THE EFFECTS OF FERTILIZER TREATMENTS ON THE RESIN CANAL DEFENSES OF SPRUCE AND INCIDENCE OF ATTACK BY THE WHITE PINE WEEVIL, PISSODES STROBI.
by
Lara vanAkker
B.Sc, University of Victoria, Victoria, British Columbia, 1996
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES (Faculty of Forestry) (Department of Forest Science)
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UNIVERSITY OF BRITISH COLUMBIA 2002
© Lara vanAkker, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced
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Department of fb^t SUSAC£
The University of British Columbia Vancouver, Canada
DE-6 (2/88) ABSTRACT
The white pine weevil, Pissodes strobi (Peck), is a serious pest of regenerating spruce (P/'cea spp.) in British Columbia. On the coast, damage by this weevil results in such severe stem defects and growth losses in Sitka spruce (Picea sitchensis), that planting this species is not currently recommended in high weevil hazard areas. In the interior of the province, hundreds of millions of interior spruce seedlings (P/'cea glauca x englemanii) are currently in weevil susceptible age classes. Past attempts to control this weevil have been unsuccessful. Current research is focused on identifying trees with genetic resistance to the weevil and identifying the defense traits which give rise to this resistance. While weevil resistance has been shown to be heritable, environmental factors such as plant nutrient status may alter the expression of defense traits. My goal was to determine the effects of fertilization treatments on the resin canal defenses in spruce and incidence of weevil attack. Fertilizer effects on tree size variables were measured to provide baselines for the comparison of treatment groups and to facilitate discussion on the effects of variation in tree size due to fertilizer treatment, on incidence of weevil attack. The constitutive resin canal systems of Sitka and interior spruce maintained under several fertilizer regimes were compared by histological examination of leader cross sections. Traumatic response intensity was compared utilizing mechanical wounding to stimulate a traumatic response, followed by histological examination of cross sections through the wounded stem. Incidence of weevil attack in response to fertilizer treatments was studied in a caging experiment in which weevils were given a choice between trees from different fertilizer treatments, and in a variably fertilized plantation, naturally infested with weevils. The effects of fertilization on constitutive resin canal variables were influenced by spruce genotype, but not resistance status. In general, there was a dilution in cortical resin canal defenses in response to fertilization, which included a decrease in resin canal density and an increase in depth and distance between inner resin canals. These effects corresponded with, and may have been due to increasing bark thickness in response to fertilization. In severely nutrient stressed trees, fertilization may also improve the ability of trees to produce a traumatic response. Fertilization increased the incidence of weevil attack in both Sitka and interior spruce. In interior spruce, more than twice as many trees were attacked in the intense fertilizer treatments than in the unfertilized control groups. The observed increase in weevil attack in the more intense fertilizer treatments may be attributed to increased host vigor, resulting in an increase in resources available for weevil feeding and oviposition and a dilution of cortical resin canals. It is recommended that further studies be undertaken to investigate the effects of fertilization on other factors such as resin composition and flow.
ii
N TABLE OF CONTENTS
ABSTRACT ii
LIST OF TABLES vi
LIST OF FIGURES viii
ACKNOWLEDGEMENTS xi
GENERAL INTRODUCTION 1 Spruce and Pissodes strobi (Peck) 1 Life history of the white pine weevil 2 Controlling weevil damage 4 Spruce resistance to the weevil 5 Plant defenses and resistance mechanisms 7 Fertilization, herbivores and plant defenses 12
FACTORS THAT INFLUENCE WEEVIL ATTACK RATES 17 Tree size 18 Constitutive resin canal defenses 19 Traumatic resin canal defenses 25
TRIAL 1. FERTILIZER EFFECTS ON INCIDENCE OF WEEVIL ATTACK AND RESIN CANAL DEFENSES IN SUSCEPTIBLE SPRUCE FAMILIES 30
METHODS 30 Tree material and fertilization 30 Data collection and analysis 32 Tree size 32 Constitutive resin canal characteristics 33 Traumatic resin canal defenses 35 Incidence of weevil attack 36 RESULTS AND DISCUSSION 37 Tree size 37 Constitutive resin canal defenses 40 Traumatic resin canal defenses 43 Incidence of weevil attack .47 CONCLUSIONS 50
TRIAL 2. FERTILIZER EFFECTS ON CONSTITUTIVE RESIN CANAL DEFENSES IN RESISTANT AND SUSCEPTIBLE SPRUCE GENOTYPES 51
METHODS 51 Tree material and fertilization 51
iii Data collection and analysis 52 Tree size 52 Constitutive resin canal defenses 53 RESULTS AND DISCUSSION 53 Tree size 53 Constitutive resin canal defenses 57 CONCLUSIONS 68
TRIAL 3. FERTILIZER EFFECTS ON INCIDENCE OF WEEVIL ATTACK AND RESIN CANAL DEFENSES OF INTERIOR SPRUCE OF UNKNOWN RESISTANCE STATUS 69
METHODS 69 Tree material and fertilization 69 Data collection and analysis 71 Tree size 71 Constitutive resin canal defenses 71 Traumatic resin canal defenses 72 Incidence of weevil attack 72 RESULTS AND DISCUSSION 73 Tree size 73 Constitutive resin canal defenses 76 Traumatic resin canal defenses 81 Incidence of weevil attack 82 CONCLUSIONS 84
SUMMARY AND DISCUSSION 85 Constitutive resin canal defenses 85 Traumatic resin canal defenses 90 Incidence of weevil attack 91
CONCLUSIONS 93
LITERATURE CITED 96
APPENDICES 110
Appendix 1. Statistical model for the analysis of fertilizer and family effects on tree size variables and constitutive and traumatic resin canal defenses in Sitka spruce seedlings, Trial 1 111
Appendix 2. Recipe for formalin acetic acid (FAA) 111
Appendix 3. Formulae for the calculation of constitutive resin canal variables: SZIN, SZOUT, AOCC, NMMS, DEP, GAP and BTHK 111
Appendix 4. Traumatic response rating system as used by Brescia (2000) 112
iv Appendix 5. Statistical model for the analysis of fertilizer, resistance class and genotype effects on constitutive resin canal variables in Sitka spruce somatic seedlings, Trial 2 112
Appendix 6. Statistical model for the analysis of fertilizer and replicate effects on constitutive and traumatic resin canal variables in interior spruce, Trial 3 113
V LIST OF TABLES Table Page 1 Abbreviation, description and unit of each constitutive resin canal characteristic measured to determine the effects of fertilization on spruce defenses 24
2 Origins of Sitka spruce parent trees for seedlings used in Trial 1 31
3 NPK fertilizer (Osmocote®, 8-9 month slow release formulation) applied to 1 year-old Sitka spruce seedlings in 1 gallon pots, in each of four treatments in a study of fertilization effects on spruce defenses. Treatments were applied in April 1998 32
4 Average height of 1 year-old potted Sitka spruce seedlings from eight weevil susceptible families at the start of Trial 1 (n=60 for each group) 37
5 Results of analysis of variance to determine the effects of four levels (0, 7, 16, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) on constitutive resin canal variables of 2 year-old potted Sitka spruce seedlings 41
6 Average constitutive resin canal characteristics observed in quarter cross sections of leaders from 2 year-old potted Sitka spruce maintained under four levels (0, 7, 16, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) 42
7 Summary of ANOVA to detect the effects of fertilization (FERT) and family (FAM) on traumatic response rating of 3 year-old potted Sitka spruce mechanically wounded to simulate weevil feeding and fertilized with three levels (0, 7, 25 gr) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) 46
8 Means and standard deviations (SD) of traumatic response rating, on a scale from 0-6, in 3 year-old potted Sitka spruce fertilized with three levels (0, 7, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation). Values with the same letter are not significantly different according to Tukey's HSD (p<.05) 46
9 Mean number and range of Pissodes strobi oviposition punctures and number of Sitka spruce trees with oviposition, failed and successful attacks and brood emergence, in three NPK fertilizer treatments (Osmocote®, 8-9 month slow release formulation, 0, 7, 25 g) (FERT) (n=18 trees for each group) 47
10 Identification, origin and resistance status of parent trees for Sitka spruce somatic seedlings used in Trial 2 51
VI 11 NPK fertilizer (Osmocote®, 8-9 month slow release formulation) applied to 1 year-old Sitka spruce somatic seedlings in 1 gallon pots, in each of four treatments, in a study of fertilization effects on spruce defenses. Treatments were applied in May 1999 52
12 Height (cm) prior to fertilization, of 1 year-old Sitka spruce somatic seedling from weevil resistant (R) and susceptible (S) genotypes 54
13 Results of ANOVA to detect the effects of fertilization, resistance status and genotype on height, leader length and leader diameter of 2 year-old Sitka spruce somatic seedlings fertilized with four levels (0, 3, 8, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) 55
14 Summary of ANOVA to determine the effects of fertilization (FERT), resistance status (RES), genotype (GENO) and their interactions, on characteristics of the cortical resin canal system in 2 year-old Sitka spruce somatic seedlings 59
15 Means of constitutive resin canal variables in 2 year-old Sitka spruce somatic seedlings fertilized with four levels (0, 3, 8, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) 66
16 Descriptions of fertilizer treatments employed in the BC Ministry of Forests Maximum Productivity research trial at Lodi Lake, BC 70
17 Nutrient sources for fertilization treatments applied at the BC Ministry of Forests Maximum Productivity research trial at Lodi Lake, BC 70
18 Summary of ANOVA for analysis of the effects of fertilization (FERT) and replicate (REP) and their interactions, on characteristics of the cortical resin canal system of interior spruce maintained under six fertilizer regimes 77
19 Average constitutive resin canal characteristics observed in quarter cross sections of interior spruce leaders maintained under six fertilizer regimes. Values with the same letter are not significantly different within columns, Tukey HSD (p<0.05) 78
20 Means and standard deviations (SD) of traumatic response rating in interior spruce treated with six fertilization regimes (n=20 for each group) ; 81
21 Summary of the significant effects of fertilization treatments varying in intensity, on constitutive resin canal systems of 2 year-old Sitka and 16 year-old interior spruce 86
vii LIST OF FIGURES Figure Page
1 Quarter cross section of a Sitka spruce leader 34
2 Heights of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.0) 38
3 Leader lengths of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.05) 38
4 Leader diameters of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.05) 39
5 Stem cross section of a Sitka spruce internode wounded with a 1 mm drill bit to simulate weevil feeding, in the spring of the second year of growth 44
6 Examples of traumatic responses ratings used in the assessment of the traumatic response intensity in cross sections of internodes of Sitka spruce fertilized with three levels of NPK fertilizer, and mechanically wounded during the second year of growth 45
7 Height of 2 year-old potted Sitka spruce somatic seedlings from putatively resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 56
8 Leader length of 2 year-old resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05). 56
9 Leader diameter of 2 year-old potted Sitka spruce somatic seedlings, from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different
viii within each genotype (Tukey's HSD, p>0.05). 57
10 Bark thickness of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 58
11 Number of inner resin canals per quarter cross section of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 60
12 Number of outer resin canals per quarter cross section of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S), fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 61
13 Size of inner resin canals in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 62
14 Percent of cross sectional bark area occupied by resin canals (AOCC) in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05). 62
15 Number of resin canals per square millimeter of bark (NMMS) in cross sections of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 63
