Zmora-Nahum et al. 2008); 6) deterioration of pathogenic toxins or other pathogenicity factors of the pathogen; and 7) parasitism involving the use of enzymes such as cellulases, chitinases, and P-1,3 glucanases that can degrade pathogen cell walls (Hagn et al. 2008; Woo et al. 2002; Lorito 1998; Boehm et al. 1993). Many mechanisms may be involved in the general suppression of diseases by compost (Yogev et al.
2006). Pathogens that have smaller propagules, such as Pythium and
Phytophthora spp., have small nutrient reserves, and thus are "nutrient- dependent" pathogens that are susceptible to general suppression by a multitude of microorganisms that can grow under high and low nutrient concentrations (i.e. facultative oligotrophs) (Boehm et al. 1993; Mandeibaum and Hadar 1990).
However, pathogens that have larger propagules (i.e. sclerotia), such as
Sclerotinia sclerotiorum, Sclerotium rolfssi and Rhizoctonia solani, are less susceptible to general suppression.
35 Pathogens that have larger propagules are more susceptible to specific suppression (Dianez et al. 2005; Scheuerell et al. 2005). Specific (direct) suppression refers to the action of one or few groups of microorganisms that are introduced and necessary for eradication of specific pathogens. For instance,
Penicillium, Petriella, and Trichoderma spp. are antagonists that can colonize sclerotia from Sclerotium rolfsii and inhibit their germination (Zmora-Nahum er al.
2008; Knudsen and Eschen 1991). A small number of composts (-20%) are known to have direct suppression against pathogens with large propagules, such as Rhizoctonia solani (Trillas et al. 2006). Specific suppression usually involves hyperparasitism, mycoparasitism, and induced systemic resistance (Hoitink et al.
2006; Dianez et al. 2005; Boulter et al. 2000). Endophytes, for example, are beneficial microorganisms that are known to induce systemic resistance.
Endophytes are plant symbionts, plant growth promoting rhizobacteria, and mycorrhizal fungi, such as Clonostachys rosea, Piriformaspora indica, and
Trichoderma spp. Controlling disease is only a subset of the benefits that endophytes provide plants (Harman er al. 2010). They also increase growth, and resistance to plant stressors (temperature, salt, and drought) and offer plants other benefits such as increased seed germination and nutrient uptake.
Endophytes colonize plants internally, and therefore have much longer periods of efficacy than chemical controls and non-endophytic biological controls.
Endophytes also have a greater effect on the plant than do chemical agents, which mainly affect the pathogen and not the plant.
36 Chemical control is a direct inhibition of a high proportion of target pests. It is highly specific and effective. However, specificity increases the likelihood of target pests developing resistance and efficacy is short lasting. Another drawback of chemical controls is that the application must be directed to the site of infection via topical or systemic application (Harman et al. 2010). Chemical control is for the most part prohibited in organic production systems. That is why compost utilization is so important in organic production.
1.5 Thesis objectives
Organic plant production encompasses sustainability and there is an ever- increasing consumer interest in purchasing produce derived from these types of production systems. The greenhouse vegetable production industry in Ontario,
Canada, is a fast growing industry with tomatoes as the leading crop. Adoption of organic technologies for more sustainable greenhouse production in Canada can improve the state of the Canadian greenhouse industry. However, a major challenge for organic growers involves a lack of suitable growing substrates.
Therefore, emphasis is placed on obtaining a high quality growing substrate, and coupling that with strategic irrigation and organic fertilizer application as research focusing on growing substrate fertility is important in organic production (Martin
2007; Nielson and Thorup-Kristensen 2004) particularly for growing young plants
(Prasad et al. 2004).
With a rising population and demand for food, several issues of waste management also cannot be ignored. All food production systems produce waste and this waste must be reused in a way to secure food sustainability for the
37 future. The best way to manage waste is to reduce and reuse inorganic waste and recycle organic waste. The production of high quality compost from organic waste is a major priority; however, for successful marketing, compost must be tested, must be consistent, and must have appropriate physical and chemical properties. Increasing plant growth and disease suppression are benefits of using managed waste (i.e. compost) for food production. With more research focusing on the applicability of compost as a means to reduce chemical inputs and decrease disease, more production systems are bound to adopt these practices.
Current recommendations for growing organic tomatoes in Canada are inadequate. This research, therefore, was conducted for developing such guidelines. Specifically, the objective of this study was to develop organic growing substrates that promote healthy transplant growth with regards to substrate fertility, and plant development. The main goal was to develop certifiable organic growing substrates using locally available and allowed materials. Overall, the research objectives were threefold: 1) to evaluate growing substrate materials eligible for organic production systems and appropriate for tomato transplant production; 2) to evaluate the contributions of managed waste in growing substrates as a means to replace the use of peat moss, perlite, and vermiculite; and 3) to investigate the ability of compost and an endophyte
(Clonostachys rosea) to suppress root diseases caused by Pythium ultimum.
38 CHAPTER 2: Developing Organic Growing Substrates for Tomato
Transplant Production
2.1 Introduction
Sphagnum peat moss (PM) was evaluated in this experiment because it is a principal material that is eligible for use in organic growing substrates in North
America. Canadian PM is available in large quantities and has physical and chemical properties that are suitable for crop growth, such as low bulk density, adequate air-filled porosity (AFP), high water-holding capacity (WHC), and high cation exchange capacity (CEC). CEC helps to minimize the leaching of nutrients from growing substrates by holding on to these nutrients (Raviv et al. 1998b).
However, the density and diversity of microflora and microfauna in PM is generally low, rendering PM a favourable environment for pathogen establishment and proliferation (Borrero et al. 2004; Raviv et al. 1998a; Hoitink and Fahy 1986). Also, the harvest of PM is considered by some ecologists to be unacceptable because it requires use of fossil fuels, and results in destruction of
PM bogs, which are important habitats for wildlife and major sinks for atmospheric carbon dioxide, which aid in reducing the impact of global warming
(Pill and Ridley 1998). Due to the challenges and increasing costs associated with PM (i.e. for fertilizer and pesticides), alternatives are being sought.
Coconut coir (CC) is commonly employed as an alternative to PM. CC has similar physical and chemical properties to PM including adequate water retention, good drainage, relative absence of weeds and pathogens, physical resilience with slow decomposition, and acceptable pH, electrical conductivity
39 (EC), and CEC (Arenas et al. 2002; Pill and Ridley 1998). Lately, growers in
Leamington, ON, Canada, have complained that weed seeds from CC have germinated in growing substrates but evidence that newly produced CC is a source of weeds is lacking (Personal communication with Dr. Y. Zheng,
University of Guelph, Guelph, ON, Canada). It is possible that weed seeds are blown into stockpiles of CC either immediately after harvesting or at intermediate holding sites. Nonetheless, CC is regarded as a renewable resource with few ecological drawbacks (Pill and Ridley 1998). However, processing and transportation of CC requires fossil fuels. Therefore, other materials allowed for use in organic production systems in Canada were included in this research.
Fine perlite (FP) and fine vermiculite (FV), which are often used in soilless growing substrates and are permitted in organic production systems, were also used in the present research for developing organic growing substrates. FP is a heat-treated porous mineral that provides substrates with good drainage, excellent aeration, and moderate physical strength and resilience. FV is also a heat-treated mineral that provides substrates with good aeration, good WHC, high CEC, plant available magnesium, and good thermal properties (van
Straaten 2007). Both FP and FV require fossil fuels for heat treatment and, for this reason, organic growers may prefer to minimize the use of these materials.
Zeolite minerals are naturally porous. Unlike FP and FV, zeolite requires no heat for its production. Zeolite has a high degree of hydration, high surface area, high CEC, high selectivity for cations, and high physical stability (van
Straaten 2007). Pushkina et al. (1995) reported that zeolite prolonged nutrient
40 retention in growing substrates and facilitated the consumption of nutrients by plants. Organic growers may benefit from the use of zeolite instead of FP or FV; however, relatively inert materials such as CC, FP, FV, and zeolite are likely to be as conducive as PM to plant diseases caused by pathogens (Hoitink et al.
1986). Alternative materials to the aforementioned ones include a variety of composted materials, which, for the most part, can be disease suppressive
(Hasna er al. 2007; Borrero et al. 2004).
Composted materials are thermophilically processed (microbially-worked) composts or non-thermophilically processed (worm-worked) vermicomposts
(VCs). Thermophilically processed compost is a dark, humus rich material that results from the process of microbial digestion, whereas VC is a dark and finely divided PM-like material that has a high degree of aeration, adequate drainage, and good water and nutrient retention as a result of passage through digestive tracts of worms (Arancon et al. 2004).
Organic production requires that thermophilic compost piles reach 55 to
60°C for several days and then decompose for at least six weeks under moist and aerobic conditions that are not water-logged, to stabilize nutrients, reduce pesticide residues, and kill weeds, seeds, and plant pathogens. VC must also be processed according to organic standards. Organic materials allowed for use in organic composting and vermicomposting include by-products of the processing of organic crops, source separated yard debris that is free of synthetic chemicals, and organically raised animal manure. Written documentation to provide the source of these materials and/or laboratory analysis to demonstrate freedom
41 from contaminants that are prohibited in organic production is necessary
(Government of Canada 2008b).
Compared to PM, compost is much heavier with higher levels of soluble salts, and higher pH. Compost-based substrates also have lower AFP and WHC, and are more variable in structure and composition than PM-based substrates
(Corti et al. 1998). However, numerous studies have shown that high quality growing substrates can be produced using composts. Compost-based substrates can produce plants of equivalent or better growth and quality compared to plants growing in compost-free substrates (Titarelli et al. 2009; Clark and Cavigelli
2005; Hummel et al. 2001).
The objective of the first experiment was to determine whether growing substrate fertility (i.e. capacity to nourish and support healthy plant growth) suitable for organic production of tomato transplants can be achieved using locally sourced composts such as: VC, manure compost (MC), and pine bark compost (PBC). Therefore no fertilizer was applied. Composts were incorporated into different growing substrates at either 20, 25, 30, 40, or 50% total content by volume (v/v), with either one single compost or multiple (two or three) composts making up the % total content.
2.2 Materials and Methods
2.2.1 Substrate Preparation
Nine materials eligible for use in organic production systems according to organic standards in Canada (Government of Canada 2006b) were selected
(Table 2.1)
42 Table 2.1. Abbreviations, and source information for materials used. Material (Abbreviation) Source Manure compost (MC)Z Dingo Farms, Bradford, ON, Canada Forterra vermicompost (VC) Forterra Environmental Corp., Puslinch, ON, Canada Pine bark compost (PBC) Gro-Bark Organics Inc., Milton, ON, Canada Granular zeolite (Z)y Bear River Zeolite Co., Preston, ID, United States Fine Canadian Sphagnum peat moss Fafard Peat Moss Co Ltd. City, QC, (PM) Canada Fine coconut coir (CC) Millenniumsoils Coir Inc., St. Catharines, ON, Canada Fine vermiculite US grade No. 3 (FV) Plant Products Ltd., Brampton, ON, Canada Fine perlite US grade No. 3 (FP) Canadian HydroGardens Ltd., Ancaster, ON, Canada Superfine vermiculite US grade No. 4 Plant Products Ltd., Brampton, ON, (SV) Canada ZMC = Unknown quantities of cattle, sheep, and horse manures (straw bedding included) and vegetable scraps (onion and carrot). y85% Clinoptilolite balance opaline silica.
Preliminary analyses, which included compacted bulk density (CBD), pH,
EC, and nutrient content (Table 2.2; other data not shown), were conducted to determine substrate formulations and ratios to be evaluated in this experiment.
43 Table 2.2 Compacted bulk density (CBD; g-L"1), pH, electrical conductivity (EC; uS-cm"1), and content of initial water-soluble nutrients (mg-L"1 suspension) in the individual materials (in their received state) used to formulate the growing substrates in the first experiment. +Z 3 Material CBD pH EC N03- + NH4 P04 " Manure compost 393.9y 8.3 2650 259 149 Vermicompost 570.8 8.6 1039 122 5 Pine bark compost 479.2 4.9 107 30 0 Peat moss 147.7 4.4 35 0 0 Coconut coirx 161.9 6.4 153 4 2 Fine perlite 128.3 8.1 14 4 0 Superfine vermiculite 145.0 8.0 38 4 0 Fine vermiculite 109.6 6.7 21 5 0 Granular zeolitew 882.0 7.6 97 0 0
Material K Mg Ca CI" S04 Na Manure compost 223 82 83 234 130 102 Vermicompost 128 81 84 132 16 91 Pine bark compost 86 83 83 40 22 93 Peat moss 0 0 0 4 0 80 Coconut coirx 83 0 82 57 10 86 Fine perlite 0 0 55 11 12 83 Superfine vermiculite 80 27 82 9 13 83 Fine vermiculite 53 27 55 12 14 83 Granular zeolitew 3.47 0 1.6 0 0 0.5 zNutrient concentrations were measured used the 1:5 method. yAII data are means of three replicates. xFine coir was compressed and dry when received so it required the addition of water for expansion; expanded coir was allowed to air dry to a desirable moisture content (mimicking the consistency of peat moss) before being assessed. wThe nutrient content for granular zeolite cannot be considered water-soluble as these values were obtained from the supplier as a chemical composition (%) rather than a water extraction analysis; other elements include Fe (1.3%) Cu (25 ppm), and Zn (35 ppm).
Twenty-three formulated substrates and two standard substrates used by the industry in Florida, which were included in the experiment for comparison, were prepared on February 1st 2008 (Table 2.3). The substrates were mixed v/v based on the CBD of each material. The CBD procedure required use of a metal
44 cylinder with a known volume and a packing weight and is described in EN
13040 Annex A, of the International Society of Horticultural Science (ISHS)
Centre Europeen de Normalisation (CEN) standards for chemical and physical analysis of growing media (ISHS 2003). After the mean CBD (three replicates) of each material was determined (Table 2.2), the materials were weighed according to the volume needed and added to a plastic bag. The bag was mixed by hand for five minutes, or until the resulting substrate appeared uniform. Dolomitic lime
(High Magnesium Limestone, National Lime and Stone Co. Findlay, OH, Unites
States) was incorporated into the standard substrates at a rate of 9.79 g lime/L of
PM in each substrate. The substrates were stored at room temperature in open plastic bags for more than two weeks before being used in the experiment.
45 Table 2.3 Compositions of the formulated growing substrates tested in the first experiment. Values are the percent of each material included on a volume basis based on the compacted bulk density of each material used. Substrate 1z 2 3 4 5 6 7 8 9 10 11 12 13 Manure compost / / / / 25 25 25 / / / 25 25 Vermicompost / 25 25 25 / / / 25 25 25 / / Pine bark compost / / / 25 / / 25 / / 25 / / Peat moss 50 75 25 / / 25 / ./ 25 / / 25 / Coconut coir / / 25 / / 25 / / 25 / / 25 Fine perlite / 25 25 25 25 25 25 20 20 20 20 20 Fine vermiculite 50 25 25 25 25 25 25 25 23 23 23 23 23 Superfine vermiculite / / / / / / / / / / / / Zeolite / / / / / / / 7 7 7 7 7 Substrate 14 15 16 17 18 19 20 21 22 23 24 25 Manure compost 25 / 20 / 20 / 20 / / 20 20 10 Vermicompost / 20 / 20 / 20 / 20 20 / / 10 Pine bark compost 25 10 10 10 10 20 20 10 20 10 20 10 Peat moss / / / / / / / / / / / / Coconut coir / 20 20 20 20 20 20 20 10 20 10 10 Fine perlite 20 50 50 / / 20 20 15 15 15 15 25 Fine vermiculite 23 / / 50 50 15 15 20 20 20 20 20 Superfine vermiculite / / / / / / / 10 10 10 10 10 Zeolite 7 / / / / 5 5 5 5 5 5 5 Numbers represent each substrate combination tested in the first experiment: 1 and 2 were chosen for comparison as they are considered standard substrates used in the Florida tomato transplant industry (Arenas et al. 2002). Substrates 3 to 25 were chosen based on preliminary analyses. The initial pH values for these substrates were aimed at being between 5.5 and 6.8 and the electrical conductivity values were aimed at approximately 750 ^s-cm"1. These values were considered appropriate for the production of tomato seedlings according to Vavrina (2002) and Lang (1996). Substrates 5, 6, 7, 11, 12, and 13 were used in the second experiment; substrates 2, 3, 5, 6, 7, and 18 were used in the third experiment.
2.2.2 Chemical Properties of the Substrates
Prior to the first growth experiment, the pH and EC of the individual materials were determined using the 1:5 (substrate:water) method following the procedures described by the CEN standards for determination of pH (EN 13037
Annex A) and EC (EN 13038) of growing substrates (ISHS 2003). A portable pH/
46 EC meter (Oakton pH/ Con 300; Oakton Instruments, Vernon Hills, II, United
States) was used to record the pH and EC of the suspensions. Water-soluble nutrient contents of the suspensions were determined using high performance liquid ion chromatography (DX-120, Dionex Canada Ltd; Oakville, ON, Canada).
During the growth experiment, substrates were analyzed on day 30 using the pourthru method: 1) deionized water (approximately 100 ml) was added to the plug trays one hour after irrigation (see section 2.2.3 for more information on plug trays used); 2) approximately 50 ml of leachate was collected and analyzed for pH and EC (Cavins et al. 2000). The samples were frozen (-85 °C) and kept for one to two weeks before analysis for contents of water-soluble nutrients.
2.2.3 Growth Experiment
On February 19th and February 20th 2008, untreated seeds of greenhouse tomato hybrid 'beefsteak' type (Solanum esculentum L. cv. Matrix F1; De Ruiter
Seeds Inc. Lakewood, CO, United States) were sown in 200-plug trays (plug volume = 15.6 cm3) cut to accommodate 30 plugs filled with experimental substrate. One seed was sown in each plug and covered with moistened SV. The trays were placed in a randomized complete block design (RCBD) (n = 5) in a dark germination chamber at 25°C [±1°C standard deviation (SD)] for three days.
The trays were then arranged in a RCBD in a glass greenhouse maintained at
25/ 23°C (±3/ 1°C SD) for the first three weeks and at 22/ 19°C (±3/ 1°C SD) for the following two weeks during the day/ night. The research was conducted in the
Bovey Greenhouse Complex (7.62 x 6.10 m) at the University of Guelph, Guelph
(lat. 43° 33' N, long. 80° 15' W), ON, Canada. Pests were controlled, as needed,
47 by means of Swarski sp. and Orius sp. for controlling thrips, Aphidius ervi for controlling aphids, and Encarsia formosa for controlling white fly (Biobest Canada
Ltd. Leamington, ON, Canada).
Seedlings were hand watered two to three times a day during the first week with a Dramm Fogg-It nozzle (0.5 gallon/ minute: (Dramm Corporation,
Fenwick, ON, Canada) and once or twice a day with a 1000PL Redhead water breaker (Dramm Corporation, Fenwick, ON, Canada) during the following four weeks. No fertilizer was applied to the seedlings and seedlings were watered with well water that was unadjusted for pH (pH -7.5; EC -1000 pS/ m).
2.2.4 Growth Measurements
Final germination counts were recorded at three weeks. At five weeks of growth (day 35), five seedlings chosen at random from each tray were lifted for determination of mean growth indices. Stem diameter at one cm above the substrate surface, stem height, and total leaf area were measured, using an electronic digital caliper (NSK, MAX-CAL, model 950-101 MAX-15, Japan), a ruler, and a leaf area meter (LI-3100; LI-COR, Lincoln, NE, United States), respectively. Detached leaves and stems were oven dried separately at 50°C until dry weights stabilized (>48 hours), then weighed with an analytical balance.
Roots were washed (debris was removed and bits of substrates were handpicked to remove most substrate particles), oven-dried, and weighed by the same method as leaves and stems. After seven weeks of growth (day 49) the remaining seedlings in the trays were removed with a dibble tool for evaluation of
48 the ease of seedling removal from the plug tray. A scale from one to five was used to evaluate seedling removal (one = difficult; five = easy).
2.2.5 Statistical Analysis
Means (n = 5) were determined and subjected to an analysis of variance
(ANOVA) using the General Linear Models procedures in SAS version 9.1 (SAS
Institute; Cary, NC, 2003). An analysis of residuals was used prior to ANOVA to verify the assumptions of homogeneity of variance. Outliers were removed from the data set based on Lund's tables for an approximate test for outliers in linear models (Lund 1975). When ANOVA was found to be significant (P <0.05) for a treatment effect, a Tukey-Kramer multiple means comparison test at P <0.05 was performed. A nonlinear regression was calculated to find the relationship between shoot dry weight (at harvest) and water-soluble nitrate content
(analyzed from pourthru on day 30 of the growth experiment), using Prism 5 for
Mac OS X, Version 5.0a, 2007.
2.3 Results and Discussion
The percentage of seedling emergence in the formulated substrates, at three weeks after sowing, did not differ significantly from that in the standard substrate [2 (75PM/ 25FV)]. However, germination percentages for seven substrates [12 (25MC/ 25PM/ 20FP/ 23FV/ 7Z), 6 (25MC/ 25PM/ 25FP/ 25FV),
13 (25MC/ 25CC/ 20FP/ 23FV/ 7Z), 9 (25VC/ 25PM/ 20FP/ 23FV/ 7Z), 7 (25MC/
25CC/ 25FP/ 25FV), 18 (20MC/ 10PBC/ 20CC/ 50FV), and 17 (20VC/ 10PBC/
20CC/ 50FV)] were higher than that of the other standard substrate [1 (50PM/
50FV)] (Figure 2.1). These substrates contained 20 to 25% MC or VC and 20 to
49 25% PM or CC v/v. Among the substrates with higher germination than the standard substrate [1 (50PM/ 50FV)], only one contained PBC (18); this substrate contained the greatest amount of FV (50%), whereas other substrates among those with high seedling emergence percentages contained 23 to 25%
FV and 20 to 25% FP. Among the seven substrates with high seedling emergence, five exhibited significantly easier removal from trays compared to other substrates.
Figure 2.1 Percent seedling emergence at 24 days after sowing. Data are means of five replicates istandard error. Data bearing the same letter are not significantly different by Tukey-Kramer's test at P <0.05. Standard substrates are in red.
Twelve substrates were among the best substrates for ease of removal from plug trays (Figure 2.2). Substrates 9 and 17, which were among the best in terms of germination, were not different from the top substrates for ease of removal; however, they were also not different from the worst substrates for ease
50 of removal, which were substrates 15 (20VC/ 10PBC/ 20CC/ 50FP) and 10
(25VC/ 25CC/ 20FP/ 23FV/ 7Z), respectively.
Figure 2.2 Ease of removal of transplant roots from plug trays at 7 weeks after sowing. Data are means of five replicates. Data bearing the same letter are not significantly different by Tukey-Kramer's test at P ^0.05.
Stunting and purple discoloration of the abaxial leaf surfaces were observed after three weeks, especially in tomato plants grown in the standard substrates (1 and 2) with no compost. To test whether the symptoms were caused by nutrient deficiency, a conventional fertilizer solution (20-18-20) was applied to a few seedlings. The fertilized seedlings recovered within a week indicating that stunted growth and leaf discoloration were related to nutrient deficiency as opposed to disease or other environmental factors.
51 CD § 15-
3 10-
o 5-
CO
0
8- T3 _ a F « fl s
T3 O O CC
Growing substrate
Figure 2.3 Growth responses at five weeks. Growing substrate compositions are described in Table 2.3. Data are means of five replicates. Data bearing the same letter are not significantly different by Tukey-Kramer's test at P <0.05; bold letters indicate substrates that were ranked among the top ten (see Table 2.4).
52 Despite nutrient-related growth restrictions, significant differences in stem height, stem diameter, leaf area, dry shoot weight, and dry root weight were found among treatments (Figure 2.3). The substrates were ranked from best (1) to worst (25) for each growth parameter and the sums of these rankings were added to give each substrate a final integrated ranking (Table 2.4).
Table 2.4 Substrates ranked by growth. Shoot Root Substrate Stem Stem Leaf Sum Final dry dry combination diameter height area of ranking weight weight number rank rank rank ranks rank rank 1z 12 1 2 2 1 1 7 2 6 4 1 1 2 2 10 3 13 3 3 4 4 3 17 4 7 5 5 3 3 5 21 5 5 2 6 5 5 4 22 6 25 6 7 6 6 6 31 7 18 8 8 9 9 8 42 8 23 11 12 8 7 7 45 9 2 7 11 11 11 9 49 10 1 12 4 12 14 10 52 11 11 10 17 7 8 14 56 12 3 9 9 14 12 12 56 13 16 15 16 10 10 11 62 14 9 13 10 13 13 16 65 15 17 17 14 18 16 13 78 16 19 14 18 15 15 17 79 17 21 16 20 19 17 15 87 18 10 18 13 24 20 20 95 19 15 21 19 20 19 18 97 20 22 20 22 17 18 21 98 21 14 24 21 16 21 19 101 22 4 19 15 25 22 23 104 23 8 23 23 21 23 25 115 24 20 22 25 23 25 22 117 25 24 25 24 22 24 24 119 zSee Table 2.3 for substrate compositions.
53 Analysis of pourthru leachate collected during the fourth week of growth revealed low EC and high pH (>7) in all substrates. The content of water-soluble phosphorous in the pourthru leachate of each substrate was also low, especially in substrates that contained VC instead of MC. This was likely because high pH reduces the solubility of phosphorus (Smith et al. 2004). Seedlings from substrates with the lowest amount of soluble phosphorus (Table 2.5) were the most purple, which is a sign of phosphorous deficiency (Garton et al. 1994).
However, in addition to phosphorus deficiency, nitrogen was a major nutrient involved in affecting the growth of seedlings. Nitrate was the only nutrient found to have a positive correlation with dry shoot weight (Figure 2.4). Hu and
Barker (2004b) found that the growth of tomato was correlated with nutrient accumulation, with nitrogen accumulation giving the highest correlation compared to phosphorous, calcium, magnesium, and potassium accumulation.
Nonetheless, we cannot rule out the possibility of other limited-nutrient effects because the seedlings experienced high pH in their root zone environment due to irrigation with alkaline well water. In general, as pH increases the availability of calcium and magnesium increases and the availability of phosphorous, iron, zinc, manganese, boron, and copper decreases (Bailey er al. 2007; Smith et al. 2004).
54 Table 2.5 The pH, electrical conductivity (EC; uS-cm"1), and content of water- soluble nutrients (mg-L"1 suspension) in pourthru leachate of each substrate on days 29 and 30.
N03- + z + 3 # pH EC NH4 P04 " K Mg Ca CI" S04 Na 1 7.9y 2140 14a Of 86ab 103a 91c-g 373a 285a 111a 2 7.8 2246 13a Of 85b 103a 92b-g 397a 290a 111a 3 8.1 2222 10a 1ef 101ab 95a 98a-d 388a 286a 112a 4 8.1 2274 10a 4c-f 118ab 93a 93a-g 372a 294a 113a 5 7.9 1884 14a 2d-f 107ab 90a 91c-g 308a 236a 107a 6 8.1 2250 22a 10bc 112ab 96a 89e-g 363a 290a 113a 7 8.1 2134 15a 11ab 109ab 91a 85g 294a 251a 106a 8 7.8 2796 13a 19a 124a 95a 90d-g 382a 288a 112a 9 8.1 2134 14a 2d-f 97ab 92a 99a-c 340a 226a 104a 10 8.1 2366 12a 3c-f 107ab 95a 101a 391a 287a 108a 11 8 2190 26a 3c-f 102ab 93a 100ab 382a 255a 109a 12 8 2336 19a 8b-f 108ab 95a 94a-f 356a 246a 106a 13 8 2378 21a 9b-d 111ab 95a 93a-g 398a 273a 110a 14 7.7 1940 13a 16ab 110ab 94a 94a-f 343a 267a 107a 15 8 1958 15a 2d-f 98ab 96a 97a-e 450a 265a 118a 16 8 2458 21a 13ab 99ab 95a 91c-g 334a 247a 112a 17 7.9 2164 12a 3c-f 106ab 94a 94a-f 358a 252a 110a 18 7.9 1890 13a 15ab 113ab 92a 87fg 309a 244a 107a 19 7.9 1790 11a 2d-f 100ab 91a 97a-e 351a 239a 108a 20 7.7 2166 9a 18a 112ab 94a 94a-f 374a 278a 110a 21 7.9 2370 13a 3c-f 84b 77a 96a-e 371a 206a 108a 22 7.8 2176 15a 3c-f 103ab 94a 100ab 393a 262a 109a 23 7.8 1914 17a 14ab 108ab 94a 91c-g 324a 244a 105a 24 7.7 2624 12a 19a 118ab 97a 94a-f 425a 280a 116a 25 7.9 2412 20a 8b-e 109ab 96a 97a-e 418a 268a 113a z# = substrate number; bolded data refer to the top ten substrates (see Table 2.3 for substrate compositions and Table 2.4 for substrate ranks). y Data for pH and EC are means of five replicates taken from pourthru leachate collected on two days. Data for nutrient concentrations were taken from the same pourthru leachate collected for pH and EC measurement. Therefore data are means of five replicates. If followed by the same letter (within a column) means are considered not significantly different by Tukey-Kramer's test at P <0.05.
55 100-
• 80- • CD
* • SO- 5 •
o o • .c 40" eft •
• • 10 15 20 25 30
Average N03 content (ppm)
Figure 2.4 Relationship between shoot dry weight and content of water-soluble nitrate in the substrate on day 30. Nonlinear regression: Y = -40.11 (±38.09 SD) +7.527 (±4.600 SD) x-0.1416 (±0.1311 SD) x2 (R2 = 0.4141).
Initially, VC had the highest soluble nitrogen content (Table 2.2) but it was a component of only two of the top 10 substrates. While the initial nitrogen content of substrates was greater in substrates containing VC than in substrates containing MC, after four weeks of growth, nitrogen content of substrates containing VC was in most instances lower than in substrates containing MC.
Combinations of PM, CC, and MC held more nitrogen than combinations of PM,
CC and VC. Phosphorous was more abundant in substrates containing MC
(ranging from 8 to 19 ppm) than in substrates containing VC (ranging from 1 to 3
56 ppm). Potassium, on the other hand, was relatively high among all mixtures (84 to 124 ppm) (Table 2.5).
Four out of the top ten substrates contained zeolite at a rate of 7% v/v.
Zeolite has a high affinity for cations and acts as a storage for nutrients such as ammonium, potassium, calcium, and magnesium (Armbuster and Gunter 2001); however, the cost of zeolite may be too high for use in organic production, especially if low rates of zeolite do not improve crop productivity. Positive effects associated with zeolite in the tested substrates may not have been evident in this experiment due to lack of fertilizer.
2.4 Conclusion
Differences in growth among substrates were evident and eight substrates produced transplants of equal or better quality than the standard substrates (see
Table 2.4). Four substrates among the top ten contained Z. However, tomato transplants exhibited nutrient deficiencies when grown in each of the substrates, including those in which the transplants grew larger; growth of the transplants was stunted in all substrates. The stunted growth was attributed to a combination of low nutrient availability associated with high root zone pH, a limited source of nutrients (from VC, MC, and/or PBC), and small container size (plug volume =
15.6 cm3). A second experiment was therefore justified to determine whether growing substrates containing zeolite could produce transplants of significantly better quality than the same substrates without zeolite on the basis that nutrient availability does not compromise growth.
57 CHAPTER 3: Zeolite as a Component in Organic Growing Substrates for
Tomato Transplant Production
3.1 Introduction
Zeolite is a hydrated aluminosilicate mineral with an internal structure of tunnels and cages linked as tetrahedrons that provide a large surface area on which cation exchange may take place (Armbuster and Gunter 2001).
Incorporation of zeolite in growing substrates reduces requirements for water, energy, and fertilizer (Eberl 2007; Cattivello 1995). Growth experiments using zeolite found that tomatoes grew taller when nutrient enriched zeolite was incorporated at a rate of 20% by volume (v/v) in the growing substrate (Pavlovic et al. 1998). However, superior growth of plants was observed when much lower rates of zeolite (3 to 7% v/v) were used (Cattivello 1995). Pushkina et al. (1995)
+ + claim that binding of ammonium (NH4 ) ions by zeolite reduced excessive NH4
+ uptake by plants. As tomato seedlings are prone to NH4 toxicity (Wilcox 1993), zeolite could potentially protect seedlings from this toxicity, zeolite improves structure, and biological activity and reduces damage caused by root pathogens in certain plants (Pushkina et al. 1995); however, the positive effects that these minerals may have for the production of organic transplants is still in question.
In the first experiment, substrates containing zeolite produced larger transplants than substrates that did not contain Z. However, because the first experiment was not successful due to nutrient deficiencies, a second experiment was needed to further investigate possible benefits of zeolite in growing substrates. To rectify nutrient deficiencies, successive experiments were
58 conducted using larger pots instead of plug trays and organic liquid fertilizers were applied. The objective was to further investigate whether there is any benefit of using zeolite (at 7% inclusion v/v) in organic substrates for tomato.
3.2 Materials and Methods
3.2.1 Substrate Preparation
Vermicompost (VC), pine bark compost (PBC), fine perlite (FP), fine vermiculite (FV), peat moss (PM), coconut coir (CC), and zeolite was used for the formulation of the substrates. Substrates 5 (25VC/ 25PBC/ 25FP/ 25FV), 6
(25MC/ 25PM/ 25FP/ 25FV), 7 (25MC/ 25CC/ 25FP/ 25FV), 11 (25VC/ 25PBC/
20FP/ 23FV/ 7Z), 12 (25MC/ 25PM/ 20FP/ 23FV/ 7Z), and 13 (25MC/ 25CC/
20FP/ 23FV/ 7Z) from the first experiment were mixed and stored at room temperature in open plastic bags for over three months; the same materials as in the first experiment were used (see section 2.2.1 Substrate Preparation).
3.2.2 Chemical Properties of the Substrates
The pH of each substrate was measured using the 1:5 method as in the first experiment. Elemental sulfur was added to reduce the pH of each substrate to 5.7 in accordance with the greenhouse growing media test for application of elemental sulfur to growing substrates containing 50% PM and 50% sand
[University of Missouri Extension 2008]; elemental sulfur additions were 0.7 g-L"1
(substrate 12), 0.9 g-L"1 (substrates 11 and 13), 1.0 g-L"1 (substrate 6), 1.1 g-L"1
(substrate 5), and 1.3 g-L"1 (substrate 7).
During the growth experiment, substrates were analyzed on days 31, 38, and 45 using the pourthru method (Figure 3.1): 1) deionized water (~ 100 to 150
59 ml) was added to each pot one hour after fertigation; 2) leachate (~ 50 ml) was collected and analyzed (Cavins et al. 2000) with a portable pH/ EC meter
(Oakton pH/ Con 300; Oakton Instruments, Vernon Hills, II, United States).
Figure 3.1 Demonstration of the pourthru method used to collect the leachate of a substrate, which was analyzed for pH, EC, and/or concentration of water-soluble nutrients. One hour after fertilizing plants, distilled water was poured onto the substrate surface so that approximately 50 ml of water drained from the substrate. The leachate was collected in a small clean container and analyzed with a pH/ EC meter or a nutrient analysis system.
3.2.3 Growth Experiment
Untreated seeds of greenhouse tomato hybrid 'beefsteak' type (Solanum esculentum L. cv. Matrix F1; De Ruiter Seeds Inc. Lakewood, CO, United States)
60 were incubated on moistened filter paper in a Petri dish in darkness at 25°C
[±1°C standard deviation (SD)] for three days to allow germination. Germinated seeds were moved to 200-plug trays (15.6 cm3), which were filled with substrate
2 (75PM/ 25FV) from the first experiment on April 30th 2008 (day 3), the seeds were covered with moistened superfine vermiculite, and placed in the Bovey
Greenhouse Complex, University of Guelph (see section 2.2.3 Growth
Experiment) at 22/ 19°C (± 3/ 1°C SD) during the day/ night. Seedlings (2 to 3 leaf stage) were transplanted, on May 20th 2008 (day 23), to 10-cm pots (volume
= 426.1 cm3) filled with the experimental substrates (5, 6, 7, 11, 12, and 13 from the first experiment; see section 2.2.1 Substrate Preparation), and arranged in a randomized complete block design (n = 5; experimental unit = one plant) also at
22/ 19°C (± 3/ 1°C SD) during the day/ night in the same greenhouse.
VC tea was used to help improve plant growth through disease suppression and improved nutrient cycling (Ingham and Alms 1999). Seedlings were sub-irrigated with deionized water or VC tea prior to true leaf emergence and irrigated with VC tea twice before transplanting using a watering can. VC tea was brewed using Forterra VC (Forterra Environmental Corp., Puslinch, ON,
Canada) in a compost tea system (Compost Tea System25™ - Growing
Solutions Inc., Eugene, OR, United States). After true leaf emergence, irrigation with deionized water (using a 1000PL Redhead water breaker, Dramm, Canada) and fertigation with Organic Gem® Liquid Fish Fertilizer (Advanced Marine
Technologies, New Bedford, MA, United States) and BlackEarth Organic Liquid
Hume (Black Earth Humates Ltd., Edmonton, AB, Canada) occurred
61 approximately every two to three days (applied directly to the substrate surface with a 50 ml container). The nutrient concentration (mg-L"1 solution) of the fertilizer solution, calculated from the manufacturers information, was 150 total N,
66 P, 332 K, 89 Ca, 10 Mg, 59 Na, 37 Fe, 300 C, and 197 CI (5 ml liquid fislvL
H20"1 + 10 ml liquid hume-L H20"1) prior to transplanting, and 300 total N, 131
P, 664 K, 178 Ca, 20 Mg, 118 Na, 74 Fe, 600 C, and 393 CI (10 ml liquid fish-L
H20"1 + 20 ml liquid hume*L H20"1) after transplanting up until the fifth week of growth after which only deionized water was applied on day 41 through to day
46 as needed. The pH of the fertilizer solution was adjusted with acetic acid
(Apple cider vinegar; Bio-Ag Consultants & Distributors Inc; Wellesley, ON,
Canada) or sodium bicarbonate.
3.2.4 Growth Measurements
After five and six weeks of growth (day 39 and 46) stem diameters, stem heights, leaf areas, shoot dry weights and root dry weights were measured (See section 2.2.4 Growth Measurements for more details).
3.2.5 Statistical Analysis
The same statistical analyses used to compare means in the first experiment were used in this experiment (see section 2.2.5 Statistical Analysis for details).
3.3 Results and Discussion
At both harvests, no significant differences in aboveground growth were observed; however, shoot and root dry weights of seedlings growing in substrate
11 (25VC/ 25PBC/ 20FP/ 23FV/ 7Z) were significantly lower than those for
62 seedlings growing in substrate 5 (25VC/ 25PBC/ 25FP/ 25FV) (Figure 3.2). Dry weights of shoots and roots, respectively, from substrate 11, were 31 and 29% lower than the controls (substrate 5) at first harvest and 26 and 28% lower at second harvest. These results indicated that the addition of zeolite to the organic growing substrates had no beneficial effect on growth of the tomato transplants.
'*>»%. Dry shoots = 0.8 ± 0.08 g 1 Dry roots = 0.4 ± 0.05 g 1 Figure 3.2 Top view of selected transplants from second experiment; shoots (stem + leaves) of transplants growing in substrate 11 with zeolite (right) were an average 31% lighter than shoots of transplants growing in substrate 5 without zeolite (left) at first harvest (day 39). Picture was taken on day 37.
Although zeolite has been shown to increase the pH- and nutrient- buffering capacity of growing substrates, by means of elevating cation exchange capacity (CEC) (Armbuster and Gunter 2001), at a low incorporation rate this effect may be limited for several reasons. The zeolite used in this experiment had a CEC between 150 and 180 meq-100 g"1. This is similar to the CEC of PM, which is usually around 100 to 200 meq-100 g"1. The CEC of humus is usually around 200 meq-100 g'1 and the CEC of clay soils is usually around 55 to 65
63 meq-100 g"1. However, CEC not only varies because of the chemical attributes of the growing substrates, it also varies because of bulk density (BD). Clay soils, for instance, are heavier than PM and humus. And, the volume needed to weigh out
100 g of PM or humus is much larger and contains much more surface area than that of the volume for 100 g of clay. Consequently, the CEC of clay is much lower than that of PM, however, if given the same volume, the CEC of clay, which would be independent of its weight, would be much higher than the same volume of PM (Tripepi 1998a). The same principle can be applied to that of the heavy Z, which had a relatively high CEC but a low incorporation volume due to its high
BD. It is therefore necessary to consider the volume and weight of each substrate component that provides CEC. zeolite is nearly six times heavier than peat; therefore, 100 g of zeolite will supply a substrate with ~6X less of its calculated CEC compared to 100 g of peat. Similarly for cost, zeolite may be cheap (US$30 to 70/ t Z) compared to vermiculite (US$75 to 85/ t FV and
US$225 to 250/1 coarse vermiculite) (van Straaten 2007) but zeolite minerals are seven to eight times heavier than vermiculites making it that much more expensive. The cost of compost (CAN $10 to $20/ t composted yard waste;
Personal communication with K. Osborne, Gro-Bark Organics Inc., Milton, ON,
Canada) is also less than the cost of Z. Composts are not only more affordable than Z, they also have the ability to provide substrates with valuable nutrients that can support the growth of healthy microbial communities, which may provide plants with some protection from pests (Subler et al. 1998; de Brito Alvarez et al.
1995), and increase nutrient cycling, which releases nutrients at a rate plants
64 require and in available forms (Ingham and Alms 1999). In addition, the CEC of compost may be adequate for nutrient and pH buffering capacity rendering zeolite unnecessary in substrates that contain compost. The pH of the substrates were within the range given by Vavrina (2002) throughout the majority of the experiment; however, in all substrates except 5 and 11, which contained the highest amount of compost (25VC/ 25PBC...), compared to other substrates
(25MC/...), pH decreased to values that are not optimal for tomato growth (<5.5) towards the end of the experiment. A high amount of compost resulted in a higher BD in these substrates; therefore these substrates had a higher surface area than the other substrates. Substrates 5 and 11 had higher CEC as a result of the higher amount of compost included, and therefore, had more pH-buffering capacity for this reason (Argo and Fisher 2008; Tripepi 1998a). These results therefore suggest that compost may be used instead of zeolite to elevate CEC.
3.4 Conclusion
The rate at which zeolite was included in the substrates was not high enough to increase growth by means of nutrient-holding capacity (a.k.a. higher
CEC), which is an attribute of Z. Higher inclusion rates of zeolite are not recommended due to high shipment cost, therefore, zeolite is not necessary as a component in organic growing substrates for tomato transplant production.
Instead, compost may be used to increase the nutrient-holding capacity and pH- buffering capacity of organic tomato transplant growing substrates.
65 CHAPTER 4: Developing New Organic Growing Substrates for Tomato
Transplant Production
4.1 Introduction
As indicated earlier in this thesis, growing substrates that allow commercial organic production of vigorous and healthy tomato transplants are lacking. The present experiment evaluates and compares transplant growth in newly formulated substrates and substrates that gave superior results in transplant growth in previous experiments. In the first experiment (Chapter 2), 25 growing substrates were tested for tomato transplant production in plug trays
(plug volume = 15.6 cm3). Ten of the substrates gave superior results in transplant growth compared to other substrates, four of these substrates contained granular zeolite (Z) at a rate of 7 or 5% by volume (v/v). Nutrient deficiencies developed in transplants in the first experiment, so a second experiment was devised to determine whether substrates containing zeolite (7% v/v) could increase growth when using 10-cm pots rather than plugs and with organic fertilizer applications (Chapter 3). Overall, no benefits were found when zeolite was included compared to when zeolite was not included.
In this chapter, practical information on substrate formulations that could be used by growers of organic tomato transplants is presented. The objective was to further evaluate the effectiveness of amending growing substrates with vermicompost (VC), manure compost (MC), pine bark compost (PBC), peat moss
(PM), and coconut coir (CC), for promoting good development, growth, and
66 health of transplants. Seedlings were started in plug trays and later transplanted to 10-cm pots (same as in Chapter 3) as this resulted in better growth than when seedlings were grown entirely in plug trays (Chapter 2). A liquid fish fertilizer was also applied to supply nutrients and to avoid nutrient deficiencies.
4.2 Materials and Methods
4.2.1 Substrate Preparation
No zeolite was used because it lacked effectiveness in both previous experiments. Therefore the 5 leading substrates from previous experiments, excluding one of the standard substrates and excluding any substrates that contained zeolite were tested again in this experiment including: substrate 2
(75PM/ 25FV); substrate 3 (25VC/ 25PM/ 25FP/ 25FV); substrate 5 (25VC/
25PBC/ 25FP/ 25FV); substrate 6 (25MC/ 25PM/ 25FP/ 25FV); substrate 7
(25MC/ 25CC/ 25FP/ 25FV); and substrate 18 (20MC/ 10PBC/ 20CC/ 50FV).
The substrates were mixed v/v and stored in open plastic bags at room temperature for over two weeks. A commercial organic sunshine mix (OSM -
Special Organic Blend, Sun-Gro Horticulture Canada Inc., Vancouver, BC,
Canada) was included in place of the standard substrate from previous experiments (50PM/ 50FV) and five additional substrates were included for a total of twelve substrates. All substrates are presented as percent content v/v in
Table 4.1.
67 Table 4.1 Compacted bulk densities (CBDs; g-L"1) and compositions of the growing substrates tested. Values are the percent of each material included on a volume basis based on the CBD of each material used. Manure bark Substrate CBD Vermicompost Pine Peat moss compost compost z y 1 135±0.2 / / / 75 2 246 ±1.5 / 25 / 25 3 319 ±2.0 / 25 25 / 4 240 ±3.6 25 / / 25 5 226 ±4.6 25 / / / 6 269 ±0.9 20 / 10 / 7 290 ±2.4 12.5 12.5 12.5 / 8 282 ±3.2 / 20 20 / 9 219 ±0.7 20 / / 20 10 279 ±7.7 20 / 20 / 11 224 ±1.7 / 20 / 20 OSM 128 ±2.8 / / / 80 Coconut Coarse Fine Superfine Fine perlite Substrate coir perlite vermiculite vermiculite 1 / / / 25 / 2 / / 25 25 / 3 / / 25 25 / 4 / / 25 25 / 5 25 / 25 25 / 6 20 / / 50 / 7 12.5 / 20 20 10 8 20 / 20 20 / 9 20 / 20 20 / 10 20 / 20 20 / 11 20 / 20 20 / OSM / 20 / / / "Numbers represent each substrate combination tested in the third experiment: 1 = standard substrate (substrate 2 in Experiment 1); 2 to 6 = previously evaluated combinations (substrates 3, 5, 6, 7, and 18 respectively in Experiment 1); 7 to 11 = new combinations; OSM = organic sunshine mix. The Florida industry standard for tomato transplant production (substrate 1) and the commercial OSM were included as comparisons. yData are means of three replicates ±standard error.
The same materials used in the first experiment (Table 4.2) were used in the third experiment (see section 2.2.1 Substrate Preparation for source details).
Substrates were mixed based on the compacted bulk density (CBD) of the individual materials (see section 2.2.1 Substrate Preparation for more details)
68 except for the commercially available substrate OSM. All substrates, except comparison substrates 1 (75PM/ 25FV), and OSM (80PM/ 20 coarse perlite), were amended with elemental sulfur (Canadian HydroGardens Ltd., Ancaster,
ON, CA) to lower pH to the optimal range (5.5 to 6.8) for transplant growth
(Vavrina 2002; Larrea 2005). Elemental sulfur was added two weeks before transplanting following rates recommended by the University of Missouri (MU)
(MU Extension 2008): 0.6 g-L"1 (substrate 3), 0.7 g-L"1 (substrates 2, 8, and 11),
0.8 g-L"1 (substrates 4 and 10), 0.9 g-L"1 (substrates 6, 7, and 9), and 1.3 g-L"1
(substrate 5). Substrate 1 required the addition of dolomitic lime at a rate of 4 g lime/L PM (Prasad et al. 2004). The OSM was used as is.
Table 4.2 Compacted bulk density (CBD; g-L"1), pH, electrical conductivity (EC; pS-cm"1), and content of water-soluble nutrients (mg-L"1 suspension extract) of each individual material (in received state) used to make the substrates. +Z a Material CBD pH EC N03" + NH4 P04 " Manure compost 393.9y 8.3 2650 259 149 Vermicompost 570.8 8.6 1039 122 5 Pine bark compost 479.2 4.9 107 30 0 Peat moss 147.7 4.4 35 0 0 Coconut coirx 161.9 6.4 153 4 2 Fine perlite 128.3 8.1 14 4 0 Superfine vermiculite 145 8 38 4 0 Fine vermiculite 109.6 6.7 21 5 0 Material K Mg Ca CI" S04 Na Manure compost 223 82 83 234 130 102 Vermicompost 128 81 84 132 16 91 Pine bark compost 86 83 83 40 22 93 Peat moss 0 0 0 4 0 80 Coconut coir 83 0 82 57 10 86 Fine perlite 0 0 55 11 12 83 Superfine vermiculite 80 27 82 9 13 83 Fine vermiculite 53 27 55 12 14 83 zNutrient concentrations were measured using the 1:5 method. yAII data are means of three replicates. xCoconut coir was compressed and required the addition of water for expansion; expanded coconut coir was allowed to air dry to a more desirable moisture content (mimicking the consistency of peat moss) before being assessed.
69 One week before transplanting, Spanish River Carbonatite (SRC), a growing substrate amendment that supplies over 80 trace minerals (Chatham-
Kent Organic Epicentre Dresden, ON, Canada) was added to the growing substrates at the recommended rate of 0.56 g-L"1 (g SRC/ L substrate). The SRC was first passed through a 60-pm sieve before mixing into a substrate.
4.2.2 Growth Experiment
Untreated seeds of greenhouse tomato hybrid 'beefsteak' type (Solanum esculentum L. cv. Matrix F1; De Ruiter Seeds Inc. Lakewood, CO, United States) were germinated on moistened filter paper in a Petri dish in darkness at 25°C
(SD ±1°C). After 3 days, the germinated seeds were planted in 15.6 cm3 cells of a plug tray containing substrate 1 (75PM/ 25FV), covered with moistened superfine vermiculite, and placed in a glass greenhouse in the Bovey Complex at the University of Guelph (see section 2.2.3 Growth Experiment for more details) at 25/ 22°C (SD ±3/ 1°C) during the day/ night. After 14 days, the seedlings were transplanted to the experimental substrates in 10-cm pots (pot volume = 426.1 cm3) and positioned on a bench in the same greenhouse at 22/ 19°C (SD ±3/
1°C) during the day/ night). Plants grown in the various treatment substrates were arranged in a randomized complete bloc design (RCBD) with five replications. A border of transplants placed around the plants within the RCBD formed a buffer zone to reduce edge effects. The blocks were systematically rotated each week to remove positional bias and harvested after 32 and 39 days.