16 Average distance between inner resin canals (GAP) in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 64
17 Average depth of inner resin canals (DEP) in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer.
ix Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05) 65
18 Average depth of inner resin canals (DEP) in leaders of 2 year-old Sitka spruce somatic seedlings, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different (Tukey's HSD, p>0.05) 65
19 Average height of 16 year-old interior spruce trees fertilized with six fertilizer treatments in three replicates (n=64 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05) 74
20 Average leader length of 16 year-old interior spruce trees fertilized with six fertilizer treatments in three replicates (n=64 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05) 74
21 Average leader diameter of 16 year-old interior spruce trees fertilized with six different fertilizer treatments in three replicates (n=10 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05) 75
22 Effects of fertilization and replicate on the number of outer resin canals in quarter cross sections of interior spruce leaders grown under six fertilizer regimes. Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05) 78
23 Percent of interior spruce trees with weevil damage in each of six fertilizer treatments. Treatments with the same letter are not significantly different (Tukey's HSD, p>0.05) 83
X ACKNOWLEDGEMENTS
This thesis is a manifestation of the cumulative support of many people, without which its completion would not have been possible. I offer my heartfelt thanks to Dr.
Rene Alfaro for his supervision, inspiration and guidance, and for the opportunity to work at the Pacific Forestry Centre. I thank Kornelia Lewis for her advice and guidance as a role model throughout my early days at PFC. To my committee, Drs. Rene Alfaro,
John McLean and Sally Aitken, I owe my gratitude for their encouragement, constructive criticisms and for keeping me on track. I extend my thanks to my fellow graduate students and lab mates, Dave Brescia, Paula Vera, James McCormick and Angus
Shand, for many insightful discussions on science and how to do it, and for their advice and camaraderie.
I am grateful for an exceptional field assistance team. Tia Heeley, George Brown,
Tony Ibaraki and Michelle Meier, were an efficient and accurate data and sample collecting force. Many thanks go to Leslie Manning and Terry Holmes who provided support in the microtechnique lab, to Barbara Hendel in the PFC library and to the folks who maintained the seedlings at PFC. I thank Dr. Rob Brockley for the opportunity to study his weevil infested Maximum Productivity Trials.
Funding for this study was provided by Forest Renewal Projects HQ96251-RE titled "Development of a bioassay for spruce weevil resistance in Sitka spruce and interior spruce" and TOR02001-02, titled "Nutrition and Fertilization of Interior Forests", and a partial VanDeusen Scholarship.
Finally, I would like to thank my family for their encouragement, support, and interest in my weevil work, and my husband Mark for his patience, love and sense of humour. LV
xi GENERAL INTRODUCTION
Spruce and Pissodes strobi (Peck).
The white pine weevil, Pissodes strobi (Peck) (Coleoptera: Curculionidae) also known as the Sitka spruce weevil, Engelmann spruce weevil and the spruce weevil, and known in the past as Pissodes sitchensis Hopkins and P. engelmanni Hopkins, is the most serious threat to spruce (P/'cea spp.) reforestation in British Columbia (BC), causing millions of dollars in losses to industry (Hall 1994). Damage by this weevil, as a result of repeated destruction of the terminal leader, causes growth losses and stem defects. After an attack, lateral branches compete for dominance, producing crooks and forks in the stem that reduce lumber quality and stand yield (Alfaro 1989a, 1994).
Although P. strobi does not kill its host, stunted trees are often suppressed by competing vegetation (Alfaro 1982). Depending on infestation levels, white pine weevil attack can reduce the yield of host trees by as much as 40% (Alfaro et al. 1997b), significantly increasing the length of time required to reach free-to-grow status. Heavily attacked plantations often require rehabilitation (Hall 1994). Although Sitka spruce is a valuable species, planting Sitka spruce is not currently recommended in areas on the
BC coast that are susceptible to heavy white pine weevil attack.
Spruce (Picea spp.) species are very important to the British Columbia forest industry. In the 1999/00 fiscal year, they comprised 22% of the billed annual harvest in the interior (11,902,000 cubic meters) and 2.5% (632,000 cubic meters) on the coast
(Ministry of Forests 1999/2000 Annual Report). From 1993 to 1999 over 90 million seedlings of various spruce species were planted annually, mostly in BC's interior (MoF
Annual Reports). As these large, mainly pure, spruce plantations become established and start to grow, hundreds of millions of spruce seedlings will be in age classes
l susceptible to the white pine weevil. In January 2002, the BC Ministry of Forests estimated there were as many as 47,000 hectares of pure spruce plantations (Sitka,
Engleman, and white spruces and their crosses as well as Norway spruce, P/'cea abies
(L.) Karsten,) in weevil susceptible age classes (BC Ministry of Forests 2002).
Life history of the white pine weevil
There are 29 described species of Pissodes in North and Central America. All are associated with conifers and feed on the phloem and outer wood of stems and branches (Langor 1998). While most Pissodes species attack weakened and recently dead material, some species, such as the white pine weevil and lodgepole terminal weevil (P. terminalis Hopping) attack only living trees, causing considerable damage.
Other species such as P. nemorensis Germar and P. schwartzi Hopkins, which usually attack recently dead or dying trees, have been known to attack and kill seedlings
(Langor 1998). The white pine weevil, P. strobi is considered to be the most destructive species of this genus and more research has focused on this species than any other in this genus.
The white pine weevil is distributed from coast to coast across North America, with the exception of the Queen Charlotte Islands in British Columbia. Its range extends as far north as the Yukon in Canada and as far south in the United States as central
Colorado in the west and northern Georgia in the east (Langor and Sperling 1995).
While it has been shown to feed on a variety of conifer species (Alfaro and Borden
1982), in BC the white pine weevil attacks mainly spruce species (Sitka spruce, P/'cea sitchensis (Bong.) Carr., Engelmann spruce, P. engelmannii Parry, white spruce, P. glauca (Moench) Voss, and interior spruce crosses (P. engelmannix P. glauca)) (Kiss
2 and Yanchuk 1991). In the east, the weevil attacks mainly pine (Pinus ) species
(Wallace and Sullivan 1985), primarily damaging eastern white pine (Pinus strobus L).
Other eastern hosts include Jack pine (P. banksiana Lamb.), white spruce, Norway spruce (Picea abies (L.) Karst.) and black spruce (P. mariana (Mill.) B.S.P.). Trees between the heights of 1.5 and 9 m tall are the most susceptible to attack (Turnquist and Alfaro 1996).
The white pine weevil life cycle is completed in one year and requires 888 degree days above a threshold temperature of 7.2 °C for development on the BC coast
(McMullen 1976b). In the spring (April/May), overwintered adults emerge and crawl or fly to host trees to feed on phloem and cortical tissues. Mating occurs from late April into
June and oviposition takes place from the first half of May into July. Eggs are deposited in feeding punctures at the top of terminal leaders and are covered with a fecal plug.