The transplants were fertilized two days immediately after the first true leaves emerged using a diluted fertilizer solution (Organic Gem® Liquid Fish
70 Fertilizer: Advanced Marine Technologies, New Bedford, MA, United States) and
Liquid Organo Hume (Black Earth Humates Ltd., Edmonton, AB, Canada). The pH of the fertilizer solution was adjusted with acetic acid (apple cider vinegar;
Bio-Ag Consultants & Distributors Inc; Wellesley, ON, Canada) or sodium bicarbonate. At first the fertilizer solution was adjusted to pH/ EC of 5.37/ 683 ps-cm"1 (on day 10) and 5.49/ 820 pS-cm"1 (on day 11); low pH fertilizer was applied to adjust the pH of the seedling substrate pH (which measured >7 by pourthru on day 9). The concentration of nutrients (mg-L"1 solution) of the seedling fertilizer solution was 150 total N, 66 P, 332 K, 89 Ca, 10 Mg, 59 Na, 37
Fe, 300 C, and 197 CI (5 ml liquid fish The nutrient concentration (mg-L"1 solution) of the full rate fertilizer solution (applied after transplanting) was 300 total N, 131 P, 664 K, 178 Ca, 20 Mg, 118 Na, 74 Fe, 600 C, and 393 CI (10 ml liquid fish H20"1) and the average pH was 5.8 ±0.02, and average EC was 1189 ±37 pS-cm"1. The full rate fertilizer was applied approximately twice a week after transplanting. Transplants were fertilized twice weekly, taking care to avoid the foliage and applying 50 ml to each pot, and irrigated on alternate days using a 1000PL Redhead water breaker. The transplants were sub-irrigated with VC tea in the first week (day 3) and overhead irrigated with VC tea in the fourth week (day 23). VC tea was brewed using Forterra VC (Forterra Environmental Corp., Puslinch, ON, Canada) in a compost tea system (Compost Tea System25™ - Growing Solutions Inc., Eugene, OR, United States). VC tea was used based on reported 71 effects in enhancing microbial activity in the root zone, suppressing disease, increasing nutrient cycling and improving plant growth (Ingham and Alms 1999). 4.2.3 Chemical Properties of the Substrates The pH and EC of the standard seedling substrate used in plug production was measured (on day 9) via the pourthru method (described in section 2.2.2 Chemical Properties of the Substrates) revealing a substrate solution pH of 7.1, which is higher than the optimal range (5.5 to 6.8; Vavrina 2002). The pH of the seedling fertilizer solution was therefore adjusted to have lower pH. Table 4.3 Initial pH and electrical conductivity (EC; pS-cm"1) of the growing substrates before and after adding elemental sulfur on May 30th 2008 (day 5). Date 29-May-08 9-Jun-08 17-Jun-08 18 days after One day before sulfur 10 days after sulfur sulfur addition addition (day 4) addition (day 15) (day 23) Substratez PH EC PH EC PH EC 1y 5.62±0.009x 45±1.3 6.12±0.027 37±0.3 6.47±0.023 n/a 2 6.86±0.015 366±11.3 6.60±0.06 360±2.8 6.9±0.045 n/a 3 6.83±0.003 403±3.8 6.46±0.01 428±0.3 6.73±0.006 n/a 4 7.22±0.017 859±15.6 6.70±0.04 791±7.4 6.88±0.040 n/a 5 8.15±0.042 818±7.8 7.67±0.03 918±3.8 7.49±0.017 n/a 6 7.29±0.013 839±11.4 6.99±0.01 866±7.3 6.90±0.009 n/a 7 7.37±0.031 679±8.5 7.04±0.01 738±4.5 6.93±0.012 n/a 8 6.99±0.031 419±10.0 6.81±0.01 445±5.6 6.64±0.012 n/a 9 7.25±0.030 841±13.3 6.97±0.02 822±7.5 6.89±0.024 n/a 10 7.12±0.023 835±9.2 6.79±0.01 854±7.7 6.71±0.010 n/a 11 7.01±0.007 390±8.5 6.90±0.02 377±2.8 6.85±0.006 n/a OSMw n/a n/a n/a n/a 6.04±0.031 300 zEach number represents a substrate (see Table 4.1 for substrate compositions); OSM = organic sunshine mix. yDolomitic lime was added to substrate 1 at a rate of 4 g lime-L"1 PM. XAII data are means of three replicates ±standard error except EC for OSM on day 23, which is the value of one reading. Values were measured using the 1:5 method. wThe OSM was not amended with pH adjusters. The pH and EC of the transplant substrates were determined prior to and after the addition of elemental sulfur (Table 4.3) to see the change in pH. One 72 day before transplanting, the water-soluble nutrient content of each growing substrate was determined (Table 4.4) as described in the first experiment using an ion chromatography system (see section 2.2.2 Chemical Properties of the Substrates for more details). Throughout the experiment, the pH and EC of the substrates were monitored by the pourthru method (see section 3.2.2 Chemical Properties of the Substrates for more details). Table 4.4. Content of initial water-soluble nutrients in the substrates (mg-L"1 suspension extract) one day before transplanting. 2 +y 3 Substrate N03" + NH4 P04 " K Mg 1 6±0.3ix 0±0.0f 8±0.2e 7±0.0d 2 54±0.3e 7±0.1f 26±0.5d 8±0.1cd 3 7±0.3i 4±0.1f 29±0.1d 10±0.1ab 4 108±1.6a 120±3.3a 62±1.1ab 9±0.1bc 5 99±1.8b 94±2.6b 67±1.5a 9±0.1bc 6 63±1.1d 81±2.1c 57±2.4bc 10±0.2a 7 40±0.5f 43±0.7e 52±1.4c 10±0.2ab 8 7±0.5i 4±0.3f 30±1.1d 9±0.0bc 9 83±0.4c 85±2.1c 53±1.1c 8±0.0cd 10 24±0.8h 62±1.0d 55±2.6c 9±0.0bc 11 36±0.2g 5±0.0f 27±0.9d 8±0.0cd OSM 7±0.4i 0±0.0f 8±0.1e 10±0.5a Substrate Ca CI" S04 Na 1 5±2.5b 8±1.0d 7±0.3d 6±0.6f 2 9±0.2a 52±0.5abc 62±0.8cd 29±0.1e 3 12±0.2a, 52±0.7abc 149±1.3ab 34±2.1de 4 9±0.2a 84±0.9a 106±3.1d 52±1.8ab 5 9±0.1a 74±15.5ab 135±1.8b 58±1.2a 6 10±0.2a 52±15.3abc 138±2.7ab 49±4.0abc 7 10±0.2a 34±0.1bcd 140±1.6ab 48±2.7bc 8 10±0.1a 33±7.4cd 131±0.8bc 41±0.6cd 9 9±0.1a 71±0.6abc 98±1.8d 52±0.4ab 10 10±0.0a 59±12.3abc 155±1.4a 57±1.8ab 11 8±0.0ab 43±0.2bcd 55±0.4c 32±0.5de OSM 11±0.6a 10±0.4d 114±12.7cd 15±0.8f zEach number represents a substrate (see Table 4.1 for substrate compositions); OSM = organic sunshine mix. yNutrient concentrations were measured using the 1:5 method. xData are means of three replicates ±standard error. Data followed by the same letter are not significantly different by the Tukey-Kramer test at P <0.05. 73 4.2.4 Growth Measurements The following growth response measurements were made at both harvests (days 32 and 39): stem diameter (one cm below the cotyledon); stem height; the number and total area of the true leaves; and fresh and oven dry weights (50°C >48 hours) of the stems and the true leaves (see section 2.2.4 Growth Measurements for more details about the instruments used for these measurements). On days 33 and 40, the roots of each transplant were washed to remove substrate and weighed for fresh weights using an analytical balance, then oven dried (50°C >48 hrs) and weighed again when weights stabilized. 4.2.5 Leaf Chlorophyll Content and Gas Exchange Measurements At the first harvest (day 32), two chlorophyll content indexes were taken from the youngest fully expanded leaves of transplants using a portable chlorophyll content index meter (CCM-200, Opti-Sciences, Tyngsboro, MA, USA). On the same day, leaf net photosynthetic rates were measured on the youngest fully expanded leaves using a portable infrared gas analyzer photosynthesis system (LI-COR 6400, LI-COR, Lincoln, NB, United States). During each measurement, leaf chamber reference CO2 concentration was 400.3 ±0.08 pmol-mol"1, the block temperature was kept at 27.4 ±0.10°C, vapor pressure deficit was 1.2 ±0.05 kpa, and the light source was red and blue with a photosynthetically active radiation of 400.6 ±0.20 pmol-m"2-s"1. 4.2.6 Statistical Analysis The same statistical analyses used to compare means in the first experiment were used (see section 2.2.5 Statistical Analysis for details). 74 4.3 Results and Discussion Use of vigorous and healthy transplants is critical for the production of healthy and high yielding tomato crops (Russo 2005). In general a good transplant is sturdy, has a well-established root system, is green, and free of pests (Vavrina 2002). Properly produced transplants are grown under minimal stress and should ensure healthy and productive mature plants that establish well and grow quickly after being planted into their final growing substrate (Larrea 2005). Transplants that are overly stressed from establishment into a new growing environment may suffer from delayed maturity and reduced yields (Vavrina 2002); however, transplants may be hardened by withholding water and nutrients for short periods of time to reduce transplant shock. If transplants have to be transported over long distances, thicker stems are critical for physical support. Generally, 12 to 17 cm tall tomato transplants with straight thick stems and well-developed leaves (flat and green) are preferred (Garton et al. 1994), while ideal transplants are not yet flowering, are 15 to 16 cm tall and wide, and have shoots that weigh approximately 100 g (Peet and Welles 2005). Absolute guidelines describing at what age transplants are ready do not exist. However, research has shown that tomato transplants between the ages of two and 13 weeks old can produce comparable yields in the field (Vavrina and Orzolek 1993). Transplanting at early stages of growth can reduce transplant shock but young transplants (two to four weeks old) may be difficult to remove from plug trays. Older transplants (seven to nine weeks) are larger and more likely to flower earlier and produce early yields but lower total yields, especially in 75 winter months (Peet and Welles 2005). Transplanting at 5 to 7 weeks is recommended for field production (Vavrina and Orzolek 1993; Schrader 2000) and transplanting at three to six weeks old is recommended for greenhouse production (Peet and Welles 2005). The transplants in this experiment were harvested at four and a half and five and a half weeks. Nielsen and Thorup-Kristensen (2004) suggested that organic growing substrates containing animal waste compost have the potential to supply most of the nutrients needed for healthy transplant growth and could limit the need for fertilizer additions; however, Larrea (2005) found a need for fertilizer despite the inclusion of a VC in her organic substrates. Based on substrate fertility analyses and growth responses, Larrea's (2005) findings are supported here. Substrate 4 (25MC/ 25PM/ 25FP/ 25FV) initially had more phosphate 3 (P04 ") and potassium (K) than the other substrates (Table 4.4), had adequate pH and EC throughout the experiment (Table 4.5), and resulted in the best transplant growth (Figure 4.1); however, when PM was replaced with CC in substrate 5 (25MC/ 25CC/ 25FP/ 25FV), significant differences were rarely observed for stem diameter, stem height, leaf number, leaf area, and rootshoot ratio. Substrate 9 (20MC/ 20PM/ 20CC/ 20FP/ 20FV) also produced comparable transplants to those in substrates 4 and 5; and substrates 2 (25VC/ 25PM/ 25FP/ 25FV) and 11 (20VC/ 20PM/ 20CC/ 20FP/ 20FV) also produced good quality transplants comparable to that in the best substrates (substrates 4 and 5); however, when PM included in substrates 2 and 11 was replaced by PBC in substrates 3 (25VC/ 25PBC/ 25FP/ 25FV) and 8 (20VC/ 20PBC/ 20CC/ 20FP/ 76 20FV), stunted development was evident. Effects of higher bulk density (BD), lower air-filled porosity at container capacity (AFP), lower total porosity (TP), nutrient leaching, and possible nutrient immobilization may have been involved in decreased growth observed in these substrates. In addition a combination of high pH and low EC in substrates 3 and 8 (Table 4.5) may have limited nutrient availability and resulted in growth disorders associated with chlorophyll content. However, when lower rates of PBC were used, as in substrate 6 (20MC/ 10PBC/ 20CC/ 50FV), transplant growth was comparable to that in the best substrates. Table 4.5 The pH and electrical conductivity (EC; pS-cm"1) of the growing substrates, measured on days 24, 31, and 38. Day 24 31 38 Substrate z PH EC PH EC PH EC 1 6.3 aby 931f 6.0bc 861e 5.7cde 759c 2 6.1bc 2845cde 5.9cdef 2429bcd 5.7bcde 1363abc 3 6.5a 2353def 6.5a 2190cde 6.2a 1085c 4 5.7de 5288a 6.0cd 3930ab 5.8bcd 1806ab 5 5.4f 5098ab 5.7f 3179abcd 5.7cde 1433abc 6 5.6def 6034a 5.8cdef 4646a 5.8bcde 1889a 7 5.7de 4540abc 6.0cd 3660abc 5.9bcd 1453abc 8 6.3ab 1966def 6.2b 2413bcd 6.0ab 1436abc 9 5.5ef 4888ab 5.7ef 3324abcd 5.8bcd 1287abc 10 5.8d 4830ab 5.9cde 2978bcd 5.9bc 1255abc 11 5.9cd 3358bcd 5.8def 2757bcd 5.6e 1141abc OSM 6.2ab 1513ef 5.9cdef 1841de 5.7de 1233abc cacn numoer represents a suusuam ^see i auits *+. i IUI suusuaie uumpu&iuuiis;, OSM = organic sunshine mix. yAII data are means of five replicates. Values were measured using the pourthru method. Substrates 3 and 8, which contained 20% or 25% of each VC and PBC, consistently produced smaller nutrient deficient transplants with significantly thinner and shorter stems, significantly lower number of leaves and leaf areas, and significantly higher rootshoot ratios (Figure 4.1). 77 Figure 4.1 Growth responses measured at first (left) and second (right) harvest. Data are means of five replicates. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. 78 Reis et al. (1998) found that good transplant growth was possible in 100% PBC when the composting process took 20 weeks compared to a grape marc compost only taking 16 weeks. It is important that compost maturity is reached prior to using compost for plant production to avoid nutrient immobilization (Lopez et al. 1998). In our case the PBC may have been immature rendering + 3 nutrients unavailable for plants; the water-soluble N (N03"+NH4 )-P (P04 >K content of the PBC (30-0-86) was much lower than that of the VC (122-5-128) and the MC (259-149-223) (Table 4.2). Also initial N and P levels (Table 4.4) in substrates 3 and 8, which contained 20% or 25% of each VC and PBC, were much lower than most of the other substrates. Higher rates of fertilizer may have been needed to overcome nutrient immobilization in substrates containing >20% PBC (Nelson 1998). Substrates 2, 4, 5, 6, 9, and 11 produced transplants with significantly thicker and taller stems, with more leaves, greater leaf areas, and lower rootshoot ratios (Figure 4.1). These substrates contained VC and PM, or MC, and supported healthy transplant growth. At second harvest all transplants except transplants grown in substrates that contained 20 or 25% VC and PBC produced transplants with heights greater than is recommended (Garton et al. 1994); height control measures such as brush strokes, impedance (Garner and Bjbrkman 1997 and 1996), withholding fertilizer (mainly P and N), and reducing the amount of far-red (700-800 nm) light reaching the plants (Evans and McMahon 2004) may be used to keep transplants at optimal heights. 79 According to Hu and Barker (2004a), the growth of tomato correlates more with N accumulation and least with P accumulation. Nutrient contents of substrates, determined by pourthru analysis on day 38, revealed that all 12 substrates had soluble N content in pourthru leachate that were not significantly different from one another (Table 4.6). Table 4.6 Content of water-soluble nutrients in the substrates (mg-L"1 pourthru leachate); measured on day 38. 2 +y Substrate N03" + N04 PO^" K Mg 1 56 ±3.5ax 603±71.4abc 92 ±2.8c 81 ±0.8a 2 42 ±2.1a 263 ±35.4d 105±4.8c 81 ±0.7a 3 44 ±2.4a 274±21.9d 110±5.3bc 80 ±0.6a 4 50 ±2.0a 727 ±78.1a 158±13.6a 85 ±2.5a 5 46 ±7.4a 432 ±92.3abcd 131 ±21.4abc 83 ±4.6a 6 45 ±4.1a 620±101.3abc 152±10.1ab 85 ±2.4a 7 39 ±10.6a 389±100.3bcd 127±20.4abc 82 ±3.0a 8 52 ±3.9a 367±14.8cd 120±2.9abc 82 ±0.2a 9 54 ±10.8a 482 ±63.5abcd 127±2.4abc 80 ±0.3a 10 41 ±8.2a 471 ±64.3abcd 124±4.0abc 80 ±0.8a 11 35 ±4.8a 264 ±63.5d 96 ±4.3c 79±1.4a OSM 47 ±13.8a 706±139.9ab 90 ±3.4c 84 ±2.2a Substrate Ca CI" S04 Na 1 84±1.1a 59 ±5.7ab 18 ±1.3b 108±19.1a 2 94 ±2.3a 46 ±2.0ab 519±37.4a 108±5.6a 3 86±1.4a 38±1.4ab 338 ±78.9ab 76 ±8.7a 4 94 ±2.9a 58 ±3.4ab 547 ±165.6a 152±14.8a 5 91 ±8.6a 40 ±4.7ab 511 ±241.9a 88 ±20.7a 6 88 ±3.0a 62 ±2.6ab 545 ±73.5a 134 ±12.4a 7 89 ±4.9a 47±10.4ab 466±187.9ab 100 ±30.1a 8 92 ±0.7a 49 ±4.0ab 506 ±23.2a 100±5.7a 9 84±1.2a 51 ±4.4ab 321 ±14.7ab 97 ±5.4a 10 84±1.7a 45 ±4.7ab 254 ±25.5ab 89 ±10.8a 11 89 ±4.5a 36 ±4.5b 376 ±98.0ab 88 ±22.0a OSM 85 ±2.3a 64 ±7.1a 176±40.0ab 118±58.8a zEach number represents a substrate (see Table 2 for substrate compositions); OSM = organic sunshine mix. yNutrient concentrations were measured using the pourthru method. xData are means of three replicates ±standard error. Values followed by the same letter are not significantly different by the Tukey-Kramer test at P <0.05. 80 3 Substrate 4, however, had significantly more P04 " and K than the OSM and substrates containing VC (1, 2, 3, 7, 8, 11). Substrates did not differ in magnesium, calcium, and sodium content but the OSM had significantly more chloride than substrate 1 and substrates 2, 4, 5, 6, and 8 all had more sulphate than substrate 1. In general substrates containing >12.5% MC had higher nutrient contents on day 38 in pourthru leachate than substrates that did not contain MC (see Table 4.1 for substrate compositions). Substrates provide physical support for plants, to hold water and nutrients, and to allow gas exchange to occur in the rhizosphere. The physical properties of growing substrates [BD, TP, water-holding capacity at container capacity (WHC), and AFP] are important. Very heavy substrates reduce the growth and development of tomato (Wilcox 1993). Whereas lighter substrates are preferred over heavier substrates for handling, shipping, and reducing worker fatigue (Tripepi 1997), and also because lighter substrates tend to have higher TP, WHC, and AFP, which benefit root growth and plant development (Strojny and Nowak 2004). However, substrates with very low BD are not preferred because of loss of physical support (Bunt 1983). Substrates (3, 6, 7, 8, and 10) were heavier in terms of CBD than substrates (OSM, 1,2,4, 5, 9, and 11) (Table 4.2). Slow, uninterrupted growth is ideal for transplant production but difficult to attain. Growers attempt to maintain optimum lighting, moisture and nutrient inputs to encourage healthy growth (Vavrina 2002). In general, a balanced and adequate nutrient availability, in any crop, lowers plant stress, improves plant vigor, and decreases the risk of disease. Balanced and adequate nutrient 81 availability was assumed in this study when substrates were capable of supporting the growth of healthy looking transplants. Nonetheless, the EC and pH of the substrates were important factors involved in nutrient availability. MC had the highest nutrient concentration compared to VC. All other materials used in this experiment had EC ranging from 14 to 153 pS-cm"1 and individual water- soluble nutrient contents lower than 100 ppm (Table 4.2). While a higher EC is an indication of higher nutrient content, a higher pH (>6.8) results in a loss of nutrient availability, as some nutrients are not easily available for plant uptake at high pH (Smith et al. 2004). VC had the highest pH and MC had the second highest pH of all materials included in this study (Table 4.2). All substrates except controls contained either one of either VC or MC or both (Table 4.1). Initially, the pH values measured for the experimental growing substrates (except comparison substrates 1 and OSM) were all higher than is recommended for tomatoes [5.5-6.8 (Vavrina 2002)]. Elemental sulfur was added to lower the pH (Larrea 2005). Regardless, the pH of the substrates remained high throughout the growth experiment (see Table 4.3 and 4.5), ranging from 6.47 for substrate 1 (75PM/ 25FV) to 7.49 for substrate 5 (25MC/ 25CC/ 25FP/ 25FV). The rate of pH change with sulfur is slow (MU Extension 2008), therefore, substrates needed more than two weeks for pH change to occur. The addition of SRC may also have had an effect on pH stabilization. SRC acts as a neutralizer in acidic soils. It is likely that both the elemental sulfur's slow reaction time and the SRC's neutralizing effect on pH were involved in the prevention of adequate pH decrease prior to the use of the substrates in growth experiments. 82 While initial pH values of the growing substrates were high, after several fertilizer applications, most substrates had acceptable pH and were maintained within the recommended range (5.5-6.8) throughout the remainder of the experiment (Table 4.5); substrate 5, however, had a pH of 5.4 on day 24, which indicates a dramatic drop in pH from 7.7 (on day 15 prior to transplanting). This drop in pH could be a result of low pH-buffering capacity in this substrate or a result of a higher addition of elemental sulfur due to its high initial state pH (8.2). Substrates with the highest pH (6.3-6.5), during the growth experiment, were substrates 3 and 8, which contained 20% or 25% VC and PBC; the pH of these substrates were maintained at or above 6.0 (Table 4.5). Similar to results from our second experiment (Chapter 3), there was a resistance to pH change in substrates containing higher amounts of compost. Overall, the fertilizer solution applied directly to the substrates maintained the pH of the substrate solution at adequate levels throughout the experiment regardless of the initial pH of the substrates; however, EC values measured initially and in pourthru leachate were not as stable as pH throughout the experiment. The recommended range of EC for seedlings measured in a 1:5 (substrate:deionized water) suspension extract is between 100 and 400 pS-cm"1 (Lang 1996). The initial EC readings of the growing substrates used in this experiment, which were measured one day before transplanting (day 15), ranged from 37 pS-cm"1, for substrate 1, to 918 pS-cm"1, for substrate 5 (Table 4.3). All substrates that had high EC (>700 pS-cm"1) contained MC at a rate of 12.5 to 83 25% (substrates 4, 5, 6, 7, and 9); these substrates generally had higher initial sate contents of water-soluble nutrients (Table 4.4). It is recommended that the EC of the applied fertilizer be adjusted to -1000 pS-cm"1 at the beginning of seedling growth and progressively increased to 2500 to 3500 pS-cm"1 at the time of planting, to promote vegetative growth and strong cell walls (Juneau et al. 2006). Our fertilizer solution was adjusted to <1000 pS-cm"1 for plugs and >1000 pS-cm"1 for transplants that were three to six weeks old. At the first pourthru collection (day 24), substrates containing MC (substrates 4, 5, 6, 7, 9, and 10) all had EC readings from pourthru leachate higher than 4000 pS-cm"1. This was likely due to (1) inadequate leaching and (2) the substrates drying out too quickly allowing salt accumulation to occur at the substrate surface. Leaching was therefore monitored more closely, aimed at leaching of 20 to 40% of applied volume (Juneau er al. 2006), and fertigation was halted for a week prior to the next pourthru measurement. By day 31 (second pourthru analysis) substrates 4, 6, and 7 still had high EC readings between 3660 and 4646 pS-cm"1. However, by the third pourthru analysis (day 38) all EC readings were between 759-1889 pS-cm"1 (Table 4.5); fertigation was restarted on day 31. Substrates were properly leached in the last two weeks of the experiment and salt accumulation in the substrates was reduced. Overall it was concluded that the cause of the high EC in pourthru leachate was due to a combination of inadequate leaching and inclusion of MC in the growing substrate. Synthetic sources of nitrogen (N) can be applied successfully to tomato transplants using 200 to 400 ppm in as little as five applications beginning after 84 first true leaf emergence to as many as 46 applications beginning one week after seeding (Reviewed by Vavrina et al. 1998). We applied a conservative amount of organic fertilizer (300 ppm N) 11 times and were able to produce transplants of good quality. Transplants from substrates that contained 20 or 25% of each VC and PBC (3 and 8), however, showed signs of nutrient deficiency halfway through the growth experiment. Stunted development, chlorotic leaves, and purple discolouration of the abaxial leaf surfaces were observed for transplants growing in these substrates. Slight purplish colouring at the base of stems and on the underside of leaves are signs of carbohydrate accumulation which help improve survival; however extensive purpling is a sign of phosphorus deficiency which delays early growth (Garton et al. 1994). Reported visual symptoms and subsequent water-soluble nutrient analysis on day 38 (Table 4.6) revealed a plausible phosphorus deficiency in these substrates. These substrates did not contain any MC, which contained more phosphorus than other materials included in the experiment (Table 4.2). Substrates that contained VC and PBC (3 and 8) also had higher pH (>6.3) throughout the experiment (Table 4.5); high pH possibly limited the availability of other nutrients in these substrates leading to other growth compromising disorders (Smith et al. 2004). Substrates containing VC and/or PBC (3, 8, 10, and 11) produced transplants with significantly less chlorophyll content in their leaves compared to the other substrates. Low chlorophyll content in the leaves, often described as chlorosis or yellowing of the leaves, has been attributed to salt stress (Al- aghabary er al. 2005), nutrient deficiency or toxicity (nitrogen, magnesium, or 85 iron) (Wilcox 1993), and extreme pH and EC, as pH and EC relate to nutrient availability (Smith et al. 2004). In this study excess salt was not the cause of low chlorophyll content index as substrates with lowest chlorophyll contents (Table 4.7) were not among substrates with the highest EC readings (Table 4.5). Instead low chlorophyll content was most likely a result of high pH and low EC. The optimal pH range for healthy plant growth is dependent on fertilizer input; higher fertilizer inputs are required at higher pH (> 6.3) to avoid chlorosis (Smith et al. 2004). Iron deficiency, which may be caused by high pH and low substrate N, might have been the cause of low chlorophyll content in substrates 3 and 8. Consequently, substrates that contained both VC and PBC (3 and 8) produced transplants with significantly lower leaf photosynthetic rates (11 to 12 pmol C02 m"2 s"1) in comparison to the other substrates (Table 4.7). Table 4.7. Leaf chlorophyll content index and photosynthetic rate on day 32. Leaf photosynthesis rate 2 y 2 1 x Substrate Chlorophyll content index (pmol CQ2 m" s" ) 1 33±1.9abcw 16.3±1.06ab 2 30 ±0.5abcde 15.2±0.77ab 3 24±1.0f 10.5 ±1.21c 4 33 ±0.8ab 14.3±1.59abc 5 32±1.4abcd 15.5±0.54ab 6 28±1.4bcdef 15.3±0.99ab 7 28 ±0.5abcdef 15.4±0.54ab 8 27±1.8def 12.3±1.08bc 9 31 ±1.2abcde 14.7 ±0.75ab 10 26 ±0.4ef 16.7±0.48a 11 28 ±0.9cdef 16.2±0.60ab OSM 33±1.0a 16.0±0.27ab zEach number represents a substrate (see Table 4.1 for substrate compositions); OSM = organic sunshine mix. yDate are means of five replicates (each replicate is the average of two sub- samples) ±standard error (SE). xData are means of five replicates ±SE. wValues within a column followed by the same letter are not significantly different by the Tukey-Kramer test at P <0.05. 86 Substrates that had higher water-soluble macronutrient contents measured in pourthru leachate on day 38 were substrates 4, 5, 6, 9, and 10, which contained MC with either PM (4), CC (5), PBC (6), PM and CC (9), or PBC and CC (10), respectively (Table 4.5). Although most of these substrates supported healthy transplant growth based on growth responses measured in this experiment (4, 5, 6, and 9), substrate 10 had comparable nutrient content, and leaf photosynthetic rate yet did not produce good transplant growth. Whereas substrates which contained VC and PM (2) or VC and CC (11), produced good transplant growth but were not among the substrates with highest macronutrient content in pourthru leachate on day 38. Nonetheless, substrates containing PBC did not support plant growth as well as other substrates regardless of nutrient content measured on day 38. Substrate 9, which contained 20MC/ 20PM/ 20CC/ 20FP/ 20FV often produced transplants of better quality than substrate 10, which contained 20MC/ 20PBC/ 20CC/ 20FP/ 20FV (especially at first harvest; Figure 4.1). The initial state nutrient contents (e.g. N and P; Table 4.4) in substrate 9 were much higher than in substrate 10, even though there was no difference in nutrient content measured at the end of the experiment (Table 4.6). This indicates that nutrient immobilization (especially with N) may have occurred in substrates containing PBC (Lopez et al. 1998). Substrates containing VC and PM (2 and 11) often produced transplants that were not significantly different than transplants from the best substrate which 3 contained MC and PM (4), but had significantly less water-soluble P04 " and K in 87 pourthru leachate than other substrates. This was likely due to the exclusion of MC in this substrate. MC had the highest source of plant available nutrients. Greater growth effects associated to the use of composted materials are more likely a result of a combination of factors including the presence of plant growth regulators, and the activity of beneficial microorganisms (Subler er al. 1998) which compete with disease organisms for nutrients or act as antagonists (De Brito Alvarez et al. 1995), and increase nutrient cycling by releasing nutrients at a rate plants require in plant available forms (Ingham and Alms 1999). Many studies looking into the influence of different types of compost and compost extracts on growth and disease suppression also attribute this plant growth- enhancing phenomenon to the above-mentioned factors (Szczech 1999; Atiyeh et al. 2000; Al-Dahmani et al. 2003; Arancon et al. 2004; Arancon et al. 2003; Termorshuizen et al. 2006). 4.4 Conclusion This study found that MC was better as compost for supplying tomato transplants with nutrients throughout the growth experiment; however substrates containing VC and PM were also good substrates that supported healthy transplant growth. The top substrates were substrates 2 (25VC/ 25PM/ 25FP/ 25FV), 4 (25MC/ 25PM/ 25FP/ 25FV), 5 (25MC/ 25CC/ 25FP/ 25FV), 6 (20MC/ 10PBC/ 20CC/ 50FV), 9 (20MC/ 20PM/ 20CC/ 20FP/ 20FV), and 11 (20VC/ 20PM/ 20CC/ 20FP/ 20FV). 88 CHAPTER 5: Using Processed Organic Waste in Growing Substrates for Tomato Transplant Production 5.1 Introduction The main constituent in growing substrates today is peat moss (PM) and the standard growing substrate for vegetable transplant production in Florida, United States, includes two seasonal variations: 1) 70 to 75% PM and 25 to 30% fine vermiculite (FV) by volume (v/v) in the summer time, and 2) 50 to 60% PM and 40 to 50% FV v/v in the winter time (Arenas et al. 2002). PM is allowed in organic systems in Canada (Government of Canada, 2008b), but, considerable concern over its use exists because it is an expensive resource that is non renewable, therefore, the harvesting of PM poses a threat to the environment (Pill and Ridley 1998). PM bogs serve as a major sink for atmospheric carbon dioxide, therefore, are essential for the mitigation of climate change (Favoino and Hogg 2008; Pill and Ridley 1998; Raviv et al. 1998b). PM is also expensive mainly because it requires a supply of fertilizer (Ozores-Hampton and Mardones 2006). For these reasons PM alternatives are sought. PM alternatives such as processed organic wastes (i.e. composts and vermicomposts) are especially important in organic and sustainable production systems because they recycle important plant nutrient sources and reduce the use of PM, pesticides, and fertilizers needed for plant growth (Agmad et al. 2008; Clark and Cavigelli 2005; Chen et al. 1992; Raviv et al. 1998a). Composts have been successful constituents in growing substrates for tomato (Zaller 2007; Diaz- 89 Perez et al. 2006), therefore, this experiment evaluated different composts as components of organic tomato transplant growing substrates. The popularity of compost use in Canadian horticulture and agriculture is increasing, especially with growing concerns for environmental protection and sustainability (OCO 2008) and the expansion of the composting industry (Lefebvre et al. 1994). Composting is an effective way of disposing waste hygienically when produced following product quality standards and regulations for compost feed stocks and the composting process that are provided by several Canadian organizations (see section 1.3 Compost). Compost feed stocks may contain a variety of wastes, which include: food scraps (Agmad et al. 2008); yard waste (Tuitert et al. 1998); animal manure (Escuadra and Amemiya 2008; Guitierrez-Miceli et al. 2007); green manure (Escuadra and Amemiya 2008; Yogev et al. 2006); wood products (Escuadra and Amemiya 2008); paper waste (Vallad et al. 2003); coffee waste (Escuadra and Amemiya 2008); sugarcane factory residues (Theodore and Toribio 1995); winemaking refuse (Dianez et al. 2006; Dianez et al. 2005); mineral deposits (Termorshuizen et al. 2006); and other materials; but, composts are often produced as combinations of several waste products (Yogev er al. 2006). Municipal biosolids can be composted (Chen and Nelson 2008; Castillo et al. 2004; Wilson et al. 2003) but are not allowed in organic production as compost feedstock. Documentation or proof that no synthetic products or restricted materials were used in the feedstock must accompany any compost that is to be used in organic production (Government of Canada 2008a and b). 90 Anaerobic digestion is a type of bioenergy system. Its product is another form of decomposed organic waste. Anaerobic digestion may one day become more globally accepted as an energy source as we transition from today's fuels, which include oil and natural gas deposits, to tomorrow's renewable energy sources, such as solar energy, wind energy, and bioenergy (Liedl et al. 2004). This experiment was therefore also intended to evaluate anaerobically digested cattle manure (ADCM) as a component of organic growing substrates. Over 25 organic growing substrates for tomato transplant production have been evaluated in this research thus far. Equal parts v/v of manure compost (MC), coconut coir (CC), fine perlite (FP), and FV achieved better transplant growth than other combinations, and comparable growth to the best substrate, which included equal parts of MC, PM, FP, and FV. To avoid the use of PM in future experiments we selected the substrate that contained CC for further investigation (see Chapter 4). The goal of this experiment was to investigate the use of several different sources of compost and one bioenergy product in organic growing substrates for tomato transplants. It was hypothesized that different organic matter could be equally capable of providing adequate support for tomato transplant growth. Our main objective was to determine whether ADCM and composts from different sources could be equally supportive of plant growth and in the case that they were not, aim to explain why this was the case. 5.2 Materials and Methods 5.2.1 Substrate Preparation Materials eligible for use in organic production systems, according to 91 organic standards (Government of Canada 2008b), were used (Table 5.1). Table 5.1. Abbreviations, and source of materials used in this experiment. Material (Abbreviation) Source Forterra vermicompost (VC) Forterra Environmental Corp., Puslinch, ON, Canada Manure compost (MC)Z Dingo Farms, Bradford, ON, Canada Manure compost with eggshells (MC+E)y Dingo Farms, Bradford, ON, Canada Worm Power Gold worm castings (WC) Worm Power, Avon, NY, United States Anaerobically digested cow manure (ADCM) Prof. Vern Osborne, University of Guelph, Guelph, ON, Canada Yard waste compost (YWC) Gro-Bark Organics Inc., Milton, ON, Canada Fine coconut coir (CC) Millenniumsoils Coir Inc., St. Catharines, ON, Canada Fine vermiculite US grade No. 3 (FV) Plant Products Ltd., Brampton, ON, Canada Fine perlite US grade No. 3 (FP) Canadian HydroGardens Ltd., Ancaster, ON, Canada Superfine vermiculite US grade No. 4 (SV) Plant Products Ltd., Brampton, ON, Canada Fine Canadian Sphagnum peat moss (PM) Fafard Peat Moss Co Ltd. City, QC, Canada ZMC = Unknown quantities of cattle, sheep, and horse manures (straw bedding included) and vegetable scraps (onion and carrot) yMC+E = MC + eggshells. Composts (Figure 5.1; see Table 5.1 for abbreviations) were received in the following order: MC and MC+E (September 29th 2008), WC (October 8th 2008), VC and YWC (October 10th 2008), and ADCM (October 20th 2008). Composts were stored at 10°C in open plastic bags until mixed on October 23rd 2008 (VC and MC, and MC+E) and October 24th 2008 (WC, YWC, and ADCM). 92 Figure 5.1. From left to right:manure compost, manure compost with egg shells, anaerobically digested cattle manure (top), yard waste compost, Forterra vermicompost, and Worm Power Gold worm castings (bottom). The compacted bulk densities (CBDs) of the individual materials used in this experiment were determined following the procedures described in section 2.2.1 Substrate preparation (i.e. CEN standards for CBD; ISHS 2003). According to the CBDs of the materials, substrates were mixed v/v (Table 5.2) and amended with Spanish River Carbonatite™ at 0.56 g-L"1. The pH of the substrates was determined (see details in section 2.2.2 Chemical properties of the Substrates) and elemental sulfur (Canadian HydroGardens Ltd., Ancaster, ON, Canada) was added to each substrate (see Table 5.2 for substrate formulations) according to this pH measurement. Rates of elemental sulfur additions were 1.6 g-L"1 (substrate 1) or 1.5 g-L"1 (substrates 2, 3, 4, 5, and 6), following recommendations from the University of Missouri (MU) (MU Extension 93 2008) to reduce the pH of each substrate to 5.7 within the optimal range (pH 5.5 to 6.8) for tomato transplant growth (Vavrina 2002). Table 5.2 Initial pH, electrical conductivity (EC; pS-cm"1), and compacted bulk density (CBD; g-L"1) of the individual materials (in their received state) used. Substrate compositions are also presented in this table. Values for substrate compositions are the percent of each material included on a volume basis based on the CBD of each material. Material pHz ECZ CBD Vermicompost 8.8 ±0.05y 946 ±14.6 556 ±2.1 Manure compost 8.6 ±0.01 1890 ±7.7 638 ±6.0 Worm castings 8.7 ±0.03 4407 ±118.6 454 ±3.3 Anaerobically digested manure 8.8 ±0.09 347 ±8.0 237 ±1.1 Manure compost with eggshells 8.9 ±0.02 835 ±69.3 696 ±3.2 Yard waste compost 8.8 ±0.05 395 ±2.0 503 ±4.5 Coconut coir 6.7 ±0.11 170 ±0.2 185 ±1.2 Fine perlite 8.2 ±0.29 24 ±11. 4 122 ±2.0 Fine vermiculite 7.6 ±0.24 41.4 ±0.6 125 ±0.3 Substrate compositions Material 1 2X 3 4 5 6 Vermicompost 25 0 0 0 0 0 Manure compost 0 25 0 0 0 0 Worm castings 0 0 25 0 0 0 Anaerobically digested manure 0 0 0 25 0 0 Manure compost with eggshells 0 0 0 0 25 0 Yard waste compost 0 0 0 0 0 25 Coconut coir 25 25 25 25 25 25 Fine perlite 25 25 25 25 25 25 Fine vermiculite 25 25 25 25 25 25 zMeasured using the 1:5 method. yData for pH, EC, and CBD are means of three replicates ±standard error. xOptimum substrate combination (25MC/ 25CC/ 25FP/ 25FV) previously evaluated. Substrates were stored in open plastic bags for three weeks prior to being used in transplant growth experiments. Growblock (GB), made of 100% CC substrate (850 cm3) and proposed for organic certification (Jiffy Products N.B. Ltd. Shippegan, NB, Canada), was used as the comparison substrate. Seedling 94 substrate (75PM/ 25FV) was prepared and used for plug production (see more details on preparation of this substrate in section 4.2.1 Substrate Preparation). 5.2.2 Growth Experiment Untreated seeds of greenhouse tomato hybrid 'beefsteak' type (Solanum esculentum L. cv. Matrix F1; De Ruiter Seeds Inc. Lakewood, CO, United States) were germinated on moistened filter paper in a Petri dish in darkness at 25/ 22°C [±3/ 1°C standard deviation (SD)] during the day/ night from October 31st 2008 (day 1) to November 3rd 2008 (day 4) then they were sown in a 200-plug tray (15.6 cm3) filled with seedling substrate (75PM/ 25FV), covered with moistened superfine vermiculite, and placed in a glass greenhouse in the Bovey Complex at the University of Guelph (see section 2.2.3 Growth Experiment). Seedlings grew in the greenhouse at 25/ 22°C (±3/ 1°C SD) during the day/ night for two weeks prior to transplanting to substrates in 10-cm pots (pot volume = 426.1 cm3) or growblocks (volume = 850 cm3) on November 17th 2008 (day 18). Transplants were placed in the same greenhouse at 21/ 19°C (±3/ 1°C SD) during the day/ night in a randomized complete block design (RCBD) with four replicates (replicate = one transplant) for each of three harvests. A border of transplants placed around the plants within the RCBD formed a buffer zone to reduce any edge effects. Transplants were harvested on days 28, 38, and 46 for final growth measurements (see section 5.2.4 Growth measurements below). A mixture of Organic Gem® Liquid Fish Fertilizer (Advanced Marine Technologies, New Bedford, MA) and Liquid Organo Hume (Black Earth Humates Ltd., Edmonton, AB) prepared at a concentration (mg L"1 solution) of 95 300 total N, 131 P, 664 K, 178 Ca, 20 Mg, 118 Na, 74 Fe, and 393 CI" (10 ml liquid fistvL H20"1 + 20 ml liquid hume»L H20"1) was used as fertilizer. Seedlings were fertilized twice (days 12 and 17), and transplants were fertilized six more times (days 21, 25, 29, 32, 36, and 42): vermicompost tea was applied once as a foliar spray on day 29 (pH/ EC = 8.0/ 585 pS-cm"1) and twice in combination with fertilizer on days 17 and 32 [pH/ EC = 5.8/ 1263 pS-cm"1 (±0.01 / 50.9 pS-crrf1 SE)]; fertilizer was applied on its own on days 12, 21, 25, 36, and 42 [pH/ EC = 5.8/ 808 pS-cm"1 (±0.01 / 59.0 pS-cm"1 SE)]. 5.2.3 Chemical Properties of the Substrates The initial pH and EC of each individual material used in the substrates were determined prior to use on day 14 following the 1:5 method. The 1:5 suspension extracts were analyzed for nutrient content using an ion chromatography system. The initial pH of each substrate was determined, same as for individual materials, using the 1:5 method (refer to section 2.2.2 Chemical Properties of the Substrates for further details). During the production phase the pH and EC of each substrate was measured using the pourthru method (Cavins et al. 2000) on days 25, 32, and 36 (refer to section 3.2.2 Chemical Properties of the Substrates for further details). A portable Oakton pH/ Con 300 Meter (Oakton Instruments, Vernon Hills, II, United States) was used to record the pH and EC of the 1:5 suspension extracts and the pourthru leachates. 5.2.4 Growth Measurements The following growth response measurements were taken at each harvest (days 28, 38, and 46). Stem diameter was taken one cm below the cotyledon 96 using an electronic digital caliper (ABSOLUTE Digimatic Caliper 500-135, Model #CD-4"BS, Mitutoya Corporation, Mitutoyo Canada Inc. Mississauga, ON, Canada); transplants were cut at the height of the cotyledons and the stem height above the cotyledons was measured with a ruler. True leaves were removed, counted, and the total leaf area was measured with a leaf area meter (LI-3100; LI-COR, Lincoln, NE). Finally stems and true leaves were weighed separately for fresh weights using an analytical balance and then oven dried (50°C >48 hours) and weighed again when weights stabilized to get the dry weights (except at first harvest). Roots were stored in a cold room (4°C) and washed (except at first harvest) on day 39 (at the second harvest) and on days 47 and 48 (at the third harvest). Stems remaining below the cotyledon were cut off at the site where colour change occurred and stem height below the cotyledons was measured with a ruler. Both the stem heights below the cotyledon and roots, which were washed to remove substrate debris, were weighed separately for fresh weights using an analytical balance then oven dried (50°C >48 hours) and weighed again when weights stabilized. Stem height is therefore the sum of stem heights both above and below the cotyledon and shoot refers to the sum of both stems and leaves. 5.2.5 Statistical Analysis Means (n = 4) were determined and subjected to an analysis of variance (ANOVA) using the General Linear Models procedures in SAS version 9.1 (SAS Institute; Cary, NC, 2003). An analysis of residuals was used prior to ANOVA to verify the assumptions of homogeneity of variance. Outliers were removed from 97 the data set based on Lund's tables for an approximate test for outliers in linear models (Lund 1975). When ANOVA was significant (P <0.05) for a treatment effect, a Tukey-Kramer test at P <0.05 was performed to compare means. 5.3 Results and Discussion A good transplant is green, straight, sturdy, and between 12 and 17 cm tall and wide; it has a well-established root system, and is free of pests (Peet and Welles 2005; Garton er al. 1994; Vavrina 2002). According to these characteristics for a good transplant, all substrates in this experiment, with the exception of the ADCM substrate, produced acceptable tomato transplants. The GB substrate most often produced the largest, leafiest, and heaviest transplants with the lowest rootshoot ratios because the volume of the GB substrate was double that of the other substrates (Kemble et al. 1994). For this reason, it was not scientifically sound to compare transplants from the formulated substrates in 10-cm pots to transplants from the GB substrate in 10-cm fabric blocks because the transplants from the GB substrate had an obvious growth advantage due to a larger rooting zone. The purpose of including the GB substrate as a comparison substrate was for practical reasons. The GB substrate is a commercially available substrate that is placed in block-shaped fabric by the manufacturer. It is recommended as an alternative to rockwool blocks for transplant production. We therefore included the GB substrate as a comparison knowing that transplants grown in this substrate would have an advantage over transplants grown in the formulated substrates and the objective was therefore to see if the formulated substrates from this experiment could produce transplants 98 of equal quality to transplants from the GB substrate despite the obvious advantage that these transplants had due to growth in a larger volume of growing substrate. All in all, transplants grown in formulated substrates were comparable to transplants grown in the GB substrate at first, second, and third harvest. At first harvest, transplants from the MC+E substrate had more leaves than transplants from the YWC substrate and were taller and had thicker stems than the transplants from the ADCM substrate. Transplants from the VC substrate, the MC substrate, and the WC substrate also had thicker stems than transplants from the ADCM substrate. At second harvest, transplants from all substrates except the WC substrate and the ADCM substrate had stem thicknesses, and numbers of leaves that were not significantly different from transplants with the thickest stems and the most leaves, (i.e. transplants from the GB substrate). All substrates except the MC+E substrate produced transplants that had comparable heights to the tallest transplants grown in the GB substrate. At third harvest, transplants from the GB substrate had thicker stems and more leaves than transplants from all substrates except transplants from the WC substrate only, and transplants from the GB substrate were also taller than transplants from all substrates except the WC and MC+E substrates (Figure 5.2). 99 Figure 5.2. Growth responses at first, second, and third harvest of the fourth experiment. Data are means of four replicates ±standard error. VC =vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC + E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. Data bearing the same letter.are not significantly different by the Tukey-Kramer test at P <0.05. In terms of leaf area, at second harvest, transplants from the GB substrate had significantly higher leaf areas than transplants from all other substrates except the YWC substrate and the MC+E substrate. Whereas at third harvest, the GB substrate had transplants with significantly higher leaf areas than transplants from all other substrates except the WC substrate (Figure 5.3). 100 Figure 5.3. Leaf area at first, second, and third harvest. VC =vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC + E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. Data are means of four replicates ±standard error. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. At second harvest several statistically significant differences in growth were observed. Shoots of transplants from the GB, MC+E, and YWC substrates were heavier than those from the VC, MC, WC, and ADCM substrates and roots of transplants from the MC+E, GB, YWC, MC, and VC substrates were heavier than roots of transplants from the WC and ADCM substrates. At third harvest, shoots of transplants from the GB and WC substrates were heavier than those from the MC+E, YWC, VC, MC, and ADCM substrates and roots of transplants from the YWC and VC substrates were heavier that those from the ADCM substrate. Rootshoot ratios of transplants from the GB substrate were lower than those of transplants from all substrates except the YWC, VC, and MC substrates (at second harvest) and the WC substrate (at third harvest). At third harvest, rootshoot ratios of transplants from the WC substrate were also lower than those from the YWC and ADCM substrates (Figure 5.4). 101 Harvest 2 (day 38) Harvest 3 (day 46) 4UU-I 1500-1 a 300- ab ', aub 1000- bc 0) be be b b b b 200- I 1 1 ° 500- o 100- j JZ 0- i 50-| 250- ab ab 40 200- ab ab a 30' 150- s 20' 100- 10- 50- 0' 0- 0.20-| 0.4- ab ab ab be be 0.15-1 0.3- ab 0.10- 0.2- 5 0.05- 0.1- 0.00- •T- -T- 0.0- r& ,-i ^\A^\^^\^ * .rf.4? .n<.4? .4 4 ro «T .4* ^ J? JF J? J? J? c$ .<& .oV .<$' .<&J .0$ N .# o# ,c># o#* o ,# c f- Figure 5.4 Root and shoot dry weight and root:shoot ratio at second and third harvest. Data are means of four replicates ±standard error. VC =vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC+E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. Initial pH values for the composts were between 8.6 and 8.9, and EC values were highest for the WC, followed by the MC, the MC+E, the VC, and the 102 ADCM (Table 5.2). Although the initial pH of each substrate was high (pH 8.2 to 8.5), the pH of the substrates measured by the pourthru one week after transplanting (day 25) was within the optimal range for tomato transplant growth (5.5 to 6.8; Vavrina 2002) in all substrates except the ADCM substrate (pH 4.3). At the second pourthru (day 32), pH measurements of all substrates, except the GB substrate and the VC substrate, had lower than optimal pH. At third pourthru (day 36), pH measurements of all substrates, except the GB, VC, and WC substrates, had lower than optimal pH. The ADCM substrate had extremely low pH measurements at each pourthru (Table 5.3). The low pH of the ADCM substrate was expected, as low pH is an indicator of decomposition that occurs under low aeration, whereas high pH is characteristic of well-aerated decomposition (Boulter et al. 2000; Hoitink et al. 1997). The EC of the ADCM substrate was also low compared to other substrates at first, second, and third pourthru, but the GB substrate had the lowest EC measured by pourthru throughout the experiment. The WC substrate had the highest EC at first pourthru and biggest difference in EC (a decrease of 5.5 mS-cm"1) from first pourthru to third pourthru (Table 5.4). Transplants growing in the ADCM substrate showed signs of nutrient deficiency (stunted growth and purple colouring of leaves) by day 38; this substrate had the lowest pH and EC measured by pourthru. Low EC directly points to a low nutrient content and low pH indicates a possible reduction in the availability of certain nutrients. Purple colouring of leaves is a sign of phosphorous deficiency, which delays early growth (Garton et al. 1994). It is 103 possible that phosphorous was a limiting nutrient in the ADCM substrate as it had a low phosphorous content (~20 ppm). However, low pH in soilless substrates is more likely to negatively affect the availability of calcium, and magnesium (Baley 2005; Figure 5.5). Water-soluble nitrogen content however was low in the ADCM compared to other composts (Table 5.5). Table 5.3 The pH of each substrate, taken initially on day 14 (prior to the addition of elemental sulfur), and subsequently on days 25, 32, and 36. Initial pH2 Pourthru pHy Substrate Day 14 Day 25 Day 32 Day 36 1(25VC/...)X 8.5 ±0.03w 5.9 ±0.03 5.7 ±0.03 6.0 ±0.02 2(25MC/...) 8.2 ±0.09 5.6 ±0.05 4.8 ±0.08 4.9 ±0.14 3(25WC/...) 8.5 ±0.02 6.2 ±0.19 5.3 ±0.07 5.7 ±0.09 4(25ADCM/...) 8.3 ±0.06 4.3 ±0.20 3.7 ±0.08 3.3 ±0.03 5(25MC+E/...) 8.5 ±0.01 5.6 ±0.07 4.8 ±0.07 5.1 ±0.03 6(25YWC/...) 8.4 ±0.04 5.9 ±0.03 5.3 ±0.11 5.4 ±0.01 GB(100CC) n/a n/a 5.7 ±0.27 5.6 ±0.17 Initial pH values were measured using the 1:5 method. yPourthru pH values were measured using the pourthru method. XVC = vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC + E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. wData are means of three replicates ±standard error. Table 5.4 Electrical conductivities (mS-cm"1) measured on days 25, 32, and 36. Substrate Day 25 Day 32 Day 36 1 (25VC/ 25CC/ 25FP/ 25FV)Z 5.7 ±0.68y 5.4 ±0.30 3.0 ±0.30 2(25MC/ 25CC/ 25FP/ 25FV) 5.9 ±0.32 6.1 ±0.54 3.9 ±0.33 3(25WC/ 25CC/ 25FP/ 25FV) 9.3 ±0.73 7.6 ±0.88 3.8 ±0.42 4(25ADCM/ 25CC/ 25FP/ 25FV) 3.3 ±0.17 3.4 ±0.06 2.3 ±0.13 5(25MC+E/ 25CC/ 25FP/ 25FV) 4.3 ±0.10 5.9 ±0.34 3.7 ±0.38 6 (25YWC/ 25CC/ 25FP/ 25FV) 4.1 ±0.38 5.0 ±0.54 2.8 ±0.09 GB(100CC) n/a 1.6 ±0.37 1.5 ±0.10 ZVC =vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC + E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. yValues were measured using the pourthru method; data are means of three replicates ±standard error. 104 Figure 5.5 Nutrient solubility as it is affected by pH change in a soilless growing substrate containing Sphagnum peat moss, pine bark compost, fine vermiculite, and sand. The pH range recommended for most greenhouse crops is highlighted. The width of the band relates to the availability of each nutrient (wider band = more nutrient availability). [This image was taken from The North Carolina State University website (www.ncsu.edu) under the title:Substrate pH and Water Quality, written by Douglas A. Bailey, Paul V. Nelson, and William C. Fonteno. The image was scanned from the image that was displayed online at: http://www.ces.ncsu.edu/depts/hort/floriculture/plugs/ph.pdf Copyright 2010 North Carolina State University. Published by NC Cooperative Extension, which is based at North Carolina's two land-grant institutions, NC State University and NC A&T State University, all rights reserved. 105 Nutrient contents of the different composts were variable. The WC had the most available nitrogen and the YWC had no available nitrogen. The MC +E had the most phosphorous followed by the MC and the WC. Potassium contents were also highest in the WC the MC and the MC+E. Magnesium and calcium contents were under 30 mg-L"1 in all composts. WC had the highest content of water- soluble chlorine, sulfate, and sodium. The ADCM and the YWC had the lowest total content of water-soluble nutrients and the growblock was not analyzed for nutrient content (Table 5.5). Table 5.5. Content of initial water-soluble nutrients (mg-L"1 suspension) in the processed organic wastes (in their received state) used to mix the substrates- 3 Processed organic waste NQ3" + NH/ PQ4 " K Mg Vermicompost 36±1.0Z 2 ±1.0 211 ±5.5 7 ±0.3 Manure compost 171 ±2.2 58 ±1.4 586 ±6.5 14 ±0.0 Worm castings 678 ±14.1 57 ±0.9 1149 ±28.0 17 ±0.4 Anaerobically digested cattle manure 28 ±0.7 21 ±0.7 52 ±0.6 12 ±0.3 Manure compost with eggshells 91 ±2.3 114 ±2.0 280 ±7.1 12 ±0.3 Yard waste compost 0±0.0 2 ±1.4 90 ±1.8 7 ±0.1 Processed organic waste Ca CX SQ4 Na Vermicompost 25 ±0.7 183 ±4.4 6 ±0.2 44 ±2.9 Manure compost 15 ±0.1 238 ±2.5 142 ±3.8 48 ±4.5 Wormcastings 29 ±0.5 627 ±14.3 155 ±1.7 211 ±13.9 Anaerobically digested cattle manure 16 ±0.2 15 ±0.3 0 ±0 17 ±1.3 Manure compost with eggshells 21 ±0.1 31 ±0.9 32 ±0.5 28 ±1.6 Yard waste compost 20 ±0.5 47 ±0.5 3 ±0.1 15 ±1.3 zNutrient concentrations were measured using the 1:5 method; data are means of three replicates ±standard error. Clark and Cavigelli (2005) tested different organic growing substrates made from two composts: 1) food residue mixed with yard waste compost and 2) straw horse bedding compost. Although the two composts had very similar 106 carbon to Nitrogen (C:N) ratios, total N contents and bulk densities, results were different for these two substrates. Substrate containing the compost made from food residue mixed with yard waste produced a comparable crop to that of the PM control. The substrate containing compost made from straw horse bedding however, produced lower quality crops. Growth reduction were attributed to net N immobilization in the straw horse bedding compost compared to that in the food residue mixed with yard waste compost, which showed net N mineralization. Similarly when an immature compost with high C:N is applied to soil, the high C levels cause the N to be immobilized by microbial biomass and also results in a reduction in organic N compared to organic C. This is why nitrogen deficiency is often observed when growing plants in immature compost with high C:N (Boulter et al. 2000). It is possible that the ADCM may have been immobilizing nitrogen. The ADCM seemed to contain a lot of animal bedding (straw); the colour or the ADCM was the colour of straw and the consistency was very fluffy (see Figure 5.1). Unfortunately we do not know the amount of straw used in the making of the ADCM so we can only speculate that the ADCM's C:N may have been high and causing nitrogen to be immobilized. However, the dramatic drop in pH of the ADCM substrate may have also been a cause of poor growth in this substrate. Compost pH is generally initially acidic (pH <6) due to the presence of organic acids that are formed during the early stages of decomposition (Day and Shaw 2001). As composting proceeds the pH reaches near neutral as these acids are converted to methane or carbon dioxide and released to the 107 atmosphere, thereby lowering the C:N (Day and Shaw 2001; Boulter et al. 2000). During decomposition, compost pH generally rises because of the decomposition of organic nitrogen (i.e. protein, amino acid, etc.), which releases ammonia (a weak base). The pH of the ADCM was initially quite alkaline (8.8). But once mixed, the pH of the ADCM substrate quickly dropped to below 4.0, which is an indicator that the ADCM may not have been stable. Most of the organic carbon in mature compost is in the form of humic substances. Humic substances are resistant to further degradation and have a high pH buffering capacity, which helps stabilize the pH of the finished compost (Hsu and Lo 1999). Reis et al. (1998) found that good transplant growth was possible in 100% pine bark compost when the composting process took 20 weeks compared to a grape marc compost only taking 16 weeks; these results support the fact that the maturity of a compost will have an effect on growth therefore, it is important that compost maturity is reached as a mature compost will help to avoid nutrient immobilization (Lopez et al. 1998). Although C:N ratio, N immobilization, and N mineralization was not measured, the low pH and EC observed for the ADCM substrate suggests that nutrient deficiencies caused poor transplant growth. The chlorophyll content index of transplants from the ADCM and MC+E substrates, at second harvest, were higher than the chlorophyll content index of transplants from the VC substrate (Figure 5.6). Raviv et al. (1998a) found that chlorophyll content was higher in substrates that contained compost compared to substrates that contained PM and FV. Low chlorophyll content in the leaves has been attributed to salt stress (Al-aghabary et al. 2005), nutrient deficiency or 108 toxicity (nitrogen, iron, or magnesium; Wilcox 1993), and extreme pH and EC, as they relate to nutrient availability (Smith et al. 2004). The optimal pH range for healthy plant growth is dependent on fertilizer input; higher fertilizer inputs are required at higher pH (>6.3) to avoid chlorosis (Smith et al. 2004). However, transplants growing in the VC substrate did not show signs of nutrient deficiency and chlorophyll content index was not significantly different among all substrates at the third harvest. If elevated pH and nutrient deficiency was the cause of lower chlorophyll content in transplants from the VC substrate at second harvest, these deficiencies were corrected in time for the third harvest. Interestingly, while nutrient deficiencies were observed in transplants from the ADCM substrate, the chlorophyll content in the leaves of these transplants did not seem to be affected. Harvest 1 (day 28) Harvest 2 (day 38) Harvest 3 (day 46) 20- is 16- 20- le 14- 18- s' 12- 16- 0- -T- -T- -T- 10' -T- A .V* X!