Larvae commence feeding as soon as they hatch, consuming the phloem beneath the bark in a downward direction. As they grow, the larvae aggregate and feed in synchrony, forming a feeding ring as they move downwards, completely girdling and killing the leader. Damage becomes evident as the needles change color and the current year's leader droops in a distinctive "shepherd's crook". In August, after completing five larval instars, pupation occurs in chip cocoons in the xylem or pith. The pupal stage lasts about two weeks and adults emerge in the early fall, chewing their way out through the bark. Adults overwinter in the duff beneath host trees or on tree boles under moss or bark scales close to the ground (Silver 1968). In BC's interior, weevil development is initiated later in the year depending on spring temperatures.
Weevil development in white spruce requires 785 degree days above a threshold temperature of 7.2 °C (McMullen 1976b, Mcintosh 1997).
3 Controlling weevil damage
Minimizing damage by the weevil has proven to be an extremely difficult task.
The following methods have been investigated or attempted in the past: clipping infested leaders (Lavallee 1989; Rankin and Lewis 1994), pheromone trapping (Hibbard and Webster 1993), pesticides (McMullen 1976a; Fraser and Heppner 1993; Fraser and
Szeto 1994; Retnakaran and Jobin 1994; Naumann et al. 1996), biological control
(Alfaro and Borden 1980; Alfaro et al. 1985; Hulme 1989; Kenis and Mills 1994; Hulme
1994), and silviculture (McLean 1989; Alfaro and Omule 1990; Taylor et al. 1994; 1996).
Despite much effort, none of these methods has proven to be operationally practical and economical as well as effective.
Alfaro et al. (1995) proposed an integrated pest management system to reduce damage by this weevil. The system proposes the use of a combination of tactics based on silviculture and genetic resistance, with other tactics (such as clipping and pesticide application) added as needed, when economically feasible and proven to be effective.
Assessment of weevil damage and continuous monitoring and forecasting of weevil population density are imperative under this system, to evaluate the need for a given tactic.
Current guidelines established in the BC Forest Practices Code require that holders of operational plans conduct pest incidence surveys and propose management strategies to reduce risks to forest resources (BC Forest Practices Code, Jan 2002).
The importance of identifying highly susceptible sites prior to planting is emphasized and for areas lacking weevil survey data, the guidelines outline methods of determining weevil hazard levels based on survey procedures or developmental requirements for
4 the weevil.
In the Prince Rupert Forest Region of BC, general weevil risk warnings have been incorporated into species selection tables (BC Forest Practices Code, Terminal
Weevil Guidebook). In certain ecosystem associations, where Sitka spruce is not the preferred crop species, inclusion of Sitka spruce is recommended, but not as pure stands. A more detailed hazard rating system has been developed for the Vancouver
Forest Region and species selection guidelines are provided for each hazard level. In medium and high hazard levels only up to twenty and ten percent of the total stocking respectively, should be Sitka spruce. Recommendations for managing weevil damage in established stands include: pruning, leader clipping, stem injected pesticides (when approved and feasible) the use of resistant stock, mixed planting and in areas with severe damage, species conversion.
A decision support tool is currently being tested that combines the BC Forest
Service Tree and Stand Simulator (TASS) with the Spruce Weevil Attack (SWAT) model, to determine weevil impacts in stands grown under different stand tending regimes and which now incorporates the possibility of using genetically resistant planting stock (Alfaro et al. 1997b).
Spruce resistance to the weevil
A severe infestation in Sitka spruce at Green Timbers (Surrey, British Columbia) in the 1930's and 40's left only two trees unattacked amongst hundreds planted (Silver
1968; Alfaro and Wegwitz 1994). These two trees and the observation of apparent differences in tolerance of weevil attack among Sitka spruce provenances (Alfaro and
Ying 1990; Mitchell et al. 1990; Ying 1991) increased interest in genetic control and
5 stimulated research to identify mechanisms of resistance. In 1984 the BC Ministry of
Forests established a small scale clonal trial to test provenances for weevil resistance with the goal of producing operational quantities of resistant trees. This test provided evidence of provenance and clonal differences in resistance to weevil attack (Ying
1991). Genetic variation resistance to the weevil was also reported in interior spruce in
1991 (Kiss and Yanchuk 1991).
The BC MoF has established fourteen trials to date, and in conjunction with the
Canadian Forest Service, private forest companies and personnel from Washington and
Oregon Forest Services, almost 30,000 Sitka and white spruce trees have been screened for weevil resistance (Alfaro et al. 2001). Spruce genotypes with heritable resistance have been found and are currently being produced for inclusion in seed orchards and BC reforestation programs. This work is summarized on the website: www.pfc.forestry.ca/entomology/weevil/resistance/resistance_e.html.
Identification of the mechanisms responsible for weevil resistance is important for the development of tree breeding programs aimed at producing improved genotypes with several resistance traits. Over the last decade, research has focused on the many facets of the weevil/host interaction with the goal of identifying resistance mechanisms that can be used for screening genetic material. A number of avenues have been explored including; physical constitutive defenses such as bark thickness, needle density, density of bark resin canals, density of sclereid cells (Kriebel 1954; Stroh and
Gerhold 1965; Overhulser and Gara 1981b; Harris et al. 1990; Tomlin and Borden
1994a; Alfaro et al. 1997a; Brescia 2000; Grau et al. 2001; O'Neill era/. 2002), inducible defenses such as the formation of traumatic resin canals (Alfaro 1995; Alfaro et al.
1996a, Tomlin et al. 1998; Brescia 2000; O'Neill et al. 2002), the chemistry of
6 constitutive and traumatic resin (Nault et al. 1999; Tomlin et al. 2000; Nault and Alfaro
2001; Martin et al. 2002), tree phenology (Hulme 1995; Alfaro et al. 2000), inhibition of reproductive maturation (Sahota era/. 1994; Leal et al. 1997; Sahota etal. 1998a,
1998b), chemical attractants and deterrents (Alfaro etal. 1979, 1980, 1981, 1984; Alfaro and Borden 1982, 1985; Tomlin and Borden 1996; Tomlin etal. 1997) and visual stimuli, such as leader length (Silver 1968; VanderSar and Borden 1977, Wood and
McMullen 1983; King et al. 1997). Current evidence indicates that resistant spruce genotypes are protected against weevil feeding and oviposition by an array of defense mechanisms that occur simultaneously (Alfaro et al. 2002).
Plant defenses and resistance mechanisms
The evolution of phytophagous insects in the Carboniferous period marked the beginning of an evolutionary "arms race" between plants and insects. The exploitation of plants by insects resulted in the evolution of plant defenses, which in turn selected for insect traits that enabled them to overcome plant defense mechanisms (Ehrlich and
Raven 1964; Berryman 1988). With the evolution of plant defenses, genetic resistance to herbivores evolved, and has been defined in a number of ways. Beck (1965), described resistance as "the collective heritable characteristics by which a plant species, race, clone, or individual may reduce the probability of successful utilization of that plant as a host by an insect species, race, biotype or individual" while Painter
(1958) defined resistant plants as those "...that are inherently less damaged or less infested than others under comparable environmental conditions in the field..."
Berryman (1988) listed three ways in which plant resistance mechanisms can operate: 1) avoid or escape the attack, 2) do nothing and tolerate the attack, 3) employ
7 defensive tactics. Avoidance or escape from herb-ivory is likely the least costly method of defense and can occur as a result of temporary or permanent invisibility to the pest
(Feeny 1976). Plant apparency or susceptibility to discovery may be influenced by size, growth form, phenology, secondary chemistry and relative abundance, and is determined both by genotype and also by various environmental influences which act on the phenotype (Feeny 1976). Tolerance is described as the ability of plants to survive under levels of infestation that would kill or severely injure susceptible plants
(Painter 1958). Plants doing nothing and tolerating the attack therefore bear only the cost of lost parts due to herbivory.
Defensive tactics can include physical barriers to insect feeding, repellents, deterrents and toxic compounds (Berryman 1988) or interference with the development and reproduction of herbivores (Feeny 1976). Defensive tactics can be classified as constitutive (pre-formed) defenses, and inducible defenses. Constitutive defenses include a multitude of chemicals and structures (thorns, trichomes, resin canals) that exist in plants growing under normal conditions, regardless of the presence of herbivores. Inducible defenses are those that develop in response to attack, such as necrosis, increased metabolic activity and the synthesis and/or production of new compounds at the wound or infection site (Cates and Alexander 1982).
Plants, insects and their interactions have evolved over history to exhibit great diversity, which enables both plants and insects to occupy a wide variety of environments. A vast array of plant defense mechanisms have evolved, and yet some plant species are better defended than others. As a result, a number of theories have arisen to explain the apparent trends in both mechanisms and levels of plant defense. A premise commonly adopted is that recognized as the optimal defense theory (Rhoades
8 1979). Since then, other theories have emerged, which attempt to explain the influence of environmental factors on the evolution of plant defenses and the phenotypic expression of defense traits.