> .<* .V?* A? ,* 0* A .v; .v.- .^ .v.- .v A v ^ .^ .^ .^ .^ .A^ ^ y-i ,-i ®y- ^i j-.si ,A.^ ,-.^i V^& ^ -« -^ -^ -^ •<* - J* J* ,/ A* J* A ^ * / ^ «/ Figure 5.6 Chlorophyll content index at first second and third harvest. Data are means of four replicates ±standard error. VC =vermicompost, CC = coconut coir, FP = fine perlite, FV = fine vermiculite, MC = manure compost, WC = worm castings, ADCM = anaerobically digested cattle manure, MC + E = manure compost with eggshells, YWC = yard waste compost, GB = growblock. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P<0.05. 109 Compared to other substrates in the experiment, it was the WC substrate that produced transplants that were most often not significantly different from the transplants growing in the GB substrate, which was an all-CC-based substrate that did not contain any compost (see Figures 5.2 and 5.3). In addition, transplants growing in the VC, MC, MC+E and YWC substrates were often not significantly different from transplants growing in the WC substrate. As in our study, Diaz-Perez et al. (2006) found organic substrates containing compost included at less than 50% were also successful at producing tomato transplants of equal quality to comparison substrates that did not contain compost. Raviv et al. (1998a) also tested substrates with and without compost for organic lettuce seedling production and found that seedlings growing in compost-based substrates were often heavier and taller than seedlings grown in non-compost- based substrates. 5.4 Conclusion Overall, the WC substrate, which contained the most plant-available nitrogen, produced the best transplants. The VC, MC, MC+E and YWC substrates also produced transplants of similar quality to those from the WC substrate. Only the ADCM substrate was significantly inadequate for tomato transplant production. Poor growth in the ADCM substrate was attributed to low nutrient content (low EC), low pH, and possible N deficiency due to nitrogen immobilization. 110 CHAPTER 6: Rate of Inclusion of Manure Compost in Organic Growing Substrates for Tomato Transplant Production 6.1 Introduction Research for sustainable systems, (i.e. organic systems) is needed so that sustainable production methods may be commonplace alternatives to conventional practice. As a natural carbon sink, peat moss (PM) is harvested from precious ecosystems; this is considered a non-sustainable practice (Pill and Ridley 1998). PM also has a low initial plant nutrient content, and can be conducive to plant diseases (Raviv et al. 1998b; Hoitink and Fahy 1986). Alternatives to PM include (not exclusively) a variety of industry wastes, such as coconut coir (CC) and wood fibers, and a variety of composted wastes, such as food waste compost, yard waste compost, and animal manure compost (Guitierrez-Miceli et al. 2007; Gruda and Schnitzler 2004; Yogev et al. 2006; Pill and Ridley 1998; Lamanna et al. 1991). Compost, not only contributes to fertility, but also structure, porosity, organic matter, water retention, cation exchange capacity, pH buffering, and disease suppression (Boulter et al. 2000; Hsu and Lo 1999). Composting is the breakdown of organic matter by microorganisms. It is an environmentally friendly, biological, and aerobic process that ultimately converts a waste product into a more stable product that can be more useful, and affordable than PM (Boulter et al. 2000; Cheuk et al. 2003); therefore, compost is a good alternative for PM. The suitability of compost for use in growing substrates is based mainly on effects of water retention and aeration (Hoitink and Fahy 1986); however, high 111 salt content and lack of uniformity in composts are still problems that need to be overcome (Bugbee 2002; Ozores-Hampton et al. 1999). Typically, less than half the volume (i.e. 20 to 30%) of a growing substrate should be comprised of compost (Larrea 2005); however, due to wide variation in type and quality, studies evaluating the use of compost in substrates for tomato production have found differing results. Animal manure compost at inclusion rates of 33.3% in soil substrates (de Brito Alvarez et al. 1995) and 20 to 40% in soilless substrates (Termorshuizen et al. 2006; Nielson and Thorup-Kristensen 2004), and animal manure vermicompost at inclusion rates of 25 to 50% in soil substrates (Gutierrez-Miceli et al. 2007) and 8 to 40% in soilless substrates (Diaz-Perez er al. 2006; Larrea 2005; Atiyeh et al. 2000; Subler et al. 1998; Wilson and Carlile 1989) have been recommended for tomato production to increase yields, reduce the incidence of disease, or improve growth. More than 25 organic growing substrates were evaluated for tomato transplant production and equal parts by volume (v/v) of CC or PM, manure compost (MC), fine vermiculite (FV), and fine perlite (FP) produced better transplants than other substrates studied. The substrate combination including CC was evaluated in this experiment because of the environmental obstacles associated to the use of PM. In this experiment, we evaluate different inclusion rates of MC in the chosen organic growing substrate; CC was therefore replaced in part with MC so that the percent inclusion rate of MC was 25, 35, 45, or 50% v/v (Table 6.1). The aim of this experiment was to find the optimal rate of inclusion of MC for organic tomato transplant production. 112 Table 6.1 Initial pH, electrical conductivity (EC; pS-cm"1), and compacted bulk density (CBD; g-L"1) of the individual materials (in their received state) used. Substrate compositions are also presented in this table. Values for substrate compositions are the percent rate of each material included on a volume basis based on the CBD of each material used. Material pHz EC CBD Manure compost 8.6±0.01y 1890 ±7.7 638 ±6.0 Coconut coir 6.7 ±0.11 170 ±0.2 185 ±1.2 Fine perlite 8.2 ±0.29 24 ±1.4 122 ±2.0 Fine vermiculite 7.8 ±0.24 41 ±0.6 125 ±0.3 Substrate compositions Material 1x 2 3 4 Manure compost 25 35 45 50 Coconut coir 25 15 5 0 Fine perlite 25 25 25 25 Fine vermiculite 25 25 25 25 zValues for pH and EC were measured using the 1:5 method. yData for pH, EG, and CBD are means of three replicates ±standard error. xOptimum substrate combination previously evaluated. 6.2 Materials and Methods 6.2.1 Substrate Preparation Materials that were used in the fourth experiment (chapter 5) were used in this experiment. See section 5.2.1 Substrate Preparation for source details of MC, CC, PM, FP, FV, and superfine vermiculite. The compacted bulk densities (CBDs) of these individual materials were determined (see section 6.2.5 Physical Properties of the Substrates below for details); according to the CBD of each material, substrates were mixed v/v and amended with Spanish River Carbonatite™ at 0.56 g-L"1 (see section 4.2.1 Substrate Preparation for more information). The pH of each substrate was determined as in the first experiment (described in section 2.2.2 Chemical Properties of the Substrates) and elemental sulfur (Canadian HydroGardens Ltd., Ancaster, ON, CA) was added to each substrate depending on the initial pH measured, at rates of 1.4 g-L"1 (substrates 113 with 35 and 45% MC), 1.5 g-L"1 (substrate with 50% MC), or 1.6 g-L"1 (substrate with 25% MC), following recommendations from the University of Missouri (MU) (MU Extension 2008) to lower the pH to 5.7 within the optimal range (5.5 to 6.8) for tomato transplant growth (Vavrina 2002). Substrates were stored in open plastic bags for three weeks prior to being used in transplant growth experiments. Growblock (GB substrate), which was made of 100% CC substrate (850 cm3) and proposed for organic certification (Jiffy Products N.B. Ltd. Shippegan, NB, Canada), was used as the comparison substrate. Seedling substrate (75PM/ 25FV) was prepared and used for plug production (see more details on preparation of this substrate in section 4.2.1 Substrate Preparation). 6.2.2 Growth Experiment The same methods, as in the fourth experiment (chapter 5), to produce seedlings and transplants were used. Transplants were placed in a greenhouse in RCBD with four replications for each of three harvests. Transplants were harvested on days 28, 38, and 46 for final growth measurements (see section 6.2.4 Growth measurements and Leaf Chlorophyll Content Index below). Transplants were irrigated and fertigated the same as in the fourth experiment (see section 5.2.2 Growth Experiment). 6.2.3 Chemical Properties of the Substrates The initial pH and EC of the individual materials were determined on day 14 (Table 6.1) following the 1:5 method. The 1:5 suspension extracts were analyzed for nutrient content using ion chromatography. The initial pH of each substrate was determined, same way as for individual materials, using the 1:5 114 method (refer to section 2.2.2 Chemical Properties of the Substrates for further details on the 1:5 method, and the nutrient content analysis system used). During the production phase, the pH and EC of the substrates were measured using the pourthru method (Cavins et al. 2000) on days 25, 32, and 36 (refer to section 3.2.2 Chemical Properties of the Substrates for further details on the pourthru method using 10-cm pots). A portable Oakton pH/ Con 300 Meter (Oakton Instruments, Vernon Hills, II, United States) was used to record the pH and EC of the 1:5 suspension and pourthru leachate. 6.2.4 Growth Measurements and Leaf Chlorophyll Content Index Stem diameters, stem heights, leaf numbers, leaf areas, shoot fresh and dry weights, and root fresh and dry weights were measured as described in section 5.2.4 Growth Measurements. Leaf chlorophyll content index was measured as described in section 5.2.5 Leaf Chlorophyll Content Index. 6.2.5 Physical Properties of the Substrates The CBDs of the individual materials and the substrate combination were determined following the procedure described in the Centre Europeen de Normalisation (CEN) standard manual for determination of laboratory CBD (EN 13040 Annex A) of substrates (ISHS 2003). Oven-dry weight bulk density (BD), total porosity (TP), water-holding capacity at container capacity (WHC), and air- filled porosity at container capacity (AFP), of each growing substrate were determined using the North Carolina State University (NCSU) porometer (Figure 6.1) in accordance with guidelines published in the "Horticultural Substrates Porometer Manual" (Fonteno and Harden 2003) and other details (Fonteno 115 1996). Determination of the physical properties mentioned above was conducted on substrates having a mass wetness (g water/ g substrate) of 1.32 [substrate 1 (25% MC)], 1.2 [substrate 2 (35% MC)], and 1.1 [substrate 3 (45% MC) and substrate 4 (50% MC)]. Figure 6.1 Demonstration of the North Carolina State University porometer. A substrate is placed in the metal cylinder [Dimensions (cm) = 7.6 (top diameter), 7.6 (cylinder height), 10 (base plate diameter); Volume = 347.5 cm3], which is then placed in the white funnel. Water is added to the funnel while the cylinder base plate is in the open position. Once the substrate is fully saturated, the base plate is turned in the closed position and water surrounding the cylinder is drained and discarded. The base plate is then turned in the open position and the water is drained, collected, and measured in a graduated cylinder. The saturated substrate still in the cylinder is weighed, then dried at 105 °C, and weighed again. Water-holding capacity at container capacity (WHC) is defined as (wet weight - dry weight) + 347.5, air-filled porosity at container capacity (AFP) is the volume of water drained from the cylinder + 347.5, and total porosity (TP) is WHC + AFP. Images were recreated from images on the North Carolina State University website (www.ncsu.edu), North Carolina State University Horticultural Substrates Lab, NCSU Porometer Information, maintained by Bill Fonteno. Available online at: http://www.ncsu.edu/project/hortsublab/diagnostic/porometer/index2.html Copyright 2010 North Carolina State University. Published by NC Cooperative Extension, which is based at North Carolina's two land-grant institutions, NC State University and NC A&T State University, all rights reserved. 116 6.2.6 Statistical Analysis Data were tested for normality and homogeneity of variance. Any outliers were removed using Lund's table for an approximate test for outliers (Lund 1975). The data set was subjected to analysis of variance using the General Linear Models procedures in SAS version 9.1 (SAS Institute; Cary, NC, 2003) and a multiple means comparison test was conducted [Tukey-Kramer; P <0.05; n = 4 (transplant growth measurements and chlorophyll content index); n = 3 (substrate physical properties analysis)]. The same statistical tests were conducted excluding the data for the control substrate to see if there were differences among the experimental substrates. Nonlinear regression model was calculated to find the relationship between shoot dry weight and percent MC, at second and third harvest, using Prism 5 for Mac OS X, Version 5.0a, 2007. 6.3 Results and Discussion A good transplant is green, straight, sturdy, and between 12 and 17 cm tall and wide; it has a well-established root system, and is free of pests (Peet and Welles 2005; Garton er al. 1994; Vavrina 2002). The GB substrate produced the largest, leafiest, and heaviest transplants with the lowest rootshoot ratios. As was explained in Chapter 5, this is because the volume of the GB substrate was double that of the other substrates, and increasing substrate volume is correlated with increasing plant growth (Kemble et al. 1994). For this reason, it was not scientifically sound to compare transplants from the formulated substrates in 10- cm pots to transplants from the GB substrate in 10-cm fabric blocks, because the transplants from the GB substrate had an obvious growth advantage due to a 117 larger rooting zone. The purpose of including the GB substrate as a comparison substrate was therefore for practical reasons. The GB substrate is a commercially available substrate that is placed in block-shaped fabric by the manufacturer. It is recommended as an alternative to rockwool blocks for transplant production. We therefore included the GB substrate as a comparison knowing that transplants grown in this substrate would have an advantage over transplants grown in the formulated substrates. The objective was therefore to see if the formulated substrates could produce transplants of equal quality to transplants from the GB substrate despite the obvious advantage the transplants grown in the GB substrate would have. All in all, transplants grown in formulated substrates were comparable to transplants grown in the GB substrate for some of the growth parameters measures at first, second, and third harvest. However, in order to determine treatment effects among formulated substrates independent of data recorded for transplants from the GB substrate, statistical analysis was also conducted omitting data from the GB substrate. At first harvest, no difference in number of leaves, stem height, or stem diameter were observed; however, when data from the GB substrate was omitted from the statistical analysis, transplants from the substrate that contained 35% MC had thicker stems than transplants from the 50% MC substrate. At second harvest, transplants from the 45% MC and the GB substrate had the most leaves, transplants from the GB substrate were taller than all other transplants, and had thicker stems than stems of transplants from the 25, 35, and 50% MC substrates. At third harvest transplants from the GB substrate had thicker stems 118 and were taller than all other transplants. When data from the GB substrate was omitted from statistical analysis, transplants from the 35% MC substrate were taller and thicker than those from the 25% MC substrate and stems of transplants from the 45% MC substrate were thicker than those from the 25% MC substrate (Figure 6.2). Harvest 1 (day 28) Harvest 2 (day 38) Harvest 3 (day 46) 4- 8- ID A a A A a / B A 3 3 !3 0,B A a A a a ft V) a ab B a S' CD 3- i b t > a 6- b A 6 fi CO 1 1 \ 6- o 2- 4- CD Xl 4- E • Z3 1- 2- Z 2- iiti 6- A 15- 25- a A A 20- A AB AB i a 3 o : a 4- 10- * b b * B b b h F| | . 15- t> h a „ 0) A jr : 10- E 2- 5- M • (/) 5- • 1 • i i 1 1 1 1 1 t 6- 2 0- 3 8- A a AB R A A a B A AB a A a b A a B i b b b A L a ' h h h u 1E", 1 5- , 6- , 4- h A 1 CU E 1 0- 4- CO •D 2- ; E ; CD 0 5- 2- 55 uu J 1 1 1 n* A* >n* G° / / / / 4? J/ /* c/ /*/ ^ / -/ J J J #J £•/ $>/ ^ / A> A° ^ ^ / Figure 6.2 Growth responses at first, second, and third harvest. MC = manure compost, CC = coconut coir, FP = fine perlite, VF = fine vermiculite, GB = growblock. Data are means of four replicates ±standard error. Data bearing the same lowercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when all data was analyzed. Data bearing the same uppercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when the GB data was omitted from the analysis. 119 Zaller (2007) showed that tomato variety is an important factor to consider when choosing the proportion of vermicompost (VC) to add to a growing substrate. Three tomato varieties at seedling stage in commercial PM-based growing substrates amended with different rates of VC (0, 20, 40, 60, 80, and 100% v/v) were studied and results showed that seedling heights of one variety were lowest in substrates containing between 20 and 60% VC, whereas overall maximum growth was achieved with -20% VC in another variety of tomato, and growth steadily increased with increasing proportions of VC in the other variety. The leaf area of transplants from the 35% MC substrate was higher than the leaf area of transplants from the 50% MC substrate at first harvest and when data from the GB substrate was omitted from analysis, the leaf area of transplants from the 35% MC substrate were higher than those of transplants from the 45% MC substrate. At second harvest, the leaf area of transplants from the GB substrate was higher than the leaf area of transplants from the 25, 35, and 50% MC substrates. At third harvest the leaf area of transplants from the GB substrate was higher than the leaf areas of all other transplants (Figure 6.3). 120 Harvest 1 (day 28) Harvest 2 (day 38) Harvest 3 (day 46) A 20-1 a 200-1 600-1 a AB a ab ~T 15- ab B o 150- A A A A A a b 400- * b b b I b A ab A b b I 100- 1 n fare a (er r ft i 1 S 1 A afl 8 i 200- 3 5- - I ,^ ^ Figure 6.3 Leaf area at first, second, and third harvest. MC = manure compost, CC = coconut coir, FP = fine perlite, VF = fine vermiculite, GB = growblock. Data are means of four replicates ±standard error. Data bearing the same lowercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when all data was analyzed. Data bearing the same uppercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when the GB data was omitted from the analysis. In terms of chlorophyll content, at second harvest (when data for transplants from the GB substrate was included and omitted) transplants from the 50% MC substrate had higher chlorophyll content indexes than transplants from the 35% MC substrate. However, there were no significant differences in chlorophyll content at first or at third harvest (Figure 6.4). 121 Harvest 1 (day 28) Harvest 2 (day 38) Harvest 3 (day 46) A AA a 20n 25- A 2°1 A A A A CD A a a A "O , a a ^ B AB C ab . ab ab 20- ^ 15- 15 Si j, i a El " c h 1 a i * 15- ; i i i 8 10- 10- x; 10- a. 2 5- 5- o 5- JZ O 0^ rt ,^ ^ ^ ^ ^ ^ ^ ^ ^ # ^ J? ^ ^ ^ ,4 ,4 ,4 ,4 & ,4 J? ,4 <4 & ,4 ,4 ,4 4 & J> J? J> J> co# J" J> J? J> J> J" J? / / / Figure 6.4 Chlorophyll content index at first, second and third harvest. MC = manure compost, CC = coconut coir, FP = fine perlite, VF = fine vermiculite, GB = growblock. Data are means of four replicates tstandard error; data bearing the same lowercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when all data was analyzed. Data bearing the same uppercase letter are not significantly different by the Tukey-Kramer test at P <0.05 when the GB data was omitted from the analysis. At second and third harvest there were no significant differences among treatments for root dry weights. Shoot dry weights, however, for transplants from the GB substrate, at second harvest, were heavier than those of the 25, 35 and 50% MC substrates, and shoot dry weights of transplants from the GB substrate, at third harvest, were heavier than transplants from the 25, 45, 50% MC substrates (Figure 6.5). Similar patterns were observed for fresh weights (data not shown). 122 Harvest 2 (day 38) Harvest 3 (day 46) 400- 1500-1 o> a §• 300- A Jf A A ab A (I 1000- A A A CD b b T b ab 1 3 200- i n 1 A i 1 1 I 8 ioo- :| i 500- \ SZ ;l \ CO i \ r\- " 1 1 1 1 1 u | | i | | A 40-1 . A A 300-i a A A a a M . a A CD a T T | a A -§. 30- H A 0 H 200- A a cn CD 3 20- J. "O 1 |: 100- 8 1°- ;i cc 1 n_ 1 u 1 1 1 II A g 0.20- A A A a 0.4- 2 ab ab ab T * A h a A A t, 0.3- h r a ab b 1 o 0.2- I | I t root : shoo n ; \ cn ;I 1 '5 0.05- ;! 0.1- i S { \ £• : : Q ^™ |: :• ^ /" ^ ^ Gc/ ^ ^ /" 4^ J? J? co J? J? jf J> c/ c/ jf J? Figure 6.5 Root and shoot dry weight and rootshoot ratio at second and third harvest. MC = manure compost, CC = coconut coir, FP = fine perlite, VF = fine vermiculite, GB = growblock. Data are means of four replicates ±standard error. Data bearing the same lowercase letter are not significantly different by the Tukey-Kramer test at P <0.05. When data from the GB substrate was omitted from statistical analysis of shoot and root dry weights, there were no differences among substrates 123 containing different rates of compost. Diaz-Perez et al. (2006) investigated organic substrates at different inclusion rates of compost (10, 20, 30 40, and 50%) for tomato transplant production and also found that shoot and root dry weights were not affected by the different inclusion rates of compost. However, when tomato transplant growth responses were evaluated using different tomato varieties (Zailer 2007), shoot dry weights for two varieties growing in plug cells were significantly affected by the proportion of VC added to the PM-based growing substrates. Shoot dry weights of seedlings growing in substrates with the most VC (80 and 100%) were lower, which is similar to our results. Shoot dry weights of these two varieties and another variety of tomato growing in 11-cm pots, however, were not affected by different proportions of VC added to PM- based growing substrates nor were shoot dry weights of these plants when grown in a field. In addition, root dry weights were affected by the different proportions of VC that were included in the PM-based growing substrate, but only for one tomato variety, which was growing in 11-cm pots, and only when this variety was statistically analyzed individually. The root dry weights of that variety were 30% lower in growing substrates containing 100% VC; these results are also similar to ours (Zailer 2007). When included in statistical analysis, transplants from the GB substrate exhibited significantly lower fresh and dry weight rootshoot ratios than the substrates including different rates of compost. Rootshoot ratios for transplants from the GB substrates were 0.16 and 0.11 for fresh and dry at second harvest and 0.20 and 0.17 for fresh and dry at third harvest, respectively (Figure 6.5). 124 Dry weight root:shoot ratios for the GB substrate were 47% lower than those of substrate 4 (50% MC) at second harvest and >35% lower than those of the 45, 35, and 25% MC substrates, at third harvest. No significant differences in fresh weight and dry weight root:shoot ratios were noticed among substrates including different rates of compost when data for transplants from the GB substrate was omitted from statistical analysis. Diaz-Perez et al. (2006) also found no difference in dry weight root:shoot ratios among substrates with different inclusion rates of compost. The dry root:shoot ratios calculated in the latter study were between 0.20 and 0.25 whereas the dry root:shoot ratios of transplants from the compost amended substrates in this study ranged from 0.14 (45% MC) at second harvest to 0.27 (25% MC) at third harvest. Individual analysis of different tomato varieties, in Zaller's (2007) experiment, revealed that root:shoot ratio was significantly affected by different proportions of VC in PM-based growing substrates for seedlings in plug cells and field plants of one variety and for seedlings in plug cells of another variety. Shifts in biomass allocation from shoots to roots (resulting in higher root:shoot ratio) occurred when seedlings were growing in substrates containing >40% VC (Zailer 2007). We found similar results at second harvest but the exact opposite trend at third harvest (Figure 6.5). Transplant performance was not further evaluated over longer periods but it has been shown that fruit quality of tomatoes may be altered by the growing substrate used to grow the transplants and that tomato variety is an important factor to take into consideration when choosing the proportion of compost to add to a growing substrate. The quality of transplant growing substrate may also have 125 implications for pest and disease resistance as well as for the susceptibility of adult plants to abiotic stress after transplanting (Zailer 2007). Nonetheless substrates are primarily meant to provide physical support for plants, to hold water and nutrients, and to allow gas exchange to occur in the rhizosphere (Strojny and Nowak 2004; Heiskanen 1997). The physical properties of growing substrates (i.e. BD, TP, WHC, AFP) are very important when choosing a growing substrate. Very heavy substrates reduce growth and development of tomato (Wilcox 1993), and are a hassle for substrate handling and shipping, and increase worker fatigue (Tripepi 1997; Fonteno 1996); however, substrates with extremely low BDs are not recommended because of loss of physical support (Bunt 1983). The CBDs of the individual materials in their received state are presented in section 6.2.1 Substrate Preparation (see Table 6.1); and for the mixed substrate combinations, CBDs were: 331 ±11.3 g-L"1 SE (25% MC), 399 ±3.9 g-L1 SE (35% MC), 493 ±2.3 g-L"1 SE (45% MC), and 499 ±9.6 g-L1 SE (50% MC). A statistical test was not conducted on CBDs but these measurements followed the same pattern as for the BDs calculated using the NCSU porometer. The BDs were significantly different (Table 6.2); however, all substrates used in this study provided adequate physical support for tomato transplant growth. 