The optimal defense theory is based on two assumptions. The first is that there is a cost associated with the production of defenses, a trade-off between defenses and growth. Therefore, resources will be allocated to defense in ways that optimize that investment. The second assumption is that organisms evolve and allocate defenses in the way that maximizes individual inclusive fitness. In addition, it is assumed that herbivory is the main selective force influencing quantitative patterns of secondary metabolism (Rhoades 1979). Adopting the concepts of the optimal defense theory,
Berryman (1988) predicted the types of defenses that will evolve given various selection pressures. When the impact of herbivory is small, or defensive traits are costly, none will be selected for and tolerance will be employed. If a certain degree of herbivory can be tolerated without much loss in fitness, avoidance is a reasonable strategy because, although avoidance is never completely effective because of counter-adaptations by herbivores, it is relatively cheap. According to Berryman (1988) preformed, or constitutive defenses, have a fixed, genetically determined cost. In the case of defensive chemicals, they are metabolically expensive and are therefore expected to evolve in long-lived plants that do not require fast juvenile growth or massive reproductive efforts to survive (Berryman 1988). While constitutive defenses can be built up during periods of energy surplus, the success of inducible defenses is dependent on the current availability of energy. However, while the cost of constitutive defense is fixed, the cost of inducible defenses is only incurred when defense is necessary.
Because defenses are costly, resource availability is likely to affect the types of
9 defense evolved. The evolution of quantitative chemical defenses (high-cost, dosage dependent defenses, sensu Feeny 1976) may be favored over qualitative defenses
(present in low concentrations and therefore represent low cost) depending on available resources (Coley et al. 1985; Coley 1987). For example, Coley et al. (1985) proposed that low resource availability should select for quantitative defenses because species adapted to these conditions have long-lived leaves, which are costly to replace and are exposed to herbivory for long periods of time. Species inhabiting resource-rich environments tend to shed their leaves frequently and therefore often utilize qualitative defenses. Bryant etal. (1983) proposed that in plants adapted to nutrient-limited environments, chemical defenses are largely carbon based (metabolically in• expensive), while plants evolving in nutrient-rich environments tend to utilize nitrogen based (metabolically expensive) defenses.
While plant resistance to herbivory is genetically based, the phenotypic expression of defense traits can be influenced by environmental pressures.
Factors such as light, temperature, relative humidity, plant nutrient status and air pollution have all been shown to alter the expression of resistance in some plant/herbivore systems (Kennedy and Barbour 1992). According to Loomis (1953) resources tend to be allocated to growth when conditions are favorable. Any factor that limits growth without reducing photosynthesis will tend to increase any differentiation response of which the plant is capable, such as the formation of cellular inclusions responsible for the production of secondary metabolites. Bryant et al. (1983) formulated a similar, but more specific hypothesis to explain the effects of fertilization and shade on phenotypic variation in secondary metabolism. They proposed that moderate nutrient deficiencies will limit growth with relatively little reduction in photosynthesis causing
10 carbohydrates to accumulate. Carbohydrates accumulated in excess of growth are available for allocation to carbon-based secondary metabolites. Increased nutrient availability causes a decrease in secondary metabolism as growth receives carbon allocation priority. As growth is limited by other factors, nitrogen may also be assimilated in excess of growth requirements and allocated to the production of N-based secondary metabolites. Insufficient light also affects the carbon/nutrient ratio by causing photosynthesis and carbohydrate concentrations to decline. Under low light conditions, there is a greater reduction in growth than in nutrient absorption, resulting in an accumulation of nutrients above levels required for growth. Under these circumstances one would expect an increase in nitrogen based defenses since carbon-based defenses are relatively more expensive (Bryant et al. 1983).
Observations of trends in herbivore damage under various environmental conditions, led to the formulation of the plant stress and plant vigor hypotheses, that associate plant physiological condition to insect performance. The plant stress hypothesis predicts that stressed plants will serve as better hosts for herbivores (White
1984), and is based on the assumption that plant stress results in increased nutritional value (due to increased availability of amino acids) and a reduction in the synthesis of defensive chemicals. While a significant number of studies have indicated a positive correlation between insect abundance and declining environmental conditions, this does not necessarily mean that changes in the host plant are responsible for changes in insect abundance. Direct environmental effects on the insect, such as a more favorable climate or a decrease in natural enemies, may be responsible for increases in abundance (Mattson and Haack 1987).
The plant vigor hypothesis predicts that vigorous plants are more favorable to
n certain kinds of herbivores (Price 1991). This theory is supported by incidents of damage to young, open-grown forestry trees, with reduced or no attack on mature trees
(Price 1991) and is in accordance with the hypotheses of resource allocation to defense
(Bryant etal.. 1983; Coley etal. 1985). Price (1991) proposes that the plant vigor and plant stress hypotheses are the equivalent of two ends of a spectrum, the extreme ends being herbivores that favor vigorous plants and others that favor stressed plants.
Herbivore response to plant conditions may be influenced by insect feeding guild (Price
1991; Meyer and Root 1996). According to Price (1991) the herbivore species that are most intimately involved in plant growth processes will conform the closest to the plant vigor hypothesis. Insects such as gall formers and stem borers, whose females oviposit at the site of larval feeding and whose larvae are endophytic, tend to attack vigorously growing trees and shrubs. Studies that support the plant stress hypothesis usually involve defoliators living in mature or overmature stands whose females are unselective with respect to larval feeding and whose larvae feed generally on foliage.
Fertilization, herbivores and plant defenses
Successful reforestation in BC requires that plantations reach free-growing status within a specified number of years after harvest. At sites where growing conditions are less than optimal, silviculture practices can be employed to achieve maximum yield and increase stand production. Fertilization can also be used strategically to accelerate the development of specific age classes and timber types (BC Forest Practices Code Forest
Fertilization Guidebook). According to the Forest Fertilization Guidebook of the BC
Forest Practices Code, silviculture prescriptions must contain fertilizer treatments if they are necessary for the stand to meet required growth levels. In BC's interior, fertilization
12 is a common practice, used to supplement sites with nutrient deficiencies, especially in nitrogen, due to harvesting and site preparation practices (Brockley et al. 1992). In coastal BC, fertilization has been recommended to enhance regeneration at sites where
"salal-check" has caused chlorosis and suppressed height growth, due to below ground competition (Peterson etal. 1997).
As well as improving annual growth increments, increased soil nutrient availability due to fertilization can influence crop relationships with weed species, pathogens and insect pests (Jones 1976; Scriber 1984) as well as influence wood properties (Smith et al. 1977). Depending on the treatment, fertilization can have a number of detrimental effects including stimulated growth of competing vegetation and increased susceptibility to pathogens and insect pests. A number of examples of detrimental effects exist in BC and are listed in the BC Forest Practices Code Forest
Fertilization Guidebook (1995). The fertilization of Sitka spruce stands, is recommended as a low priority as it may increase the incidence of attack by Pissodes strobi (Xydias and Leaf 1964). On Vancouver Island, a fertilizer study initiated to bring young chlorotic
Sitka spruce out of check, resulted in high incidence of weevil attack in fertilized trees
(Weetman et al. 1989). Nitrogen-fertilized Douglas-fir in the Intermountain region
(Northern Idaho, Montana, and eastern Washington) is thought to be more susceptible to armillaria root rot (Armillaria ostoyae). In the interior, sharp increases of red squirrel feeding damage on lodgepole pine have been observed after fertilization (BC Forest
Practices Code Forest Fertilization Guidebook 1995). The Forest Fertilization
Guidebook recommends consultation with local forest health specialists and stand tending foresters when dealing with these organisms in areas being considered for fertilization treatments.
13 Understanding the effects of fertilizer treatment on insect population growth is imperative to the development of effective silviculture regimes. Nitrogen is a critical element in the growth of all organisms, playing a central role in metabolic processes, cellular structure and genetic coding (Matson 1980). Many organisms respond to nitrogen fertilization with enhanced growth, reproduction and survival, which suggests that nitrogen is a limiting factor. Because animals have vastly higher nitrogen requirements relative to plants, the nitrogen content of a plant is vitally important to herbivores (Matson 1980). Fertilizer-induced physiological and morphological alterations in host plants affect host selection, survival, growth rates and reproduction of insect herbivores (Scriber 1984). However, despite a considerable amount of research, the ability to predict insect responses to host fertilization remains limited. While the majority of studies indicate positive responses by herbivores to fertilization, a large proportion of studies indicate no response and a smaller proportion indicate negative responses
(Waring and Cobb 1992). This may be explained by the preference of some herbivore feeding guilds for vigorous hosts while others prefer stressed hosts (Price 1991).
As well as increasing the availability of nitrogen to herbivores, fertilization may influence plant defenses by shifting the allocation of carbohydrates away from the formation of carbon-based defenses to growth (Bryant et al. 1983). The impact of nutrient availability on the production of secondary metabolites was reviewed by
Gershenzon (1984), who found that while nitrogen and sulfur deficiencies decreased the production of most kinds of secondary compounds containing these elements, stress usually increased concentrations of secondary metabolites.