126 Table 6.2 Physical properties of substrates. 117 „ , , , z Tota•wiail Water-holdinWater-holdinag Air-filleAir-filled wetaht...... Substrate ., V/0/y N ., /0/N .. ,0/N weight bulk porosity (%) capacity (%) porosity (%) g , -ix1 dendensitit y (g-L" L ) 1(25% MC)X 83.7±0.07w 63.5 ±2.41 20.3 ±2.40 225 ±3.5c 2(35%MC) 84.5 ±0.66 68.1 ±0.42 16.4 ±0.50 253 ±2.7b 3(45%MC) 85.1 ±0.56 68.4 ±0.87 16.7 ±1.42 292 ±6.0a 4(50%MC) 83.8 ±0.06 68.9 ±0.38 15.0 ±0.44 304 ±2.0a zEach number represents a substrate (see Table 6.1 for substrate compositions). yTotal porosity, water-holding capacity at container capacity, air-filled porosity at container capacity, and dry weight bulk density were measured using the porometer. XMC = manure compost. wData are means of three replicates ±standard error. Data followed by the same letter are not significantly different by the Tukey-Kramer test at P <0.05. In horticultural systems, substrates with lower BDs are also preferred over heavier substrates because light substrates tend to have higher TP, WHC, and AFP, which are beneficial to root growth and the development of plants (Strojny and Nowak 2004). Oxygen availability to the roots is important for root respiration, water uptake, and nutrient uptake, especially in substrates containing organic matter from composted material, because roots compete with the aerobic microorganism in compost for oxygen (Raviv et al. 2004a). Strojny and Nowak (2004) found that substrates that were better suited to plant growth had the lowest BDs (-100 g-L"1), the highest TP, and more AFP at container capacity than the other substrates being studied. Similarly, Bunt (1983) found that TP was inversely related to BD with no interaction found between the particle size distribution of the substrate and TP; however, it is generally known that increasing particle size in a growing substrate can increase root aeration because larger air pockets are formed around larger particles. In this study, the substrate containing the least amount of MC (i.e. lowest BD) did not provide 127 significantly more TP than the other substrate combinations. The lighter substrate, which contained 25% MC, did have slightly lower WHC and higher AFP than the other substrates but this difference was not statistically significant (Table 6.2). Fonteno and Bilderback (1993) found similar results when incorporating increasing amounts of polyacrylamide hydrogel to pine bark and pine bark/ sand substrates. Optimum TP of 85% has been recommended in the past (de Boodt and Verdonck 1972). But, more recently it has been reported that organically based growing substrates have between 75 and 85% TP (Fonteno 1996). All substrates studied in the fifth experiment had TPs within the range given by Fonteno (1996). Oxygen availability to the roots is important for water and nutrient uptake and root respiration, especially in substrates containing aerobic microorganisms (Raviv et al. 2004a). Heiskanen (1997) reviewed that AFP at container capacity above 10% of the total volume of the substrate is commonly considered to provide sufficient aeration for plants in mineral soils but that in PM mixtures, this value can be >40%. de Boodt and Verdonck (1972) recommended that AFP at container capacity should be above 10 to 20% and Raviv and Heinrich Leith (2008) suggested it should be within the range of 10 to 30%. Substrates studied here had AFPs between 15 and 20% (Table 6.2). The pH values for the individual materials are presented in Table 6.1 and the initial pH values of the substrates are presented in Table 6.3 along with the pH of the substrates measured by pourthru, 25, 32, and 36 days after seeding. The pH of the substrates generally increased with increasing rates of MC. This 128 was expected as MC has a pH of 8.6. Table 6.3 The pH of each substrate, on days 14 (prior to the addition of elemental sulfur), 25, 32, and 36. Initial pHz Pourthru pHy Substrate Day 14 Day 25 Day 32 Day 36 1(25%MC)X 8.2±0.09w 5.6 ±0.05 4.8 ±0.08 4.9 ±0.14 2(35%MC) 8.3 ±0.01 5.9 ±0.05 5.3 ±0.10 5.4 ±0.01 3(45%MC) 8.3 ±0.01 6.1 ±0.07 5.6 ±0.06 5.6 ±0.02 4(50%MC) 8.4 ±0.02 5.8 ±0.31 5.5 ±0.08 5.9 ±0.05 Growblock n/a n/a 5.7 ±0.27 5.6 ±0.17 initial pH values were measured using the 1:5 method (see section 2.2.2 Chemical Properties of the Substrates for more details on the 1:5 method). yPourthru pH values were measured using the pourthru method. XMC = manure compost. wData are means of three replicates ±standard error. The EC of substrates measured by pourthru also increased with increasing rates of MC (Table 6.4). At first pourthru (day 25) EC readings of substrates containing MC at different rates ranged from 5.9 to 8.7; at second pourthru (day 32) EC ranged from 6.1 to 7.3; at third pourthru (day 36) EC ranged from 3.9 to 4.6; however, the EC of the GB substrate were consistently lower (EC 1.5 to 1.6). The increase in EC as the rate of MC increased was expected; the content of water-soluble nutrients (mg N-L suspension extract"1) of + 3 the MC was 171 ±2.2 N (N03"+NH4 ), 58 ±1.4 P04 ", 586 ±6.5 K, 14 ±0.0 Mg, 15 ±0.1 Ca, 238 ±2.5 CI", 142 ±3.8 S04, and 48 ±4.5 Na. 129 Table 6.4 Electrical conductivities (mS-cm"1) measured on days 25, 32, and 36. Substrate Day 25 Day 32 Day 36 1 (25% MC)Z 5.9 ±0.32y 6.1 ±0.54 3.9 ±0.33 2 (35% MC) 6.8 ±0.35 6.2 ±0.24 4.1 ±0.03 3 (45% MC) 7.0 ±0.68 6.4 ±0.35 4.6 ±0.40 4 (50% MC) 8.7 ±0.84 7.3 ±1.05 4.5 ±0.49 Growblock n/a 1.6 ±0.37 1.5 ±0.10 ZMC = manure compost. yValues were measured using the pourthru method; Data are means of three replicates ±standard error. 6.4 Conclusion Overall, transplants growing in substrates containing MC at inclusion rates of 35 and 45% were among the best in terms of stem height, stem diameter, number of leaves, leaf area, root and shoot fresh and dry weights, and dry rootshoot ratios. We recommended that no more than 45% MC be used to grow tomato transplants based on growth responses evaluated in this experiment. The substrate with the lowest amount of composted manure (25%) had a significantly lower BD, but there were no significant differences in TP, WHC, or AFP when the inclusion rate of MC was increased from 25 to 50%. Therefore, incorporating MC at a higher rate than 25% and less than 45%, may provide better transplant growth performance without significantly compromising the root zone environment for AFP. 130 CHAPTER 8: Using Compost and Clonostachys rosea Inoculant to Suppress Pythium ultimum in Tomato Transplants 8.1 Introduction Suppression of plant disease with compost such that disease development is limited even though the host is susceptible and a virulent pathogen is present, has been primarily attributed to biological factors. Many microorganisms derived from organic sources have been recognized as suppressive agents against the development of plant- diseases (Boulter et al. 2002c; van Bruggen and Semenov 2000; Szczech 1999). Increased microbial activity is a principal benefit associated with the use of composts and other organic soil and substrate amendments in horticulture and agriculture. Elevated microbial activity has been linked to disease suppression, which has an ecological basis and is important in organic crop production. Furthermore increased microbial activity is associated with higher nutrient turnover rates. Organic production is often considered healthier for humans compared to conventional production systems because elevated microbial activity enables the reduction in use of chemical pesticides and fertilizers (van Bruggen and Semenov 2000; Drinkwater et al. 1995; Chen et al. 1992). Suppression of plant diseases by means of composts often takes effect only after considerable time (e.g. weeks or months) after the compost is applied (Escuadra and Amemiya 2008). The microbial activity in compost also may only suppress certain pathogens that affect a particular crop (Zachow et al. 2008) or may only partially control the principal pathogen that is present. In some 148 instances, microorganisms present in composts simply fail to effectively suppress disease at all. Despite these limitations, it is important that attempts are made to develop effective disease control methods that are compatible with the criteria of organic production systems. While it is known that microorganisms that occur naturally in composts may protect plants against disease, a better understanding of the microorganisms in relation to disease suppression is needed as a basis for developing use protocols of composts in disease management. Benefits of inoculating growing substrates with beneficial microorganisms produced in artificial culture alone or in combination with composts have been explored (Trillas et al. 2006; Hjeljord and Tronsmo 1998). For example, compost from agricultural waste combined with inoculum of Trichoderma asperellum suppressed Rhizoctonia solani in cucumber seedlings (Trillas et al. 2006). In addition, there are numerous other reports of the use of Trichoderma spp. and other beneficial fungi and bacteria as biocontrol agents in the root zones of plants (Abbasi et al. 2007; Hoitink et al. 2006; Huang et al. 2006; MinFu er al. 2006). Clonostachys rosea is an endophytic fungus that is known to improve the acquisition of root zone micronutrients by plants and increase plant resistance to stress and disease (Sutton et al. 2008). C. rosea was successfully used to control Pythium tracheiphilum in Chinese cabbage under field conditions (Moller et al. 2003) and to protect deleafing wounds on tomato stems against stem cankers caused by Botrytis cinerea (Sutton et al. 2002). Circumstantial evidence also suggests that the fungus influences levels of plant hormone activity. Applied as seed treatments, C. rosea was found to increase emergence rates and growth 149 of seedlings of a wide range of crop plants (e.g. peas, lentils, soybeans, wheat, barley, and tall fescue) under growth room conditions (Personal Communication with Dr. J.C. Sutton, University of Guelph, Guelph, ON, Canada). Composts have been shown to suppress diseases caused by several Pythium spp. in many different crops. Several types of compost were shown to effectively suppress root disease caused by P. irregular, P. ultimum and P. aphanidermatum in cucumber (Scheuerell et al. 2005; Dianez et al. 2005). Numerous other reports that composts suppressed Pythium diseases have been published. For instance, mortality rates of cabbage transplants caused by P. aphanidermatum were lower when the transplants were produced in growing substrates amended with compost compared to commercial peat and vermiculite substrates (Raviv et al. 1998b). Composts prepared from sugarcane factory residues also suppressed P. aphanidermatum in climatic chamber experiments (Theodore and Toribio 1995). Compost was also used to suppress P. graminicola in creeping bentgrass (Craft and Nelson 1996) and microbial communities of leaf compost played a major role in the suppression of sporangial germination of P. ultimum on cottonseeds (McKellar and Nelson 2003). Other composts suppressed P. ultimum in field tomatoes (de Brito Alvarez et al. 1995) wheat, and cucumbers (Chen and Nelson 2008). Many Pythium spp. are able to infect wide diversities of plant species. Species of Pythium frequently are the most important pathogens of seedlings prior to and after emergence. Some seedlings can survive post emergence infection by Pythium spp. but in many instances exhibit reduced vigor and poor 150 growth. Mature plants that have been infected by Pythium spp. also may continue to grow but yield poorly (Martin and Loper 1999). In view of the continued major importance of Pythium spp. as destructive pathogens in horticultural crops, further research is justified to explore practical means for their control. Pythium root rot of tomato is an important disease of greenhouse tomato leading to significant losses in greenhouses where transplants are produced. In Canada the main causal agents of Pythium diseases in tomato transplants are P. aphanidermatum and P. ultimum (Gravel et al. 2005). Given that C. rosea is able to enhance the growth of numerous kinds of plants, and that this fungus and various composts are able to suppress Pythium root rot, it is plausible that combinations of composts and C. rosea may act synergistically to improve the health and growth of tomato transplants. No reports have been made as to whether C. rosea applied alone or in combination with compost can improve the vigor of tomato transplants or suppress Pythium root rot in tomato transplants. The working hypothesis of the present work is that C. rosea and compost applied in combination will act synergistically to improve tomato transplant growth and to suppress root rot cause by P. ultimum. The objectives were to determine: 1) whether the microbial community in a manure compost (MC) is able to suppress P. ultimum in the roots of tomato transplants when applied as an amendment to the growing substrate; and 2) whether inoculation with C. rosea in the compost-amended substrate affects transplant growth directly or interactively with any effects on disease suppression by the compost. Substrate was sterilized or not by autoclaving to test this hypothesis. 151 8.2 Materials and Methods 8.2.1 Substrate Preparation Substrates contained MC, coconut coir (CC), fine perlite (FP) and fine vermiculite (FV). The seedling substrate (25MC/ 25CC/ 25FP/ 25FV) and transplant substrate (40MC/ 10CC/ 25FP/ 25FV) were prepared the same way as in previous experiments. Materials were mixed on October 30, 2009 (see section 2.2.1 Substrate Preparation for details on substrate preparation and source information of the materials) and amended with Spanish River Carbonatite™ at 0.56 g-L"1 (see section 4.2.1 Substrate Preparation for more information). Elemental sulfur was added at half of the recommended (University of Missouri (MU) Extension 2008) rate (1.11 g for every liter of MC in the substrate). The substrates were stored in open plastic bags for five months. On March 24, 2010, one day before seeds were sown in the seedling substrate, the average pH of both the autoclaved and non-autoclaved seedling substrates was 8.1 [±0.1 standard error (SE)] so a small amount of elemental sulfur (0.25 g-L"1) was added to these substrates on this day. On March 26, 2010, 19 days before transplanting, the pH values of the autoclaved and non-autoclaved transplant substrates were 7.8 and 7.9, respectively. Therefore, elemental sulfur was added (11.7 and 12.0 g-L"1 respectively) to these substrates on this day. 8.2.2 Chemical Properties of the Substrates Recommended rates of application of elemental sulfur (MU Extension 2008) needed to lower the initial pH of the growing substrates to the acceptable range (5.5 - 6.8) for tomato production, were determined according to the initial 152 pH of the MC (first application) or the subsequent pH of the seedling and transplant substrates (second application). The pH and EC measurements were made using the 1:5 method (see section 2.2.2 Chemical Properties of the Substrates for this method). The pH and EC of the seedling substrates were also measured by pourthru (using plug trays) on days 11, 13, 15, 18, and 22 of the growth experiment, and the pH and EC of the transplant substrates were measured by the modified pourthru method for a drip irrigation setup on days 26, 31, 34, 38, 52, and 59 (see section 7.2.2 Chemical Properties of the Substrates for more details on the modified pourthru method for a drip irrigation setup) The starting pHs of the autoclaved and non-autoclaved seedling substrates were 8.1 and 7.9, respectively. However, the pH of these substrates, measured by pourthru, was within the recommended range (5.5 to 6.8; Vavrina 2002). The starting pHs of the autoclaved and non-autoclaved transplant substrates, were 7.8 and 7.9, respectively. However, after transplanting, the pH of these substrates, measured by pourthru, was maintained above those recommended for production of tomato transplants. For this reason, fertilizer solutions were kept relatively acidic throughout the experiment. The ECs of the seedling substrates, measured by pourthru, were all maintained below 2500 uS-cm"1 whereas those from the transplant substrates were maintained below 3000 pS-cm"1 (except in treatment 3 on day 22). By day 38, the ECs of the transplant substrates, measured by pourthru, fell below 1000 pS-cm"1 and did not rise above 1000 pS-cm"1 afterwards. The fertilizer nutrient concentration was increased accordingly on day 52 to avoid nutrient deficiencies (Figure 8.1). 153 5-i J tSj^iS^ W%z, 0- A i ^ %K — 1-not autoclaved ^" 2-not autoclaved + P u ^^fefv. 3-autoclaved ^cN. 5- /! 4-autoclaved + P u ?-• ••••*' — 5-notautoclaved + C r V* — 6-not autoclaved + P u * C r — 7-autoclaved *C r — • 8 autoclaved + P u + C r 0- CM CJ Day 1-notautoclaved 2-notautoclaved •*• P u 3-autoclaved 4-autoclaved +P u 5-not autoclaved + C r 6-not autoclaved + P u + C r 7-autoclaved + C r 8-autoclaved *P u + C r r- m in CM C\J oO 2 IS EC pH S 8 »• •?•*»• ™ SSinioiSmSmSinS tl Figure 8.1 Top: pH measured by pourthru throughout the growth experiment. Middle: electrical conductivity (EC; pS-cm"1) measured by pourthru throughout the growth experiment. Bottom: fertilizer solution's pH and EC throughout the growth experiment. 154 8.2.3 Growth Experiment Untreated seeds of greenhouse tomato hybrid 'beefsteak' type (Solanum esculentum L. cv. Matrix F1; De Ruiter Seeds Inc. Lakewood, CO, United States) were germinated on a moistened filter paper in a Petri dish in a dark area at room temperature [22 ±2°C standard deviation (SD)] from March 22nd 2010 (day 1) to March 25th 2010 (day 4). Seeds were then planted in 15.7 cm3 cells of a plug tray containing seedling substrate (25M/ 25CC/ 25FP/ 25FV), covered with moistened superfine vermiculite, and placed in a glass greenhouse in the Bovey Complex at the University of Guelph (see section 2.2.3 Growth Experiment for more details) at 21/ 17 ±3/ 1°C SD during the day/ night for about three weeks prior to transplanting. Seedlings were transplanted to the experimental substrate in 10- cm pots (426.1 cm3) on April 14th 2010 (day 24). Transplants were placed in the greenhouse at 21/ 15 ±3/ 1°C SD during the day/ night in a randomized complete block design (RCBD) with six replicates (replicate = two transplants, one for each harvest) for each of the eight treatments (see section 8.5.4 Treatments below for more details). A border of transplants placed around the plants within the RCBD formed a buffer zone to reduce any edge effects. Transplants were harvested after 43 and 60 days of growth for growth measurements (see section 8.5.6 Growth measurements). Seedlings were irrigated and fertilized as needed by sub-irrigation. Seedlings were fertigated twice (day 11 and 15) with a half rate fertilizer solution and twice (day 18 and 22) with a full rate fertilizer solution. Immediately after transplanting, fertilizer was applied directly to the substrate surface, at a rate of 155 50 ml per pot. For transplants, the full rate fertilizer solution was applied until day 52. On days 34, 38, and 52, pourthru EC measurements indicated that the EC of the substrate was low, so the application rate of the fertilizer solution was subsequently increased. The nutrient concentration (mg-L"1 solution) of the reduced rate fertilizer, calculated from the manufacturers information, was 177 total N, 24 P, 129 K, 23 Ca, 2 Mg, 1 Na, 7 S, and 1 Fe (5 ml BioFish-L H20"1 and 5 ml Root Conditioner-L H20"1; pH/ EC = 4.4/ 1039 pS-cm"1 ±0.13/ 3.0 pS-cm"1 SE); the nutrient concentration (mg-L"1 solution) of the full rate fertilizer was 354 total N, 48 P, 310 K, 46 Ca, 4 Mg, 2 Na, 14 S, and 3 Fe [10 ml BioFish-L H20"1 and 10 ml Root Conditioner-L H20"1; pH/ EC = 4.6/ 1877 pS-cm"1 ±0.03/15.3 pS-cm"1 SE (for seedling fertigation); pH/ EC = 4.6/ 1688 pS-cm"1 ±0.03/46.8 pS-cm"1 SE (for transplant fertigation)]; and the nutrient concentration (mg-L"1 solution) of the increased rate fertilizer solution was 532 total N, 72 P, 465 K, 69 Ca, 6 Mg, 3 Na, 21 S, and 4 Fe (15 ml BioFish-L H20"1 and 15 ml Root Conditioner-L H20"1; pH/ EC = 4.6/ 2447 pS-cm"1 ±0.02/ 54.9 pS-cm"1 SE. Both Biofish and Root Conditioner are OMRI listed and 100% organic (Biofert Manufacturing Inc. Langley, BC, Canada). Organic apple cider vinegar was used to reduce the pH of the fertilizer solutions. Transplants were irrigated with deionized water using drip irrigation emitters with a flow rate of 2 L-hour"1 (Netafim® Melbourne, VIC, Australia) using one emitter per plant. Irrigation scheduling was controlled by a computer system (Argus Control Systems Ltd, White Rock; BC, Canada), which was programmed to irrigate for two minutes (~67ml) at three times (6 am, 11 am, and 4 pm) 156 everyday except days when pourthru (PT) was conducted and/or fertilizer (F) was applied (days 26 (F and PT), 29 (F), 31 (F and PT), 34 (F and PT), 36 (F), 38 (F and PT), 39 (F), 40 (F), 45 (F), 46 (F), 47 (2F), 48 (F), 51 (F), 52 (2F and PT), 53 (2F), 54 (2F), 55 (F), 56 (F), 57 (3F), 58 (2F), 59 (2F and PT); on these days, automatic irrigation at one or two times would be stopped. 8.2.4 Treatments There were eight treatments based on the seedling and transplant substrates (Table 8.1) Seedling substrate was sterilized or not sterilized and amended or not amended with inoculum of C. rosea. The seedling substrate was sterilized in 10-L lots by autoclaving for one hour each day (20 minutes at 105°C) on three consecutive days immediately prior to planting on March 25, 2010 (day 1) (Kavroulakis et al. 2005; Theodore and Toribio 1995). The seedling substrate was amended with C. rosea EV-1A (obtained from Origro Inc., Burlington, ON) on days 12 and 19 by sub-irrigation using 1 L of spore suspension in deionized water (spore concentration = 2 * 108 spores L"1; Personal communication with E. Vermolen, Aldershot Greenhouses Ltd., Burlington, ON, CA) to saturate approximately 1570 cm3 (100 plugs) of seedling substrate. The spore suspension remaining after the sub-irrigation event was discarded. The transplant substrate was sterilized by autoclaving in 10 L batches on days 19, 20, and 21, and used for transplanting on day 21. Seedlings that were started in sterilized seedling substrate were transplanted only to transplant substrates that had also been sterilized. Transplants were inoculated with 10 ml of P. ultimum mycelia suspension added to a depression in the transplant substrate, in which seedlings 157 were placed, at a dose of 12.08 mg ±0.001 mg SE (dry weight mycelia/ plant). Seedlings grown in substrates amended with C. rosea were drenched a second time with 50 ml of C. rosea spore suspension (2x108 spores/ L) immediately after inoculation with P. ultimum. Table 8.1 Treatments Treatment (seedlings) 1 2 3 4 5 6 7 8 Substrate was autoclaved • • • • C. rosea • • • • Treatment (transplants) 1 2 3 4 5 6 7 8 Substrate was autoclaved • • • • C. rosea • • • • P. ultimum • • • • 8.2.5 Pathogen A virulent strain of P. ultimum isolated from infected tomato roots was provided by the laboratory of Dr. Valerie Gravel (Universite Laval, Quebec, QC, Canada; Gravel et al. 2009). The strain was grown on potato dextrose agar (PDA; Difco Laboratories, Becton Dickinson, Sparks, MD, United States) at room temperature (22°C ±2°C SD) and transferred every two weeks to fresh medium. Mycelia on agar were then transferred to potato dextrose broth (PDA; Difco Laboratories, Becton Dickinson, Sparks, MD, United States) and allowed to grow for several days. The mycelia were, rinsed with deionized water, and blended in deionized water. The resulting mycelia suspension was then used as inoculum. The same amount of fresh agar was blended in the same amount of deionized water and used as control. Three 10 ml samples of the mycelia suspension were collected on filter paper and dried (at room temperature until weights stabilized); the resulting dry weights were recorded as the inoculum dose (dry weight basis). 158 8.2.6 Growth Measurements Transplants were harvested on days 43 and 60. At the first and second harvests, the height and diameter (ABSOLUTE Digimatic Caliper 500-135, Model #CD-4"BS, Mitutoya Corporation, Mitutoyo Canada Inc. Mississauga, ON, Canada) of the stems, leaf area (LI-3100; LI-COR, Lincoln, NE, United States), and dry shoot weight (taken after >48 hours in drying oven at 50°C until dry weights stabilized) of each transplant were measured. At the second harvest, four chlorophyll content indexes were collected from the two youngest fully expanded leaves of each transplant, using a portable chlorophyll content index meter (CCM-200, Opti-Sciences, Tyngsboro, MA, USA). Root dry weights and the number of flowers and buds were also measured and the time from transplanting to opening of the first flower was also recorded. 8.2.7 Microbiology C. rosea On day 22, root segments (~1 cm) from seedlings that were inoculated with C. rosea were plated on paraquat - chloramphenicolagar (PCA) medium (20 g commeal agar, 1 L distilled water, 200 mg chloramphenicol, and 0.1 ml paraquat) to confirm the presence of C. rosea. At the first harvest (day 43), roots were washed and rinsed with distilled water or ethanol (75%). Five root segments per plant (-1 cm) for each rinse were transferred to PCA medium in separate Petri dishes (one for water rinse and one for ethanol rinse) and incubated at 25 ±2°C SD for up to 7 days in which time the presence or absence of C. rosea was determined by microscopic examination based on the morphology of the 159 conidiophores and conidia growing on the PCA (Figure 8.2). At the second harvest, roots were washed as at the first harvest. Five root segments per plant (~1 cm) for each rinse were transferred to PCA medium (12 g agar, 1 L distilled water, 200 mg chloramphenicol, and 0.1 ml paraquat) in separate Petri dishes (one for water rinse and one for ethanol rinse) and incubated at 25 ±2°C SD for 48 hours after which the presence or absence of C. rosea was determined by microscopic examination (Figure 8.2). Percentages were calculated as: Number of transplants with positive detections of C. rosea * 100 Number of transplants harvested for this determination Pythium spp. Another five root segments per plant (~1 cm) for each rinse at the first harvest were also transferred to a Pythium selective (P5) medium (20 g cornmeal agar, 1 L distilled water, 250 mg ampicilin, 5 mg pimaricin, and 5 mg rifampicin) in one Petri dish (half plated with five water-rinsed root segments and half plated 160 with five ethanol-rinsed root segments), and placed in a growth chamber at 25 ±2°C SD for 48 hours after which the presence or absence of Pythium spp. was determined by microscopic examination. At the second harvest, five root segments (per plant) from each rinse (~1 cm) were transferred to P5 medium (same recipe as at first harvest) in separate Petri dishes (one for each rinse), and placed in a growth chamber at 25°C for 48 hours after which the presence or absence of Pythium spp. was determined by microscopic examination (Figure 8.3). Figure 8.3 Top: P. ultimum at 20X magnification growing on Pythium selective (P5) medium. Bottom: Pythium sp. at 400X magnification (mycelium running from the top left hand corner to the bottom center of the picture) from water- rinsed root segments of a transplant that was inoculated with P. ultimum), which was plated on (P5) media at the first harvest. Characteristics used to detect Pythium spp. were absence of cross walls and cytoplasmic streaming. 