Recent studies of the effects of fertilization on plant defenses have focused mainly on the production of secondary metabolites including phenolics (Muzika and
14 Pregitzer 1992; Kyto etal. 1996, 1998; Bjorkman etal. 1998; Stout et al. 1998;
Wainhouse etal. 1998; Warren etal. 1999; Keinanen etal. 1999; Viiri etal. 1999), terpenoids (Kainulainen etal. 1996; Bjorkman era/. 1998; Honkanen et al. 1999;
Jarzomski etal. 2000; Wainhouse era/. 2000), proanthocyanidins (Warren etal. 1999), resin flow and composition (Holopainen et al. 1995; Kainulainen et al. 1996; Kyto et al.
1996, 1998; Wainhouse etal. 1998; Warren ef a/. 1999; Viiri etal. 1999) and alkaloids
(Gerson and Kelsey 1999). Results indicate with few exceptions, a general decrease in secondary compounds with nutrient availability. Observing an increase in terpenoid concentration while phenolic compounds decreased, Bjorkman etal. (1991, 1998) proposed that differences in the way that groups of compounds are stored may account for their opposite responses to fertilization. Keinanen ef al. (1999) found that some phenols decreased while others increased, and suggested that different branches of the biosynthetic pathway of phenols may compete for substrates, leading to the differential accumulation of phenolic compounds. Different responses in. phenols and terpenes led
Honkanen et al. (1999) to propose that changes in growth and concentration of defensive compounds may have a biosynthetic explanation instead of an evolutionary or ecological explanation, because protein synthesis (resulting in growth) competes with phenolic synthesis but not with terpenoid synthesis.
Other recent studies have included fertilizer effects on induction of secondary metabolite production (Bryant et al. 1993; Stout etal. 1998) and have produced conflicting results. Bryant et al. (1993) found that nitrogen fertilization reduced delayed- inducible resistance to vertebrate grazing in Alaska paper birch, while Stout et al. (1998) found that polyphenol oxidase and proteinase inhibitors were not influenced by nitrogen fertilization. Stout ef al. (1998) suggested that the effects of resource availability on a
15 natural product likely depends on a number of factors including the product's rate of turnover, typical concentration, composition, pathways involved in the product's synthesis and it's role in plant defense.
While resistance to weevil attack has been shown to be a highly heritable trait
(Ying 1991; Kiss and Yanchuk 1991; Kiss etal. 1994; King etal. 1997), environmental factors may influence the performance of resistant stock (Loomis 1953; Bryant et al.
1983; Alfaro et al. 1996b). The use of fertilization as a silviculture practice in the BC forest industry stresses the need to investigate further its effects on herbivore populations and plant defenses. The physiological and morphological changes in spruce, caused by fertilization, may affect host selection by P. strobi, and influence the phenotypic expression of defense traits, affecting resistance to this weevil. In order to effectively utilize genetic resistance in weevil management programs, knowledge of the effects of fertilization on plant resistance and defense traits, is necessary to facilitate effective selection and deployment of genetically resistant spruce.
16 FACTORS THAT INFLUENCE WEEVIL ATTACK RATES
A number of tree characteristics have been found to influence weevil preference or have been correlated with host susceptibility, such as tree height, leader length and diameter (Sullivan 1961; Silver 1968; Alfaro 1989b; Alfaro etal. 1993; King etal. 1997), bark thickness (Stroh and Gerhold 1965; Tomlin and Borden 1997a), chemical composition (Alfaro etal. 1980, 1981, 1984; Harris etal. 1983; Alfaro and Borden 1985;
Tomlin and Borden 1997b; Tomlin etal. 1997; Nault etal. 1999; Nault and Alfaro 2001), distribution of cortical resin canals (Stroh and Gerhold 1965; Alfaro 1996a; Tomlin and
Borden 1997a), traumatic resin production (Alfaro 1995, 1996a; Brescia 2000; Tomlin et al. 2000; O'Neill etal. 2002), needle distribution (Sullivan 1961; Harris etal. 1990), plant stress (Lavallee etal. 1994a,b), climate and microclimate conditions (McMullen
1976a,b; Wallace and Sullivan 1985; McLean 1989; Taylor et al. 1996), predator and parasite population levels (Alfaro 1996b), antibiotic attributes (Sahota etal. 1994,1998b,
2001; Leal et al. 1997) and tree phenology (Hulme 1995; Alfaro et al. 2000; Brescia
2000).
Because weevil feeding and oviposition are limited by factors such as bark thickness, leader length and diameter (Kriebel 1954), it is expected that fertilization will enhance such host attributes and therefore, will increase tree susceptibility to weevil attack. Fertilization may also influence weevil preference and host susceptibility by increasing nitrogen content thus improving the quality of food available for weevil consumption and by shifting resource allocation away from the formation of defense compounds (Bryant et al. 1983). Increased susceptibility to weevil attack due to fertilization would also be in accordance with the plant vigor hypothesis, which suggests that vigorous plants are more favorable to certain kinds of herbivores (Price 1991).
17 In this study I investigate the effects of fertilization on various parameters related to tree and leader size and to constitutive and induced resin canal defenses of spruce.
Using the data collected herein, I explore the relationship between incidence of weevil attack and fertilizer effects on tree size and host defenses.
Tree size
Tree size can influence plant-insect interactions in a number of ways, for example, by affecting host selection or suitability. Numerous studies have explored the effects of tree size variables on the interaction between weevils and their hosts.
Spruce trees are most susceptible to weevil attack when they are about 4-5 years old (Alfaro 1982), and while they are from 1.5 to 9 m tall (Turnquist and Alfaro
1996). The susceptibility of a stand decreases as the trees reach heights above 12 m
(Harris et al. 1968; McMullen 1976a). Within a Sitka spruce stand, the greatest incidence of attack occurs in trees from three to six meters tall (Silver 1968, McMullen
1976a). Weevils seem to prefer vigorous hosts, since fast growing trees with long, thick leaders are more susceptible to attack than trees with smaller leaders (Silver 1968,
Gara et al. 1971, Alfaro et al. 1993). According to Sullivan (1961), white pine leaders 4 mm or less in diameter are rejected as hosts for the weevil. He also found that the extent of weevil attack (measured by the length of the leader in which oviposition occurred), increased in relation to increased diameter.
Comparing attack rates among spruce families, Alfaro et al. (1993) found that tall
Sitka spruce families were more susceptible to weevil attack, whereas King et al. (1997) found that more vigorous interior spruce families (families with longer leaders) were more likely to have effective resistance to weevil attack. Explaining this apparent
18 discrepancy, King et al. (1997) hypothesized that while favorable environmental effects influence individual trees to make longer, thicker leaders that are more attractive to the weevil, genotypic effects act independently to reduce susceptibility to weevil attack.
That stand growth and yield can be influenced by the use of fertilizers is indisputable. Although it is not the goal of this thesis to determine the effects of fertilization on tree size, the tree sizes obtained by the various fertilization treatments will be reported to establish base lines for the comparison of treatment groups and to facilitate the discussion of the possible influences of fertilization on susceptibility of trees to weevil attack.
Constitutive resin canal defenses
The resin canal systems in conifers are important components of constitutive or preformed, defense systems. Primary resin secreted by cells lining the resin canals, is considered to be the first line of defense of conifers against invading organisms
(Berryman 1972). Like many conifers, Picea and Pinus spp. possess well developed systems of vertical and horizontal resin canals in the xylem and cortex, which make up the primary resin canal system (Berryman 1972).
Cortical resin canals, produced by the apical meristem (Werkerand Fahn 1969) form at the base of young scales, on either side of the procambial strand (Charon et al.
1986). The initial cells of the duct undergo divisions in various directions, but mainly periclinal to the future duct cavity (Werker and Fahn 1969). The formation of the lumen is initiated schizogenously (by the separation of cells to form a space) and enlarged by lysigenous processes (by the dissolution of cells to form a space) (Charon et al. 1986).
Mature, differentiated ducts are elongated structures consisting of an intercellular space
19 surrounded by epithelial cells which are surrounded on their outer side by one to three layers of sheath cells (Wu and Hu 1997). The production of resin by secondary metabolism takes place in the epithelial cells (Werker and Fahn 1969).
In Sitka spruce, cortical resin canals form a three dimensional network in the stem, involving dichotomous branching of larger canals to form smaller ones, some fusion of adjacent canals and occasionally the spontaneous origin of small canals in the outer cortex. The size, number, distance apart and shape of resin canals varies with position in the stem. There is a continuous ring of larger resin canals located toward the inside of the cortex (inner resin canals) and smaller canals located within the sterigmata ridges (outer resin canals) (Jou 1971). Studying very young seedlings, Suzuki (1979) found that the cortical resin canal network of white spruce is similar to that of Sitka spruce. However, he did not observe outer resin canals in the cortex of either species.