161 Percentages were calculated as: Number of transplants with positive detections of Pythium spp. x 100 Number of transplants harvested for this determination Bacterial and Fungal Colony Forming Units Colony forming units (CFUs) were counted from the pourthru leachate of each substrate, collected on days 25 (one day after transplanting and P. ultimum inoculation) and 59 (one day before second harvest and 34 days after P. ultimum inoculation). Pourthru leachate was stored (4°C) for no more than 24 h before being spread onto PDA and trypticase soy agar (TSA; Difco Laboratories, Becton Dickinson, Sparks, MD). Pourthru leachate from day 25 was serially diluted by a factor of ten, 2 times, 3 times, and 4 times ([0.01], [0.001], and [0.0001], respectively) in sterilized deionized water before plating. After two days of incubation at 27°C, it was possible to resolve all colonies that formed on the PDA plates. However, for TSA plates, more than 600 colonies were often observed, therefore TSA plates were split into four sections and only one section was counted and multiplied by four to estimate the number of colonies present (up to 2500 colonies per plate). Pourthru leachate from day 59 was also serially diluted in sterilized deionized water before plating. Dilution concentrations of [1], [0.1], and [0.01] were made for PDA plated CFU counts, and dilution concentrations of [0.0001], [0,00001], and [0.000001] were made for TSA plated CFU counts. After two days of incubation at 27°C, colony counts for PDA plates from the non diluted and the [0.1] dilution were often greater than 600, so only the plates from the [0.01] dilution were counted (8 - 680 colonies per plate). For the TSA plates, 162 counts were often too high to count (>600). Most counts were made from the six times diluted [0.000001] leachate (2 to 600 colonies per plate); however, some counts from plates with the lower dilutions were made if counts were <600. Microbial Community Profile Analysis Pourthru leachate collected on days 31 and 59 were plated onto Biolog EcoPlates (Biolog Inc., Hayward, CA, United States). A Biolog EcoPlate is a 96- well MicroPlate, which contains 31 unique carbon sources (one sole carbon source per well) combined with a redox dye (terazolium violet); each well is repeated three times and water is used as the control (Garland 1997). When bacteria that were present in the pourthru leachate were able to utilize a carbon source for growth, the well turned purple. After 48 hours of incubation at 27°C, a microplate reader (Model 680, BIO-RAD, Hercules, CA, United States) was used to measure the optical density of the EcoPlates at 595 nm. On day 31, plates were filled with non-diluted pourthru leachate (100 pi well"1) from three replicates (i.e. one plate was used for each treatment), whereas on day 59, plates were filled with non-diluted pourthru leachate (100 pi well"1) from three replicates each with three sub-replicates (i.e. three plates used for each treatment). 8.2.8 Statistical Analysis SAS 9.1 (SAS Institute; Cary, NC, 2003) was used for analysis of data. The fungal and bacterial CFU data were transformed by taking the Log 10. For growth measurement data and log CFU data, an analysis of residuals was used prior to ANOVA to verify the assumptions of homogeneity of variance. Outliers were removed from the data set based on Lund's tables for an approximate test for 163 outliers in linear models (Lund 1975). When ANOVA was found to be significant (P <0.05) for a treatment effect, a Tukey-Kramer multiple means comparison test (at P <0.05) was performed to compare means. For log CFU data, interactions from the individual and combined effects of autoclaving, inoculation with C. rosea, and inoculation with P. ultimum were analyzed in proc glm using the F-test for determining whether to reject H0: that there is no interaction (at P <0.05). For microbial community analysis, data were transformed by dividing by the average absorbance for each EcoPlate to remove the effect of microbial density. Data were pooled and subjected to the mixed procedure using multivariate analysis of variance (MANOVA) to test for effects of autoclaving, inoculation with C. rosea, inoculation with P. ultimum, day, and sub-replication (data were pooled as it was not possible to perform a MANOVA test due to insufficient degrees of freedom for error when data was not pooled). Blocks were included as random effects. Interactions from the individual and combined effects of autoclaving, inoculation with C. rosea, and inoculation with P. ultimum were analyzed in proc mixed using Wilks' Lambda's approximate F-test (P <0.05). Analysis of variance for each of the 32 measures of carbon source utilization (for the pooled and the separate data from days 31 and 59) was conducted. When ANOVA was found to be significant (P <0.05) for a treatment effect, a Tukey-Kramer multiple means comparison test (at P <0.05) was performed. 8.3 Results Root Inspection Roots from P. ultimum inoculated substrates (i.e. treatments 2 and 4), which 164 were non-sterilized and sterilized substrates, respectively, appeared to be slightly smaller than roots from all other treatments, but this difference was not statistically significant at P £0.05. However, roots from transplants growing in substrates that were inoculated with P. ultimum (treatments 2, 4, 6, and 8) especially those from treatment 4 (sterilized substrate), were much more fragile and more likely to fragment than roots of transplants from substrates that were not inoculated with P. ultimum (treatments 1,3,5, and 7) (Figure 8.4). Figure 8.4 Top: unwashed and washed root systems of transplants from treatments 1-8. Bottom: all washed root systems of transplants from treatments 1-8 for measurement of root dry weights (see Table 8.1 for treatments). Growth Measurements At the first harvest, the stem diameter and height of the transplants ranged respectively from 6.0 ±0.24 mm SE (treatment 165 4) to 6.4 ±0.12 mm SE (treatment 1) and from 27.8 ±0.33 cm SE (treatment 1) to 30.7 ±0.53 cm SE (treatment 4) and did not differ significantly among treatments. The leaf area and dry weight of the transplants however, ranged respectively from 438.9 ±7.58 cm2 SE (treatment 2) to 559.7 ±17.7 cm2 SE (treatment 8) and 1.4 ±0.05 g SE (treatment 2) to 1.9 ±0.07 g SE (treatment 8) and differed among treatments (Figure 8.5). First harvest Second harvest Leaf area Leaf area 650- 1800-1 600- i 1700- 3 a L a a 550- i ab T T ri I l 2 at cm2 cm [ 1600- 500- cd cd bed I j d 1500- 450- r. iiii ii 12345678 12345678 First harvest Second harvest Shoot dry weight Shoot dry weight 2.2- 11.0-1 i 1 2.0- 10.5- a ab ab L a , L a 1.8- 10.0- 1 » 3 i [ g " 7 b t , |[ g r 1.6- 9.5- j 1| 1 " T ; 1.4- 9.0- i.i I i I • I i I 1 I Treatment 12 3 4 5 6 7!i Trcalment 1 2 3 4 5 f > 7 8 Autoclave B HI • • a B • v' Autoclave B B • • @ E3 • • C. rosea E S S HI • ^ /" »' C. rosea B @ B 0 ^ v' • • P.ullmium @ • @ • H • B » P. ultimum B ^ I3 ^ E »' B • Figure 8.5 Leaf areas and shoot dry weights at first and second harvest. Data are means of six replicates ±standard error. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. At the second harvest, no differences were found for any of the growth parameters measured (Figure 8.5). Transplant stem diameter ranged from 6.8 mm ±0.17 mm SE (treatment 2) to 7.2 mm ±0.12 mm SE (treatment 7), 166 transplant height ranged from 64.2 ±1.03 cm SE (treatment 3) to 67. 8 ±2.60 cm SE (treatment 4), leaf area ranged from 1553.0 ±52.06 cm2 SE (treatment 3) to 1660.9 ±19.04 cm2 SE (treatment 5), dry shoot weight ranged from 9.2 ±0.22 g SE (treatment 2) to 10.2 ±0.25 g SE (treatment 8), and dry root weights ranged from 2.0 ±0.10 g SE (treatment 2) to 3.4 ±0.78 g SE (treatment 7). There were also no differences in chlorophyll content indexes, which were between 31.1 ±1.08 SE (treatment 5) and 35.5 ±1.09 SE (treatment 3) and the number of days it took for the first flower to open, ranging from 58.3 ±0.21 days SE (treatment 3) to 59.7 ±0.21 days SE (treatment 1), were also not significantly different. The lowest number of buds on a cluster was 3.5 ±0.76 buds SE on the second cluster (treatment 7) and the highest number of buds on a cluster was 6.2 ±0.60 buds SE on the first cluster (treatment 5), and again no difference was found. Incidence of recovery of C. rosea from root segments Incidences of recovery of C. rosea from water-rinsed root segments taken from plants that were inoculated with C. rosea, treatments 5, 6, 7, and 8, were lower at the second harvest compared to at the first but these data could not be statistically compared for a difference. C. rosea was not recovered from transplants that were not treated with the agent (Figure 8.6). 167 rinse d r o Figur e 8 o ZZ CD 5" o o ZZ o 3 o Q. c -*> % incidence of recovery % inadcncc of reco\cry CD 3 1 n > : ~3 O > H —* CD —^ o -3 I; & 3 s I 0 CD o s a j Q. Q. o C3D CO First harvest Second harvest incidence of recovery of Pythium spp. incidence of recovery of Pylhmm spp. from cthanol-nnscd root segments from cthanol-nnscd root segments 100- 100- o 80- S 80- gj 60 •g- 60- £ " V 5 40- g ">0- 12 T3 J 20- I 20- 5? 0J T w 0- 0 ] 12 3 4 5 6 7 8 12 3 4 5 6 7 8 Trealment Treatment Autoclave BB^^BH^^ Auloclavc BB^^HH^^ C tosea BBSS • • • V c msixi mis&mssss P. ultimum E^B^B^®*' P.ulimum B^B^B^B^ Figure 8.7. Incidence of recovery of Pythium spp. from water-rinsed and ethanol- rinsed root segments of eight treatments at first and second harvest. The incidence of recovery of C. rosea from ethanol-rinsed root segments of transplants growing in substrates that were inoculated with C. rosea was greater at the first and second harvest (21 and 20% respectively) when substrates were sterilized compared to when substrates were not sterilized (8 and 12% at the first and second harvest respectively). For P. ultimum inoculated transplants, at first harvest, the incidence of recovery of Pythium spp. from ethanol-rinsed root segments of transplants growing in substrates that were sterilized was also greater than that of transplants growing in non-sterilized substrates (Figure 8.8). 169 First harvest Second harvest Outcome of C rosea inoculated treatments Outcome ol C rosea inoculated treatment 40-| 29% 29% 30- 20- 13 8% 8% 4%4% 4%4%4%4%4%4% 4% 4% 4% 4% 4 /„ 4% 4"/ 10- n n 4% 4% m Treatment oi II II n n n Treatment outcome 1 2 9 10 11 12 13 14 15 16 17 18 19 20 l\ihtum spp from water nnseB /Vf/tntffl spp fromuatcrnnscB V • H • • • • • a m • • E a a lithium spp from ethanol nnsea Pvlhmm spp trom ethanol nnseS S a a • ti a C rosea from waier nnse h irst harvest Second harvest Outcome of P uHimum inoculated treatments Outcome ofP ulltmum inoculated treatments 25- 2 % 25% 20- M% 21% 15- 13% 1 8"A 8% S /«, 8 /u 8* 10- 8% 8% 8% 8% 4% 4% 5- 4% 4% 4% 4/0 fl (1 Ircannent outcome 1 2 Treatment outcome 1 Pvthmm spp from water nnsc • • Pvthnim spp from water rinse • Pvtlmtm spp from ethanol nnsc • fg P\thtum spp from ethanol nasc • C iOSLO fromwjicrnns e a iS ( rasai from w aicr nnsc [g C rosea from ethanol nnsc |Sj g] C Kaea from ethanol nnsc @ Autoclaved ^ g) Auloclai cd E Hrst harvest Second harvest Outcome Outcome 10% 8% 8% 2% 2% 2% 2% 2% 2% 2% 2"/, 2% 2% 2°/ 2D/ n IJ JJ. IJ IJ IJ IJn n n n gang Treatment outcome 1 Treatment outcome i 2 3 4 5 6 7 8 13 14 15 16 17 18 19 20 Ptfluum spp from w filer rmsic • /Vrti 'ii spp from water rinse,' • a • e v B B • • a • v • a m Pithium spp from ethanol nnsc |5] wnspp from ethanol rinses a a a B • s a a a • B a • • B a • B C rcwea from water rinse H C rmixi from water rinsefx( a a a s B • • • B B • a • • • B • B C ro\t« from ethanol rinse a rosea from ethanol rinse® B a a B B S B a • B • • • a • Autoclaved a • • • • Autotla\ cdB ^ a B • • • B B B B • Figure 8.8 Percentages for different outcomes recorded (i.e. incidence of recovery of C. rosea and Pythium spp. from water-rinsed and ethanol-rinsed root segments) at first and second harvest Fungal Colony Forming Units (PDA) There was no significant treatment effect on fungal CFU counts from PDA. However, when sources of variation were partitioned (Table 8.2), there were significant effects of inoculation with the fungus C. rosea and inoculation with the fungus P. ultimum on fungal CFUs counted on day 25. Fungal CFU counts were greater in substrates that were inoculated with C. rosea than in substrates that 170 were not inoculated and CFU counts were lower in substrates that were inoculated with P. ultimum than in non-inoculated substrates (Figure 8.9). PDA (day 25) TSA (day 25) ab be be b b c c a a a a a a a a TSA (day 59) PDA (day 59) a ab ab ab ab ab b b Treatment 12 3 4 5 6 7 Treatment 12 3 4 5 6 7 8 Autoclave H H • • H E • Autoclave BIBSSBSSS C.ro.sea B B S B • • • C.rmea BBHE^^^^ P ultimum m • B • B / P. ultimum B^H^a^Bv' Figure 8.9 Colony forming units (CFU) counted from Potato dextrose agar (PDA) and Trypticase soy agar (TSA) on days 25 and 59. N = 24 (day 26 PDA), N = 23 (day 25 TSA), N = 24 (day 59; PDA), and N = 31 (day 59 TSA). Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. For day 59, there was a significant effect of autoclaving and a significant interaction between autoclaving and both inoculations (Table 8.2). 171 Table 8.2 Variance partition for fungal colony forming units (CFUs) taken on days 25 and 59, from potato dextrose agar (PDA). Day 25 CFUs taken from PDA Sums of (n = 3, N = 24) squares Mean F P Source of variation df (Type III) Square value value Sub-replicate 2 11.2381 5.619 1.81 0.1998 Autoclaved 2.6708 2.6708 0.86 0.3693 C. rosea 20.7521 20.7521 6.69 0.0216 Autoclaved * C. rosea 1.0892 1.0892 0.35 0.5630 P. ultimum 21.1435 21.1435 6.81 0.0206 Autoclaved * P. ultimum 1.1802 1.1802 0.38 0.5473 C. rosea * P. ultimum 2.9183 2.9183 0.94 0.3486 Autoclaved * C. rosea * P. ultimum 0.2458 0.2458 0.08 0.7825 Model 9 61.2378 6.8042 2.19 0.0909 Error 14 43.448 3.1034 Corrected total 23 104.6858 Day 59 CFUs taken from PDA Sums of (n = 3, N = 24) squares Mean F P Source of variation df (Type III) Square value value Replicate 2 1.316 0.658 5.37 0.0186 Autoclaved 0.5812 0.5812 4.74 0.0470 C. rosea 0.1346 0.1346 1.10 0.3123 Autoclaved x C. rosea 0.3995 0.3995 3.26 0.0925 P. ultimum 0.0051 0.0051 0.04 0.8408 Autoclaved * P. ultimum 0.0168 0.0168 0.14 0.7163 C. rosea * P. ultimum 0.4314 0.4314 3.52 0.0816 Autoclaved * C. rosea * P. ultimum 0.5837 0.5837 4.76 0.0466 Model 9 3.4684 0.3854 3.15 0.0270 Error 14 1.7153 0.1225 Bacterial Colony Forming Units (from TSA) Bacterial CFU counts for treatments 7 and 3 (sterilized substrates with and without inoculation with C. rosea), on day 25, were on average 15 and 8% higher than all other treatments. On day 59, counts from treatment 2 (non-sterilized with P. ultimum inoculation) were 33 and 36% higher than counts from treatment 1 (non-sterilized) and treatment 4 (sterilized with P. ultimum inoculation) respectively (Figure 8.9). When sources of variation were partitioned, a 172 significant effect of autoclaving on day 25 counts was found, but there was no significant effect of autoclaving on day 59 counts. Instead, there was a significant effect of inoculation with C. rosea, a significant interaction of autoclaving and inoculation with P. ultimum, and a significant interaction of autoclaving and both inoculations on day 59 (Table 8.3). Table 8.3 Variance partition for bacterial colony forming units (CFU) taken on days 25 and 59 from tripticase soy agar (TSA). Day 25 CFUs taken from TSA Sums of (n = 3, N = 23) squares Mean F P Source of variation df (Type III) Square value value Sub-replicate 2 3.3162 1.6581 54.09 <0001 Autoclaved 1.5550 1.5550 50.73 <0001 C. rosea 0.8277 0.8277 27.00 0.0002 Autoclaved x C. rosea 0.0096 0.0096 0.31 0.5848 P. ultimum 0.236 0.236 7.70 0.0158 Autoclaved * P. ultimum 0.7174 0.7174 23.4 0.0003 C. rosea * P. ultimum 0.0000 0.0000 0.00 0.9909 Autoclaved * C. rosea * P. ultimum 0.0969 0.0969 3.16 0.0988 Model 9 7.2984 0.8109 26.45 <0001 Error 13 0.3985 0.0307 Corrected total 22 7.6969 Day 59 CFUs taken from TSA Sums of (n = 3, N = 31) squares Mean F P Source of variation df (Type III) Square value value 0.8526 Replicate 2 1.7053 557 4.48 0.0255 Sub-replicate 2 6.6953 3.3476 17.58 <.0001 Autoclaved 0.0077 0.0077 0.04 0.8425 C. rosea 1.1420 1.1420 6.00 0.0242 Autoclaved * C. rosea 0.1278 0.1278 0.67 0.4228 P. ultimum 0.0003 0.0003 0.00 0.9685 Autoclaved * P. ultimum 1.8213 1.8213 9.57 0.0060 C. rosea * P. ultimum 0.0044 0.0044 0.02 0.8805 Autoclaved * C. rosea * P. ultimum 2.1327 2.1327 11.20 0.0034 Model 11 30.9535 2.8140 14.78 <0001 Error 19 3.6172 0.1904 Corrected total 30 34.5707 173 Microbial Community Profile Analysis Community-level physiological profiles based on patterns of sole carbon source utilization were compared and significant differences were found for 21 of the 31 carbon sources when data from both days (31 and 59) was pooled. Some trends were noticed (Figure 8.10). Microbial communities from substrates that were autoclaved and not inoculated with P. ultimum (treatments 3 and 7) exhibited significantly lower pyruvic acid methyl ester (B1) utilization and significantly higher 2-hydroxy benzoic acid (C3) and D-glucosaminic acid (F2) utilization compared to microbial communities from substrates that were not autoclaved and inoculated with P. ultimum (treatments 2 and 6). MANOVA for pooled data revealed a significant effect of autoclaving on the microbial diversity of a substrate. Autoclaved substrates (treatments 1, 2, 5, and 6) tended towards having microbial communities with lower 2-hydroxy benzoic acid (C3), L-phenylalanine (C4), and D-glucosaminic acid (F2) utilization than communities from non-autoclaved substrates (Figure 8.10). There was also a significant effect of inoculation with C. rosea on the microbial assemblage and a significant effect for an interaction between autoclaving and inoculation with C. rosea. There was also a significant effect of inoculation with P. ultimum and a significant interaction between autoclaving and inoculation with P. ultimum. The microbial assemblage was also significantly affected by time (i.e. day) (Table 8.4), therefore, the community-level physiological profiles based on patterns of sole carbon source utilization were compared for each day separately. 174 Figure 8.10. Microbial community profiles of treatments taken from pourthru leachates on days 31 and 59 (pooled data). Data are means of three replicates and three sub-replicates istandard error. Data bearing the same letter are not significantly different by the Tukey-Kramer test at P <0.05. 175 Table 8.4 Multivariate analysis of variance test criteria and exact F statistic for the hypothesis of no overall effect or interaction of inoculation with P. ultimum, inoculation with C. rosea, and substrate autoclaving on microbial community composition sampled from substrate pourthru leachates on days 31 and 59 (N=25; Numerator df = 32; Denominator df = 52). F Effect Wilks' Lambda value value P value Autoclaved 0.0828 17.99 <0001 C. rosea 0.3176 3.49 <0001 Autoclaved * C. rosea 0.4248 2.20 0.0056 P. ultimum 0.2156 5.91 <0001 Autoclaved * P. ultimum 0.2170 5.86 <0001 C. rosea * P. ultimum 0.5289 1.45 0.1160 Autoclaved * C.rosea * P. ultimum 0.5136 1.54 0.0819 Day 0.0938 15.69 <0001 Sub-replication 0.0424 6.26 <0001 Significant treatment effects were found for 6 carbon sources on day 31 and for 23 carbon sources on day 59 (Figure 8.11). As with the pooled data, similar differences in microbial community assemblages on the different days were noticed. Substrates that had been autoclaved and not inoculated with P. ultimum (treatments 3 and 7) had lower pyruvic acid methyl ester (B1) utilization and higher (3-methyl-D-glucoside (A2), 2-hydroxy benzoic acid (C3), and D- glucosaminic acid (F2) utilization compared to microbial communities from substrates that had not been autoclaved and inoculated with P. ultimum (treatments 2 and 6). 176 I DJV31 | Div59 oOJ3 y w ^-O tl 20- t 5- 1 0- w TOM iffil 20- 1 5- 1 0- 0 5- sa^^<3ib,\ t in ,5-i M 20-1 4» 1 W osJk •- 1. "Jfc h fa A % ^ a 1) &• *o "b 20- 20- o 1 5- 1 5- 1 0- y -Q| 1 0- 0 5- J,"[] 05- oo-umi¥V¥¥ nib»bfe\% N %"} =• «3 I Gl G3 20- 15- 1 0- A Ax**., 10- 1 0- 05- 0 5- 05- oo * lilfgili11i*^lti1Fr'i | f l wtew giiiill \ V^itK H2 H3 20- 20- 1 5- 1 5- 10- 0 5- Trr'f'rTT'rT TTTW 00- lifc ialsBiii n < 11 v'V .>$' 3'oA'fe N a T> f. *» 177 8.4 Discussion Results from the present investigation were in part consistent with the hypothesis. In general, Pythium spp. will destroy fine feeder rootlets and root hairs making the roots less able to efficiently utilize nitrogen and other nutrients (Martin and Loper 1999). Based on examination of the transplant roots, applications of C. rosea and MC in combination or individually seemed to improve the root system health of tomato transplants inoculated with P. ultimum. Roots from transplants growing in sterilized substrates inoculated with the pathogen but not the beneficial endophyte (treatment 4) were much more fragile and more likely to fragment than roots of transplants from all other substrates that were inoculated with the pathogen. Recovery of Pythium spp. from ethanol- rinsed root segments of transplants inoculated with the pathogen also only occurred in treatment 4, which suggests that establishment of the pathogen in the root zone is more likely to occur in sterilized substrates that are not amended with the beneficial endophyte. Furthermore the observation that incidence of recovery of C. rosea from root segments of transplants from treatments 7 and 8 tended to be higher than in root segments of transplants from treatments 5 and 6 (especially at first harvest) suggests that sterilizing a growing substrate increases the ability of the endophyte to become established in the plant root zone. It is known that the nature of a sterilized growing substrate creates an environment that is favourable for microbial colonization due to lack of microbial competition for available nutrients by newly colonizing microbes (Hointink and Fahy 1986). In fact, sterilization of growing substrates releases nutrients from recently killed 178 microorganisms and from heated organic matter thereby providing more nutrients for both microorganisms and plants (Mandeibaum et al. 1988). The latter statement is in line with the finding in this study that transplants from treatments 3, 4, 7, and 8 (sterilized substrates) were larger, at first harvest, than transplants from treatments 1, 2, 5, and 6 (non-sterilized substrates), which suggests that sterilizing a growing substrate allows plants to get a head start. Sterilized substrates inoculated with P. ultimum exhibited lower fungal CFU counts early on in the experiment. This was unexpected and perplexing as inoculations with this fungus in theory should have increased the number of fungal CFU counted from these substrates. Nonetheless, substrates that were autoclaved and inoculated with either C. rosea or P. ultimum had slightly higher fungal CFU counts than substrates that were not autoclaved and inoculated. Similarly to fungal CFU counts, higher numbers of bacterial colony forming units were also recovered from autoclaved substrates. Initially, the expectation was that sterilized substrates would have lower fungal and bacterial CFUs due to the effect of autoclaving killing off the microbial populations present. However, as the opposite was observed it was apparent that the findings were in line with our findings for incidence of recovery of Pyhtium spp. and C. rosea, which suggested that sterilized substrates are more favourable for microbial colonization than non- sterilized substrates. Therefore, it is likely that recovery of higher numbers of CFUs from autoclaved substrates was truly a phenomenon of reduced competition and increased availability of nutrients (Mandeibaum et al. 1988; Hointink and Fahy 1986). 179 Furthermore, the observation, at first harvest, that incidences of recovery of Pythium spp. from water-rinsed root segments of transplants from treatment 1 (non-sterilized substrate not inoculated) was greater than from those of treatment 3 (sterilized substrate not inoculated) suggests that the Pythium spp. recovered here may have been coming from the compost. Furthermore the observation that recovery of Pythium spp. from treatments 3, 5, and 7 (i.e. substrates that were not inoculated with P. ultimum) was greater at second harvest than at first harvest also suggests that Pythium spp. were present in the compost. Whether or not the Pythium spp. recovered from the roots of these treatments were non pathogenic can only be assumed. We observed that the roots of transplants from treatments 2, 4, 6, and 8, which had been inoculated with P. ultimum were much more fragile and likely to break (especially those from treatment 4) than roots of transplants from treatments 1, 3, 5, and 7, which had not been inoculated with P. ultimum. This observation was a key finding that suggested that non-pathogenic Pythium spp. were recovered from the root segments of transplants from treatments that were not inoculated with P. ultimum (treatments 1,3,5, and 7). The genus Pythium comprises many diverse species. Some Pythium spp. are plant pathogens while others are strict soil saprophytes, or are parasites of insects, mammals, algae, or fish (Martin and Loper 1999). Pythium spp. nonetheless are ubiquitous fungi. Thus, it could be anticipated that non-sterilized substrates, which contain compost and have higher microbial diversity, would be more likely to harbor Pythium spp. that are not plant pathogens. P. oligandrum and P. nunn are likely good biocontrol agents against phytopathogenic Pythium 180 spp. like P. graminicola, P. spinosum, P. aphanidermatum, and P. ultimum (Martin and Loper 1999). Observations of recovery of Pythium spp. from roots of transplants growing in non-sterilized substrates that were not inoculated with P. ultimum, may be attributed to the greater diversity of microorganisms from compost, which may have included non-pathogenic Pythium spp. Sterilized substrates, on the other hand, are more likely to only harbor heat resistant spp. such as Bacillus spp., which for the most part can be disease suppressive (Chun et al. 2003). Microorganisms that are not resistant to heat, such as Pythium spp., should not survive autoclaving. However, proper steps for identification of the Pythium spp. were not made. Therefore it cannot be said with certainty that non-pathogenic Pythium spp. originated from the compost nor can it be said that P. ultimum spread from P. u/f/'mtv/n-inoculated substrates to substrates that were not inoculated with P. ultimum. However the statistical analysis was not sufficiently robust to confirm the observations made. In addition, due to the lack of statistical evidence for a significant difference in aboveground growth of P. u/f/mt/m-inoculated transplants compared to the aboveground growth of transplants growing in substrates that were not inoculated with P. ultimum, it cannot be concluded that infection by P. ultimum negatively affected the growth of the transplants. Nor can it be confirmed that the Pyhtium spp., which were recovered from roots of substrates that were not inoculated with the pathogen, negatively affected the transplants. All in all, transplants were fairly young, which may explain why aboveground growth effects due to inoculation with P. ultimum were not observed. Often, root disease 181 symptoms arise under periods of stress in larger plants that exert larger requirements from their roots for water and nutrients (Martin and Loper 1999). The most significant finding that P. ultimum caused some disease in the root zone, although not statistically supported and subjective, was nonetheless an observation that suggested that some level of root disease caused by P. ultimum occurred in our experiment In terms of microbial analysis, ANOVA provided evidence for an effect on fungal and bacterial CFU counts due to significant effects of autoclaving, inoculation with C. rosea and inoculation with P. ultimum or significant interactions of these effects. Overall, CFU counts, revealed a trend towards higher microbial activity from autoclaved substrates. The expected difference in carbon source utilization of microbial communities from substrates that were inoculated with P. ultimum compared to carbon source utilization of microbial communities from substrates that were not inoculated with P. ultimum was not observed in this experiment. Chun et al. (2003) searched for carbon sources that could be used to benefit bacterial biological agents (Bacillus spp.) without providing any benefits to the pathogen (Pythium spp.) and therefore identified several carbon sources that are utilized by Pythium spp. In our study microbial communities from P. ultimum inoculated substrates (treatments 2, 4, 6, and 8), had significantly higher pyruvic acid methyl ester (B1) utilization than microbial communities from substrates that were not inoculated with P. ultimum. Chun er al. (2003) did not identify pyruvic acid methyl ester (B1) as a carbon source utilized by P. arrhenomanes, P. myriotylum, or P. 182 dissotocum. Tween 40, glycogen, and a-methyl-D-glucoside, were however identified by Chun et al. (2003), as being used by Pythium spp. Tween 40 was included as a carbon source (C1) in the microplate used in the study; however, the