The network of resin canals in spruce is in effect, a physical and chemical "mine field", which weevil adults and young larvae avoid during feeding and oviposition (Stroh and Gerhold 1965; Alfaro 1995). The inundation of egg and larval chambers by resin is a major mortality factor of the weevil (Silver 1968; Overhulser and Gara 1981; Dixon and Houseweart 1982; Therrien 1995). Both the physical and chemical properties of resin contribute to defense. The severing of resin canals results in a flow of resin which acts to flush, cleanse and protect wounded tissue (Berryman 1972). The main chemical components can act as repellents, deterrents or toxicants and are mainly monoterpenes, diterpene resin acids, sesquiterpenes, terpene alcohols and fatty acids
(Berryman 1972).
The distribution of resin canals in the cortex affects the amount of food and space available for feeding and oviposition, and may influence host selection as well as
20 weevil survival. Stroh and Gerhold (1965) observed that feeding excavations were abandoned when they came into immediate contact with outer resin canals or when difficulty was encountered while feeding around a canal. They proposed that the weevil then retreats and seeks a new site for feeding and oviposition efforts, which may be on the same or a different host tree. However, adult weevils are not always successful in avoiding resin canals as observed by Wallace and Sullivan (1985) who noted resin flow from weevil feeding punctures.
The probability of weevils encountering a resin canal depends on the distribution and size of canals in the cortex. A number of variables describing resin canal distribution have been explored and related to weevil feeding or host resistance. Stroh and Gerhold (1965) found that the width and depth of weevil feeding cavities were correlated with the depth of the inner resin canals in eastern white pine. Resistant hosts tend to have more outer resin canals than susceptible hosts (Plank and Gerhold 1965;
Tomlin and Borden 1994). Alfaro etal. (1997a) and Grau etal. (2001)found that resistant white and Sitka spruce had significantly more dense resin canals, than susceptible spruce of the same species. Tomlin and Borden (1997a) reported that resistant Sitka spruce had higher densities of outer resin canals in combination with thinner bark. Brescia (2000) found that in resistant trees, a greater percentage of the bark area was occupied by resin canals, than in susceptible trees. This was due to the combination of larger resin canals and thinner bark in resistant trees.
In spruce, the constitutive resin canal system is thought to function in defense by decreasing the available space in the cortex for feeding and oviposition, thereby deterring adults. Resin flow from severed canals causes egg, larval (mainly young larvae) and pupal mortality. The distribution and size of cortical resin canals influences
21 the efficacy of this defense system not only by influencing the amount of available space but also by influencing the quantity of resin produced. Resin flow, the concentration of resin in tissues, and resin chemistry are thought to depend on a number of factors, including the number, size and density of resin canals (Schopmeyer etal. 1953; White and Nilsson 1984; Bjorkman etal. 1991; Blanche etal. 1992; Tomlin etal. 1996).
While it has been shown that weevil resistant spruce genotypes tend to have similar bark resin canal characteristics (Plank and Gerhold 1965; Tomlin and Borden
1994; Alfaro et al. 1997a; Grau et al. 2001; Brescia 2000) and that resin canal frequency is genetically controlled (White and Nilsson 1983), the expression of resistance traits may be influenced by environmental factors (Cates and Alexander
1982). However, few studies have been undertaken to explore environmental effects on resin canal defenses. Studying constitutive and induced resinosis of Sitka spruce
Tomlin et al. (2002 in prep.) report limiting effects of overstory shading on resinosis.
Smith et al. (1977) studied the effects of fertilization on wood properties of Corsican pine and found that nitrogen fertilization increased the radial diameter and cross sectional area of xylem resin canals. Bjorkman etal. (1991) suggested that the formation of resin ducts in pine needles is limited by nitrogen availability. The effects of nitrogen fertilization on defenses in Scots pine needles was studied by Bjorkman etal.
(1998), who found that resin acid concentration was higher in fertilized trees, and was positively correlated with resin canal density. Studying fertilized Norway spruce, Kyto et al. (1996) found that the number of constitutive xylem resin canals correlated positively with tree vigor and that there were indications of a potential increase in this constitutive defense after fertilization. The size of needle resin ducts in Sitka spruce was found to be
22 positively correlated with increased tree growth due to nitrogen fertilization (Wainhouse et al. 1998). Kainulainen et al. (1996) also found higher numbers of resin canals in mature needles of nitrogen fertilized Scots pine seedlings.
While fertilization may have direct effects on the processes influencing resin canal formation, fertilizer effects on tree size and morphology also influence the size and distribution of cortical resin canals. A dilution in cortical resin canal density has been observed with increasing stem diameter within an internode (Jou 1971) and between internodes (Alfaro 1996a). Brescia (2000) also observed that the density of cortical resin canals decreases over the growing season, as shoot diameter increases.
In white spruce, the number and density of resin canals have been found to correlate positively with height growth rate (Alfaro et al. 1997a). Increased growth due to fertilization may also influence the amount of space in the cortex available for weevil feeding, by affecting bark thickness. These observations indicate that the effects of fertilization on resin canal distribution may not only be direct, by influencing resin canal formation, but also indirect by influencing tree and shoot size.
The dilution of resin canal density with increasing shoot diameter may also correspond with an increase in the average distance between inner resin canals in the cortex. While this variable has not been explored in past studies, it may have a significant effect on weevil behaviour. In order for adult weevils to avoid resin canals while feeding and forming oviposition chambers, the average distance between inner resin canals must be at least as great as the average diameter of a weevil's snout (0.4 ±
0.02 mm, Manville et al. 2002). Feeding by late instar larvae may also be impeded by inner resin canals that are separated by distances smaller than larval head capsule sizes.
23 According to allocation theories of plant defense, under favorable growing conditions, such as those created by optimal fertilization, growth receives resource allocation priority over secondary metabolism and differentiation processes (Loomis
1953; Bryant et al. 1983). Following this theory, it may be predicted that fertilization will constrain differentiation processes responsible for the development of resin canals, resulting in less dense canals in the cortex of fertilized trees. However, since growth processes would be promoted, average resin canal size, corresponding with bark growth, should be greater in fertilized trees.
The effects of fertilization on the size and distribution of cortical resin canals in spruce leaders were studied using different spruce material in each of three trials. The following hypotheses were tested: a) fertilization causes a decrease in the density of cortical resin canals, b) fertilization affects the distribution and size of resin canals in the cortex, c) fertilization affects the phenotypic expression of weevil resistance in spruce genotypes. The constitutive resin canal characteristics that were examined for each trial are listed and defined in Table 1.
Table 1. Abbreviation, description and unit of each constitutive resin canal characteristic measured to determine the effects of fertilization on spruce defenses.
Abbreviation Description Unit NIN number of inner resin canals - NOUT number of outer resin canals - SZIN size of inner resin canals mm2 SZOUT size of outer resin canals mm2 NMMS number of resin canals per square millimeter of bark No./ mm2 AOCC percent of bark* occupied by resin canals - DEP average depth of inner resin canals beneath the bark mm GAP average distance between inner resin canals mm BTHK bark thickness mm "throughout this thesis I have adopted the definition of bark given by Esau (1977). Bark: A nontechnical term applied to all tissues outside the vascular cambium or the xylem.
24 Traumatic resin canal defenses
While plants have evolved various adaptive strategies to defend against herbivores, herbivores have evolved an array of strategies to counter these defenses and facilitate the exploitation of their hosts (Panda and Khush 1995; Rausher 1996).
Alfaro et al. (1999) hypothesized that the white pine weevil has developed a number of counter-adaptive strategies for overcoming constitutive resin canal defenses in its spruce hosts, which include avoidance and deactivation of cortical resin canals. Stroh and Gerhold (1965) observed that adults which encountered the epithelial cells of a resin canal when feeding, continued excavating but reoriented the direction of feeding so that it proceeded around the canal. Thus, adult feeding as well as gregarious feeding by larvae in the cortex, consume the supportive tissues surrounding the resin canals causing collapse and de-activation of the canals (Stroh and Gerhold 1965, Alfaro
1996b; Alfaro et al. 1999).
In conifers that don't have constitutive defenses or in which constitutive defenses have been successfully avoided or de-activiated, attacking organisms may be faced with the tree's induced defenses, which are mobilized in response to trauma (Berryman
1972). The formation of traumatic resin canals in the xylem and the production of traumatic resin, is an induced defense mechanism that is widespread among the
Pinaceae and may have evolved in response to insect attack or fungal invasion
(Berryman 1972)1. Traumatic resinosis is a dynamic secretion of secondary metabolites synthesized in response to wounding (Tomlin et al. 2000). In the case of bark beetles, extensive traumatic resin production inundates beetle galleries, repelling the invading insects and causing heavy adult and larval mortality (Safranyik 1988).
1 The induced formation of resin canals in the xylem will be subsequently referred to as the traumatic response.
25 The formation of traumatic resin canals in spruce, in response to weevil attack and mechanical wounding, has been observed in white pine, Sitka, white and Norway spruces as well as interior spruce crosses (Alfaro 1995, Alfaro et al. 1996a; Tomlin
1996; Tomlin et al. 1998; Lavallee et al. 1999; Brescia 2000; O'Neill et al. 2002) and has been correlated with weevil resistance (Tomlin etal. 1998; O'Neill etal. 2002). The inundation of feeding and oviposition cavities by traumatic resin causes mortality of eggs and young larvae of white pine weevils feeding on white spruce (Alfaro 1995).
The formation of longitudinal (vertical) traumatic resin canals in the xylem of
Norway spruce stems in response to artificial wounding, was studied by Nagy et al.
(2000). They found that the initial stages of longitudinal traumatic resin canal development were visible between six and nine days after wounding. Small clusters of swollen, irregularly shaped, dividing cells appeared in the region of the cambium. The dividing cells form a callus of parenchyma cells, from which the wound resin ducts arise
(Shrimpton 1978). After eighteen days, the clustered cells had begun to differentiate into traumatic resin canal epithelial cells with dense cytoplasm and enlarged nuclei (Nagy et al. 2000). The resin canal lumens formed schizogenously in the centre of the clusters.
By day thirty-six, the epithelial cells were fully developed and had round profiles and cytoplasm rich with plastids.
Spruce species have a complex network of both longitudinal and transverse resin canals in the xylem which can arise as either constitutive or induced canals (Panshin and de Zeeuw 1980). Constitutive and induced canals are thought to develop similarly
(Panshin and de Zeeuw 1980), but differ in their distribution and in the chemistry of the resin they produce. Normally, constitutive canals occur singly in spruce species, scattered throughout the annual ring, while traumatic resin canals are produced in
26 response to injury and often occur in bands that are concentric with the annual ring boundary (Alfaro 1995). The formation of longitudinal traumatic resin canals is not limited to the immediate wound area, but has been observed to extend around the circumference of the xylem and extend throughout the wounded internode above and below the wound (Alfaro etal. 1996a). Christiansen etal. (1999), studying Norway spruce observed traumatic resin canals up to several meters above the wound site.
Resin produced in response to wounding is thought to differ in composition from constitutive resin, having higher concentrations of phenols, terpenes and diterpene resin acid, especially close to the wound site (Werner and lllman 1994; Klepzig et al. 1995;
Tomlin et al. 2000).
The intensity of the traumatic response to white pine weevil attack can be measured by the number of complete and incomplete rings of traumatic resin canals in the xylem. Response intensity varies with weevil attack category, wound intensity, phenology, host species and genotype, and has been correlated with weevil resistance
(Alfaro et al. 1996a; Tomlin 1996; Tomlin et al. 1998; Brescia 2000; O'Neill et al. 2002).
White spruce trees with failed weevil broods (oviposition occurred but brood weevils did not emerge), had much higher traumatic response than trees in which the attack succeeded (Alfaro et al. 1996a). Response intensity was also greater in resistant white spruce families than in susceptible families (Tomlin et al. 1998; Brescia 2000), with resistant trees producing multiple rings of traumatic resin canals in the xylem more frequently than susceptible trees (Tomlin et al. 1998). Among Sitka spruce genotypes,
Brescia (2000) found that the highest response occurred in early flushing Sitka spruce trees. The traumatic response of Sitka spruce is not as intense as that of white spruce
(Tomlin 1996), and in the Sitka x white spruce introgression zone, the traumatic
27 response increased with the proportion of the genome represented by white spruce
(O'Neill et al. 2002). This suggests that the relative importance of the traumatic response as a defense mechanism differs in Sitka and white spruce.
The resistance status of the tree has also been shown to be correlated with the timing of traumatic resin canal development, with resistant trees inducing traumatic canals earlier than susceptible trees (Tomlin etal. 1998). Both the timing and intensity of the traumatic response are thought to contribute to successful defense (Berryman and Ashraf 1970; Shrimpton 1978; Alfaro etal. 1996a).
The ability of a tree to respond to invading organisms using induced defenses is thought to depend in part, on tree vigor (Shrimpton 1978; Paine and Stephen 1987).
Traumatic defenses require immediate access to energy reserves (Berryman 1988;
Nebeker ef al. 1993) and the formation of complex biochemical and anatomical structures such as traumatic resin canals puts heavy demands on plant resources
(Nagy et al. 2000). These observations suggest that trees that are at an energetic disadvantage due to limited resources or attack by insects or disease, may be less capable of producing an effective traumatic response. This hypothesis seems to contradict the theory that limited resource levels promote differentiation processes and the synthesis of secondary metabolites. However, it is possible that in environments where growth is limited, the levels of photosynthate produced may not be sufficient for the production of an energy expensive traumatic response.
The goals of this thesis were to study the effects of fertilization on spruce with respect to 1) constitutive and 2) traumatic resin canal defenses, and 3) susceptibility to
Pissodes strobi attack. Reported here are the results of three trials, each conducted on different groups of spruce material subjected to different series of fertilization regimes.
28 The tree material included two groups of young potted trees (from putatively weevil susceptible Sitka spruce seedlings, and putatively resistant and susceptible Sitka spruce somatic seedlings) and a group of young interior spruce (P. glauca x P. englemanni) trees of unknown resistance status, in a plantation setting.
29 TRIAL 1. Fertilizer effects on incidence of weevil attack and resin canal defenses in susceptible spruce families
METHODS
Tree material and fertilization
This trial was initiated with the goal of determining the feasibility of manipulating the defenses of spruce against attack by the weevil using fertilizer treatments. The effects of fertilization on the constitutive and traumatic resin canal systems as well as the incidence of attack in fertilized trees, were studied. Eight families of Sitka spruce, four half-sibling2 and four full-sibling families, all considered susceptible to weevil, were utilized in this trial.
The Sitka spruce families used in this trial were deemed susceptible based on the results of field observations of weevil attack in replicated trials established by the BC
Ministry of Forests and measured by the Canadian Forest Service (Alfaro et al. 2001).
In these studies, families were considered to be resistant if less than 25 percent of the trees sustained weevil attacks, whereas they were susceptible if more than 50 percent of the trees sustained attacks (George Brown, Canadian Forest Service, Victoria, BC, pers. comm. 2002).
This study was completed over two growing seasons, from March 1998 until
November 1999. One year-old seedlings were provided by Pacific Forest Products
(now Western Forest Products), Saanich Forestry Centre in March 1998. Eight Sitka spruce families, assumed to be weevil susceptible, were used. Four of the families consisted of full-sibling (families: 36x98, 61x67, 92x118, 125x59) and the remaining four families were open pollenated (families 38, 39, 40 and 658). The origins of the parent
2 Families may be 'half sibling' where open pollination results in only the mother being known, or 'full sibling' where pollination was controlled and both parents are known.
30 trees are given in Table 2. The families used in this study ranged in origin from northern
Washington to northwestern Vancouver Island.
Table 2. Origins of Sitka spruce parent trees for seedlings used in Trial 1.
Parent No. Location Elevation (m) Latitude (°) Longitude (°) 36 Kauwinch R 75 50 12 127 15 38 Green Tbr 0 49 9 122 50 39 Green Tbr 0 49 9 122 50 40 Green Tbr 0 49 9 122 50 59 Malksope R 0 50 9 127 25 61 Malksope R 10 50 9 127 25 67 Malksope R 75 50 9 127 25 92 San Josef 152 50 37 128 3 98 San Josef 244 50 40 128 5 118 Port Eliza 229 49 52 127 7 125 Yellow Bl 213 49 52 127 8 658 Forks 120 48 4 124 18
Sixty seedlings from each family were planted into 1 gallon pots in March 1998 using the following soil mixture: 1 bale (4 cubic ft compressed) Premier Brand peat, 2 cubic ft medium washed sand, 2 cubic ft coarse vermiculite, 250g Micromax
Micronutrients, 750g General Purpose Dolomite (IMASCO Minerals INC) and maintained in an open air compound at the Pacific Forestry Centre (PFC), Victoria, BC.
In early April 1998 the seedlings in each family were randomly divided into four groups and each group was fertilized with a different level of 18-7-12 fertilizer (Nitrogen,
Phosphorus, Potassium (NPK), Osmocote®, 8-9 month slow release formulation).
Fertilizer application rates for each treatment are given in Table 3. The fertilizer was spread evenly on the top of the soil and half an inch of forestry sand was added to cover the fertilizer.
In the third week of April 1999, the forestry sand and residual fertilizer were scraped from the surface of the soil and the seedlings were re-fertilized using the same
31 methods as in the previous year.
Table 3. NPK fertilizer (Osmocote®, 8-9 month slow release formulation) applied to 1 year-old Sitka spruce seedlings in 1 gallon pots, in each of four treatments in a study of fertilization effects on spruce defenses. Treatments were applied in April 1998.
Fertilizer treatment Amount applied (g) Control 0 Low 7 Medium 16 High 25
The effects of fertilization on the following tree characteristics were studied: height, leader length and diameter, constitutive resin canal density, intensity of traumatic resin canal response, and incidence of weevil attack.