expected higher utilization by microbial communities from P. u/f/mt/m-inoculated substrates was not observed. Glycogen was also a carbon source (F1) in the microplate used in this study. Unfortunately, the expected trend of higher glycogen (F1) utilization by microbial communities from P. u/r/mt/m-inoculated substrates was not observed. a-methyl-D-glucoside was not included in the Biolog microplate used in this study. Instead the microplate contained B-methyl-D-glucoside (A2). Both a-methyl-D-glucoside and B-methyl- D-glucoside are derivatives of glucose. All derivatives of glucose in the microplate used in this experiment were B-methyl-D-glucoside (A2), N-acetyl-D- glucosamine (E2), D-glucosaminic acid (F2), and glucose-1-phosphate (G2). However no expected trends were observed for these carbon sources either. These observations are likely due to the Pythium spp. that was recovered from substrates that had not been inoculated with P. ultimum. The Pythium spp. that were observed in the substrates that were not inoculated with P. ultimum may have interfered with the microbial community profile analysis; however, the MANOVA provided some evidence for microbial shifts occurring among the different treatment effects. There was an effect on microbial assemblages due to autoclaving, inoculation with C. rosea, inoculation with P. ultimum, and the interaction of autoclaving and inoculation with P. 183 ultimum or C. rosea but no effect on the microbial assemblages due to the interaction of inoculation with C. rosea and the inoculation with P. ultimum. Observations made from the microbial analyses conducted in this experiment indicate that microbial community differences were observed among the substrates undergoing the different treatments in this experiment. The results also pointed to microbial community changes, being affected by the action of autoclaving versus not autoclaving. In a more robust study on gene sequencing for identification of microorganisms that are present in substrates undergoing P. ultimum suppression due to microbially active compost, Hagn et al. (2008) found that the presence of P. ultimum in compost-amended growing substrates induced distinct microbial shifts. When P. ultimum was introduced into the substrate, the bacterial community shifted from a y-/5- Proteobacteria- and Verrumicrobia- dominated community to an Actinobacteria- and a-Proteobacteria-dominated community and Hyphomicrobium spp. were the closest related reference sequence observed. Bacteria often associated with suppression of Pythium spp. (pseudomonads) were not abundant in this system. The explanation for this was a possible signal between the pathogen and the bacteria resulting in suppression of the bacteria. Homobasidiomycetes were the dominant fungi found in substrates without P. ultimum: they were however, not detected in P. ultimum- inoculated and suppressed substrates. Instead fungi from the phylum Ascomycota (especially the class Sordariomycetes) were the dominant fungi in P. ty/f/'mivm-inoculated and suppressed substrates and Sordaria and Chaetomium species were found as the closest related reference sequence. These fungi may 184 antagonize Pythium spp. by antibiosis. Chytridiomycota were also abundant in the P. u/f/mtvm-inoculated and suppressed substrates. Chytridiomycota comprise parasites and saprobes linked to parasitism of hyphomycetes and the production of hydrolases and cellulases, which could be involved in the breakdown of P. ultimum cells (Hagn et al. 2008). Trichoderma, a commonly used biocontrol agent against Pythium spp., was not detected in the P. u/f/'mum-inoculated and suppressed substrates from Hagn et a/.'s (2008) study. It is possible that the type of compost used did not support the growth of Trichoderma spp., as population shifts are often affected by variations in composts due to inconsistencies in composting methods (Larney and Hao, 2007; Curtis et al., 2005; Dianez et al., 2005; Larney et al., 2000; Tuitert et al., 1998; de Brito Alvarez et al. 1995). On the other hand, Scheuerell et a/.'s (2005) study on suppression of Pythium spp. with different composts found no significant relationship between individual microbial populations found in different composts and pathogen suppression. Instead they found that the suppression of P. irregulare and P. ultimum in cucumber was related to a compost's potential to support microbial activity. There is a great potential to inoculate composts with disease suppressive microorganisms for more consistent disease suppression and promotion of plant growth. Recent technologies to quantify and identify microorganisms present in suppressive composts have provided vital information leading to novel understanding of compost utilization (Hagn et al. 2008; Escuadra and Amemiya 2008; Massart and Jijakli 2007; Fracchia et al. 2006; reviewed by Hill et al. 2000; Boehm et al. 1993). Research on microbial growth, distribution, function, and the 185 nature of interactions among species during disease suppression events is still in its infancy (Hill et al. 2000). Infections are often erratic and unpredictable, especially when dealing with microbial communities to suppress infection; therefore, a deeper molecular knowledge of pathogen infection processes that can either cause disease or resistance in host plants is important (Ekengren 2008). Induced systemic plant defenses and biological, and chemical factors are likely to be involved and therefore complicate the research (Kavroulakis et al. 2005). Strategies to further improve compost utilization, which depends on its quality, its consistency, its microbial community, and its efficiency, will continuously be needed (Kuo et al. 2004). 8.5 Conclusion We recommend the use of compost and C. rosea for P. ultimum suppression. These can be used on their own or in combination. We especially recommend the use of C. rosea when using a substrate that is mostly inert, such as one that contains no compost and is largely comprised of peat, coir, perlite, or vermiculite. Our main reason for recommending the use of these biological means of controlling Pythium disease in tomato transplant production is that at first harvest only the substrate that was autoclaved, not inoculated with C. rosea, and inoculated with P. ultimum (treatment 4) had an incidence of recovery of Pythium spp. from ethanol-rinsed root segments, and this incidence of recovery was high (80%). The roots of these transplants were also more fragile than roots of every other transplant in the study. 186 CHAPTER 9: Summary and Recommendations 9.1 Summary In organic systems, quality growing substrates and fertilizers are essential; this is particularly true for young plants because transplants are often the precondition of the whole production technology. The goal of this research was to provide growers with more information and technology to ensure high quality organic tomato transplant production. Objectives were: 1) to develop organic growing substrates suitable for growing tomato transplants; 2) to evaluate various sources of compost and different inclusion rates of compost; and 3) to investigate the use of compost alone, or in combination with a beneficial endophyte (Clonostachy rosea) to suppress Pythium root rot (Pythium ultimum). Materials that are eligible for use in organic production systems were selected and preliminary analyses were conducted to determine which combinations and ratios of these materials had acceptable pH, EC, and nutrient content for growing tomato transplants. These substrates were used in several growth experiments. The following is a summary of the findings from the research: In the first growth experiment, 25 growing substrates were tested in plug trays. Poor growth and signs of nutrient deficiency (purple colour on undersides of leaves) were noticed after three weeks. These symptoms indicated possible nitrogen and/or phosphorus deficiency; however, the possibility of other nutrient deficiencies could not be ruled out because the irrigation water used was highly alkaline. High pH may also have limited the availability of iron, manganese, zinc, 187 and copper for plant uptake. The substrates were, nevertheless, ranked, from best to worst, based on growth parameters measured at harvest. The substrates that ranked among the best were tried again in successive experiments, which comprised the use of fertilizer application in pots instead of plugs. In the second experiment we included zeolite (Z) at 7% by volume (v/v) in three growing substrate formulations to examine, more accurately than in the first experiment, the difference, if any, between substrates with and without Z. Z has a high affinity for cations and acts as storage for nutrients; however, benefits were not evident in the first or second experiment because the rate at which Z was included was not high enough to increase cation exchange capacity. Higher inclusion rates of Z are not recommended due to high shipment cost. Therefore, Z was deemed uneconomical at >7% inclusion rate. In the third experiment the six best growing substrates from the first experiment (excluding any substrates that contained Z) were evaluated as well as four new growing substrates chosen based on previous research results. Several growing substrates were considered adequately supportive of tomato transplant growth, however substrates containing manure compost (MC) generally resulted in superior growth when compared to substrates containing pine bark compost (PBC) and vermicompost (VC). Lower nutrient concentrations were observed in the leachate of growing substrates that contained peat moss (PM) when compared to nutrient concentrations in the leachate of growing substrates that contained PBC and/or coconut coir (CC) as a replacement for PM. Optimal transplant growth was observed in the substrate containing 25% 188 MC, 25% PM, 25% fine perlite (FP), and 25% fine vermiculite (FV) (i.e. the 25MC/ 25PM/ 25FP/ 25FV substrate). When PM was replaced with CC, no significant differences in growth were found between the transplants from these two substrates. The 20MC/ 20PM/ 20CC/ 20FP/ 20FV substrate, the 20VC/ 20PM/ 20CC/ 20FP/ 20FV substrate, and the 25VC/ 25PM/ 25FP/ 25FV substrate also produced transplants of comparable vigor to transplants from the aforementioned substrates. However, when PM was replaced by PBC, stunted transplant development was evident. The 20MC/ 20PBC/ 20CC/ 20FP/ 20FV substrate, and the 25MC/ 25PBC/ 25FP/ 25FV substrate produced the smallest transplants with the least number of leaves and lowest leaf area. High bulk density (BD), low air-filled porosity at container capacity (AFP), high nutrient leaching, and the possibility that nutrient immobilization may have occurred due to the inclusion of a possibly immature PBC as a PM alternative, were linked to the reduced growth of transplants in these substrates. In addition a combination of high pH and low EC in these substrates may have limited nutrient availability and resulted in growth disorders, which may have caused and may have been aggravated by lower chlorophyll content, which was observed for substrates including PBC. When lower rates of PBC were used, however, good transplant growth comparable to that in the best substrate was possible. The 20MC/ 10PBC/ 20CC/ 50FV substrate was considered a good substrate for tomato transplant production. However, it was the 25MC/ 25CC/ 25FP/ 25FV substrate that was chosen for further investigation in succeeding experiments. 189 PM harvesting is considered a non-sustainable practice to some ecologists. As PM bogs are precious habitats for wildlife and a major sink for atmospheric CO2 aiding in the reduction of global warming, PM alternatives are sought. CC, which has similar physical and chemical properties to PM and is considered a renewable resource without as many ecological drawbacks as PM, was, therefore, chosen over PM for inclusion in the substrate formulation that was used in ensuing experiments. The objective of the fourth experiment was to determine whether or not processed organic wastes from different sources could be considered equally supportive of tomato transplant growth. We evaluated MC with (MC + E) and without (MC) eggshells, Forterra VC, Worm Power Gold worm castings (WC), yard waste compost (YWC), and anaerobically digested cattle manure (ADCM). Five substrates were formulated containing 25% CC, 25% FP, 25% FV, and 25% of each organic waste. Growblock, which was made of 100% CC substrate and proposed for organic certification by Jiffy Products N.B. Ltd. was used as the comparison substrate. Growblock is commercially available and recommended for transplant production. Therefore this transplant production technique was used as a comparison to the traditional 10-cm pots filled with growing substrate. However, as was expected, growblock had a growth advantage due to the larger substrate volume (0.850 cm3), which was double that of the 10-cm pot (0.425 cm3). The growblock was therefore more likely to hold more nutrients than any of the substrates in the 10-cm pots, and more likely to allow more space for root 190 growth. However, the leachate with the most plant-available nitrogen was observed for the substrate formulated with WC, which produced transplants that were often not significantly different from transplants grown in the growblock. Substrates including VC, MC, MC + E, and YWC, also produced transplants of similar quality than transplants growing in the substrate containing WC. The substrate that included ADCM was the only substrate that was inappropriate for tomato transplant production due to poor soluble nutrient content and low pH. The objective of the fifth experiment was to find the optimal rate of inclusion for MC. The substrates were selected to have increasing amounts of MC; therefore the combinations were: 1) 25MC/ 25CC/ 25FP/ 25FV, 2) 35MC/15CC/ 25FP/ 25FV, 3) 45MC/ 5CC/ 25FP/ 25FV, and 4) 50MC/ OCC/ 25FP/ 25FV. Optimum growth was often observed in substrates containing either 35% or 45% MC. An inclusion rate of 40% for MC was therefore considered optimal for producing thicker and taller transplants. Based on this experiment a 40% inclusion rate for MC was used in the sixth and the seventh experiment, which were intended to investigate the use of aged pine bark (APB) for substrate aeration and to evaluate the use of MC for disease suppression, respectively. The objective of the sixth experiment was to reduce growers' dependency on materials that require heat for expansion and thus use large amounts of fossil fuels for production. In this experiment, APB was investigated as an alternative for FP and FV. Increasing the amount of APB increased AFP; however, increased AFP, in this case, did not correlate with increased growth. Optimum growth was observed in the substrate combination that included the least amount 191 of APB (i.e. 40MC/ 25PM/ 25CC/ 10APB). The APB may have been immobilizing nitrogen; however, nitrogen drawdown index was not evaluated therefore we did not determine whether or not nitrogen immobilization was occurring in substrates that contained higher rates of APB. Other substrates that provided transplants with comparable growth were the 40MC/ 30PM/ 30CC substrate and the 40MC/ 25PM/ 25CC/ 5FP/ 5FV substrate. Overall, increased growth was inversely related to AFP and positively related to total porosity and water-holding capacity at container capacity. Microbial suppression of plant pathogens using compost and other organic amendments as alternatives to or reducers of agrochemicals is in line with sustainable plant production and the needs of the organic horticulture sector. In our seventh experiment we investigated whether disease suppression was possible with a natural microbial community from MC and/or the fungal endophyte Clonostachys rosea. The aim was to investigate whether the combination of MC and C. rosea could suppress Pythium ultimum in tomato transplants more than either one of the two applications on their own. Applications of C. rosea and MC in combination or individually seemed to improve the root system health of tomato transplants growing in substrates that were inoculated with P. ultimum. The roots of transplants growing in sterilized substrates that were inoculated with P. ultimum were much more fragile and more likely to break than the roots of transplants from all other P. ultimum inoculated substrates. Furthermore, the incidence of recovery of Pythium spp. from ethanol-rinsed root segments of transplants growing in sterilized substrate 192 that was inoculated with P. ultimum was greater than that from ethanol-rinsed root segments of transplants growing in non-sterilized substrate inoculated with P. ultimum. Incidence of recovery of C. rosea from segments of roots of transplants growing in sterilized substrate also tended to be higher than that from segments of roots of transplants growing in non-sterilized substrate, especially at first harvest. These observations suggest that sterilizing a growing substrate increases the ability of the pathogen and the endophyte to become established in the plant root zone. Overall, microbial activity tended to be higher in sterilized substrates. Fungal CFU counts and bacterial CFU counts were both higher in sterilized substrates. This finding further suggests that sterilized substrates are more favourable for microbial colonization than non-sterilized substrates. Inert materials have been described as ideal sinks for microbial colonization, in the past, due to reduced competition occurring in these substrates. Sterilization of a growing substrate also releases nutrients from recently killed microorganisms and from heated organic matter thereby providing more nutrients for both the microorganisms and plants in comparison to a non-sterilized growing substrate. Pythium spp. were recovered from all treatments at second harvest. However, the roots of transplants from substrates that had been inoculated with P. ultimum were much more fragile and likely to break (especially in sterilized substrate not inoculated with the beneficial endophyte) than roots of transplants from substrates that had not been inoculated with P. ultimum. This observation was a key finding that suggested that non-pathogenic Pythium spp. were 193 recovered from the root segments of transplants from treatments that were not inoculated with P. ultimum. The observations at the second harvest for recovery of Pythium spp. from roots of transplants from non-sterilized substrates may be due to the greater diversity of microorganisms from the MC, which may have included non-pathogenic Pythium spp. All in all, the Pythium spp. identified from the leachate of substrates that were not inoculated with P. ultimum may have interfered with the microbial community profile analysis that was conducted to investigate the microbial community profile of each treatment based on carbon source utilization; however, the multivariate analysis of variance provided some evidence for microbial shifts occurring among the different treatment effects. There was an effect on microbial assemblages due to autoclaving, inoculation with C. rosea, inoculation with P. ultimum, and the interaction of autoclaving and inoculation with P. ultimum or C. rosea but no effect on the microbial assemblages due to the interaction of the inoculation with C. rosea and the inoculation with P. ultimum. Observations made from the microbial analyses conducted in this experiment indicate that microbial community differences were observed among the substrates undergoing the different treatments in this experiment. The results also pointed to microbial community changes, being affected by the action of sterilizing versus not sterilizing the growing substrate. However, due to the lack of statistical evidence for a significant difference in aboveground growth of P. t//f/mt/m-inoculated transplants compared to the aboveground growth of transplants growing in substrates that were not inoculated with P. ultimum, it cannot be said with precision that the infection of 194 transplants with P. ultimum affected the growth of these transplants in a negative way. Nor can it be said that the Pythium spp. recovered from roots of substrates that were not inoculated with the pathogenic P. ultimum were not negatively affecting these transplants. All in all, transplants were fairly young, which may explain why aboveground growth effects due to inoculation with P. ultimum were not observed. Only the observation that roots from sterilized substrates that were inoculated with P. ultimum were more fragile compared to other roots from other treatments suggests that some effect on roots was occurring in this experiment. Therefore, it was concluded that the establishment of P. ultimum in the root zone of transplants is more likely to occur when the transplants are grown in sterilized substrates that are not amended with the beneficial fungal endophyte C. rosea. 9.2 Recommendations Overall, several growing substrate combinations containing different rates of PM, CC, FP, FV, APB, and compost (MC, VC, WC, YWC, and/or PBC) can be used to grow tomato transplants successfully in 10-cm pots with organic fertilizer applications (Table 1). We recommend the use of PM at no more than 30% (v/v) due to ecological drawbacks associated to the harvest of PM bogs. We also recommend no more than 30% CC, and no more than a combined volume of 50% FV and FP, due to the environmental impacts associated to the fossil fuels used to transport or produce these materials. Finally, according to our research, processed organic wastes may be included at 10 - 40% v/v. We found that MC between 25 and 40% was acceptable but PBC and APB at more than 10% was 195 unacceptable. Other composts evaluated in this research were not tested at over 25% v/v. Further testing is needed to determine what are the optimum rates of inclusion for the VC, the WC, and the YWC. Table 9.1 Substrates recommended for organic tomato transplants Substrates2 12 3 4 5 6 7 Composty 25 25 20 20 40 40 40 Coconut coir / 25 20 20 25 25 30 Fine Sphagnum peat moss 25 / 20 / 25 25 30 Fine perlite 25 25 20 / / 5 / Fine vermiculite 25 25 20 50 / 5 / Pine bark compost / / / 10/ / / Aged pine bark / / / / 10 / / zSubstrates were supplied with Spanish River CarbonatiteIM (Chatham-Kent Organic Epicentre in Dresden, ON, CA) at 0.56gL"1 and elemental sulfur (Canadian HydroGardens Ltd., Ancaster, ON, CA) according to recommendation (MU Extension 2008). yThe substrates that contain manure compost at 20 or 25% may have this compost substituted for Forterra vermicompost, Worm Power Gold worm castings, or yard waste compost. We recommend the investigation of nitrogen immobilization as it was not evaluated in this experiment and would be beneficial to determine whether simply increasing fertilizer inputs could overcome the negative growth effects that were seen in substrates containing higher amounts of PBC and APB. Whether the cost of adding more fertilizer outweighs the savings of excluding more expensive and environmentally harmful materials such as FP and FV will need to be investigated as well. Often, in literature, compost is not recommended at higher rates than 20%; however, composts vary widely in terms of pH, salinity, nutrient content, and physical properties. Differences are mainly attributed to the vast amount of materials that can be used to produce composts as well as the many different types of composts available, such as microbially-processed compost or worm- 196 processed VC, and the many different processes of composting available (e.g. in vessel composting, open air composting, small scale composting, etc.). When using a new batch of compost we recommend that growers try their chosen substrate on a small scale before mixing large amounts of substrate. Growers must also consider what kinds of transplants they are trying to grow when selecting the amount of compost to include in their substrate. Transplant height is often a problem for growers who plan to ship their transplants over long distances. The optimum height for tomato transplants in shipment is 10 cm. If this is the case, the grower must experiment with the amount of compost to be included in the substrate, the amount of fertilizer to be applied to the substrate, and the number of watering events. In our research, we aimed to produce larger leafier transplants but also considered the cost of applying fertilizer excessively. We found that applying fertilizer (300 mg-L"1 total N, 131 mg-L"1 P, 664 mg-L"1) two to three times a week was adequate for optimum growth and we applied water by grower's discretion (when substrates were considered dry enough to require a watering event). With the recommended substrates, our transplants were not nutrient deficient and were never allowed to reach wilting point. Growers however may produce more compact transplants for transportation if they use no more than 25% compost and reduce the number of fertilizer applications and the number of watering events. When considering the use of MC and C. rosea to suppress disease we recommend the use of C. rosea on its own especially when using a substrate that is mostly inert (such as one that contains no compost and is largely comprised of 197 PM, CC, FP, or FV) or in combination with a substrate that contains MC. Although the microbial community from the active compost may compromise C. rosea's ability to colonize the roots of the transplants, the combination of the MC's microbial community and C. rosea is better suited at preventing the colonization of roots by Pythium sp. (especially during early growth stages) than that of the MC's microbial community on its own. With that said, more research is needed to investigate ways to suppress disease with compost more effectively. The mechanisms involved in disease suppression that are associated with the inclusion of compost in growing substrates are valuable tools that still need to be uncovered and fully understood. Compost of consistent quality is essential for its widespread adoption as an alternative to conventional disease suppression; however, consistency in compost production does not necessarily ensure consistent disease suppression in practice. In order to substantiate the claim that compost can be used as a tool in disease suppression, it is first necessary to appreciate the complex nature of compost utilization. Research focusing on compost amendments in soilless organic substrates combined with microbial inoculants is crucial for better understanding of structure, function, interactions, and synergisms among microbes as potential biocontrols of plant pathogens. There is great potential to inoculate composts with disease suppressive microorganisms for more consistent disease suppression and promotion of plant growth. Naturally occurring microorganisms associated with disease suppression are: bacteria, such as Bacillus, Streptomyces, Azospirillum, Enterobacter, Flavobacterium, Pseudomonas, Burkholderia, Xanthomonas, Janthinobacterium, 198 and Flavobacterium, spp. (Boulter et al. 2002c; van Bruggen and Semenov 2000; Bashan 1998; Boehm et al. 1993; Harman 1992); actinomycetes (Huang et al. 2006; Bulluck III et al. 2002); fungi, such as Trichoderma, Gliocladium, Penicillium, Acremonium, Talaromyces, Fusarium, and Geotrichum spp. (Pantelides et al. 2009; Panina et al. 2007; Dianez et al. 2005; Dal Bello et al. 2002; van Bruggen and Semenov 2000; Hoitink et al. 1997); non-sporulating Pythium spp. (Theodore and Toribio 1995); and non-pathogenic Rhizoctonia spp. (Wen et al. 2005). New species (i.e. Subtercola pratensis and Microbacterium testaceum), however, are always being discovered (Zachow et al. 2008). Understanding how beneficial microorganisms exert their protective effects is essential for effective use of compost and microbial inoculants in the future. Creating standardized methods of compost processing and standardized tests throughout the composting process, as well as continuing research on beneficial microbial inoculants, will further increase our knowledge of composts and inoculants as tools to suppress plant diseases. Despite advances in microbial enumeration and identification, knowledge about structure and function, and interactions and synergisms among microbes is still in its infancy and the extent to which the host and the pathogen influence the microbial community is still in question. Therefore, strategies to further improve compost utilization (with and without the addition of beneficial microbial inoculants), which ultimately depends on quality, consistency, and efficiency, are still needed. 199 REFERENCES Abbasi, P.A., J. Al-Dahmani, F. Sahin, H.A.J. 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