Data collection and analysis
Tree size
Seedlings were measured in March 1998 prior to the initial fertilization treatment and again in the third week of April 1999. Height measurements were made from the root collar to the tip of the apical bud, using a measuring tape. Leader diameter was measured with a Vernier caliper, approximately 1 cm below the base of the apical bud.
To assess the effects of family and fertilization on tree size variables, analysis of variance (ANOVA) was performed using STATISTICA® software from StatSoft, Inc.
(2300 East 14th St., Tulsa, OK 74104) (for model see Appendix 1). Type III sums of squares were calculated using the general linear model. Fertilization was treated as a fixed effect. Tukey's HSD was used to detect differences between fertilizer groups within families.
32 Constitutive resin canal characteristics
After one season of growth, under the initial fertilization treatment, on March 15th
1999, two trees were randomly selected from each treatment in each family (2 seedlings x 8 families x 4 treatments = 64 samples in total), and sampled for resin canal analysis.
A section of leader approximately 4 cm long was cut from each tree in the mid-leader region and fixed in formalin alcohol acetic acid (FAA, Appendix 2) for 48 hours. Samples were then transferred to 70% EtOH for storage until sectioning. Leader cross sections approximately 60u. thick were made using a sliding microtome. Sections were stained with 0.1% aqueous Safranin and mounted in glycerin between a glass slide and coverslip. Microscopic images were videocaptured using Sigma Scan® Image Analysis
System and the following measurements were made on quarters of cross sections
(Figure 1): radius, radius to inner resin canals, radius to cambium, bark area, area and number of inner and outer resin canals.
The following variables were calculated from the measurements: average size of inner and outer resin canals (SZIN, SZOUT), percent of bark area occupied by resin canals (AOCC), number of resin canals per millimeter squared of bark (NMMS), depth of inner resin canal ring (DEP) and average gap size between inner resin canals (GAP)
(for calculations see Appendix 3) and bark thickness (BTHK). Variable abbreviations, definitions and units are given in Table 1.
To assess the effects of fertilization on constitutive resin canal variables, families were pooled together to yield 16 seedlings per fertilizer treatment. Analysis of variance
(ANOVA) was performed using STATISTICA® software from StatSoft, Inc. (2300 East
14th St., Tulsa, OK 74104) to compare means between fertilizer groups (for model see
Appendix 1). Type III sums of squares were calculated using the general linear model.
33 Figure 1. Quarter cross section of a Sitka spruce leader.
Fertilization was treated as a fixed effect. Tukey's HSD was used to detect
differences between groups. The following transformations were required to meet the
assumptions that the data were normally distributed within each group and had
homogeneous variances: AOCC = Log(AOCC), NMMS = (NMMS)1/2, SZIN = (SZIN)1/5,
BTHK = (BTHK)1/2 and GAP = (GAP)1'2.
Kruskal-Wallis ANOVAs and Kolmogorov-Smirnov two sample tests (non-
parametrics tests), were used to detect differences in number of outer resin canals
(NOUT) and depth of inner resin canals (DEP) between fertilizer treatments as no transformations were successful in creating homogeneous variances. SZOUT was not examined statistically as the number of samples from each fertilizer group varied from 2 to 14.
34 Traumatic resin canal defenses
The goal of this study was to determine the feasibility of using fertilization treatments to influence the intensity of the traumatic response in Sitka spruce considered to be susceptible to the white pine weevil.
Six families were chosen for this trial: Progeny from open pollenation of parents
39, 40, 658, and progeny of controlled crosses of 125x59, 36x98, 61x67 parents. Ten seedlings were selected at random, five for wounding and five as unwounded controls, from the control, high and low fertilizer treatments from each family (for levels of fertilizer, see Table 3). On May 28th 1999, coinciding with peak adult weevil feeding
(Silver 1968), the trees were wounded to simulate weevil feeding. A Dremmel® drill equipped with a 1 millimeter drill bit was used to make a total 36 wounds/tree (12 holes, on each of 3 sides of the leader), just through the bark of the leader, in the region where weevil egg laying would normally take place (about 4 cm below the apical bud).
In the middle of October 1999, when the trees had completed their third growing season (and had received two fertilizer treatments), 4 cm lengths of stem were sampled from the area of wounding. In order to assess the traumatic resin response, slides of stem cross sections were prepared and scanned using the same methods as for the constitutive resin canal analysis. The traumatic response was rated using a scale from 0
(no resin canal formations or preformations) to 6 (two complete rings of resin canals in the xylem) as per Brescia (2000) (Appendix 4).
Since unwounded trees rarely produce a spontaneous traumatic response
(Brescia 2000; O'Neill et al. 2002) ANOVA was conducted using only data from wounded trees, to detect the effects of fertilization and family on traumatic response rating (TRAU). Type III sums of squares were calculated using GLM in STATISTICA®.
35 Preliminary analyses indicated negligible effects of the interaction between fertilization and family groups, consequently interaction effects were not included in the model.
Fertilization was treated as a fixed effect. Examination of residuals indicated normally distributed errors for TRAU and the appropriate F-tests were conducted to assess the significance (p< 0.05) of effects. Due to mortality caused by root weevils, there were unequal sample sizes among the treatment groups. Tukey's HSD for unequal n, was conducted to detect differences between groups.
Incidence of weevil attack
A preference trial, in which trees in cages were exposed to weevils, was conducted to determine whether weevils would preferentially attack trees from certain fertilizer groups. Caging experiments do not represent true 'choice' tests, since weevils are not permitted to search for another host if none of the trees in the cage is suitable.
However, with this in mind, caging experiments can prove useful for screening host material (Klimaszewski et al. 2000; Alfaro et al. 2002).
The same six families used for the wounding trial (39, 40, 658, 125x59, 36x98,
61x67), were used for the caging trial, which was conducted over the 1999 growing season. From each of the six families, three trees from the control, low and high fertilizer treatments were randomly selected and then randomly assigned to one of three cages, so that each cage held one tree from each family in each fertilizer treatment and
18 trees in total. Leader diameter and length were measured prior to placing the trees in cages. The cages were set up in an open compound at The Pacific Forestry Centre
(PFC). A laboratory colony of weevils was initiated in the fall of 1998 by the collection of weevils emerging from infested Sitka spruce leaders clipped near Eve River, BC. The
36 colony was maintained over the winter in an open compound at PFC. On May 28
1999, one female and two male weevils were released on the soil surface at the base of each tree. In the fall, after brood weevil emergence had ceased, egg plugs and exit holes, and the number of trees with killed leaders and failed attacks were counted.
Because there were fewer than expected exit holes, the leaders were dissected in the lab in effort to determine the cause of brood mortality. No statistical tests were conducted on these data, due to small sample sizes.
RESULTS AND DISCUSSION
Tree size
Prior to fertilization, average height varied between families, from 29.2 cm to 39.7 cm (Table 4). As expected, following fertilizer treatment, trees in fertilized groups were taller and had longer, thicker leaders than trees in control groups in all families (Figures
2-4). Heights ranged from an average of 44.1 cm in control groups to 80.7 cm in high fertilizer groups. However, in a number of families, tree size was not significantly different between specific fertilizer groups (Figures 2-4).
Table 4. Average height of 1 year-old potted Sitka spruce seedlings from eight weevil susceptible families at the start of Trial 1 (n=60 for each group).
Family Height (cm) Standard Deviation 38 35.5 1.80 39 35.4 2.09 40 35.3 1.80 125X59 29.2 1.77 36X98 39.7 1.39 61 X67 38.9 2.20 92X 118 39.0 1.79 658 38.9 1.73
37 100 90 -c— 80 70 60 50 40 30 control low medium high control low medium high control low medium high 38 39 40 100 • !—~—•—rr 90 t.„.C.„4„. •E 80 > .c,....i...Dc. u 70 - h • _ DC 60 50 D> 40 30 control low medium high control low medium high control low medium high 125X59 36X98 61 X67 100 c c c 90 b 80 b i • ±1.96*SE of mean 70 .. e ±1.00*SE of mean 60 a 50 4- ; 40 —r Mean 30 control low medium high control low medium high 658 Family and Fertilizer treatment
Figure 2. Heights of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.05).
60 : ; c : i U....L4... c I • c • c 45 b 30 \ *» ^ r--"6--r-i—- t-.-b— - 15 '""ar*-•:"" f-- t— } - 0 '"""a"""Tr•" control low medium high control low medium high control low medium high 38 39 40 E 60 o c 45 — -f— tr"" = 4 rT"-nw-r " 30 • ^ 4 _. O) i \ '• c 15 : _ 0 4r i • i ; d) control low medium high control low medium high control low medium high T3 125 X 59 36X98 61 X67 0ro) 60 45 H [••*--!-*•- "T~ ±1.96*SE of meaji 30 .4-*-; •i ±1.00*SE of meaji 15 -«.-[••- \ } - • Mean 0 •*• i I control low medium high control low medium high 92X 118 658
Family and Fertilizer treatment
Figure 3. Leader lengths of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.05).
38 control low medium high control low medium high control low medium high E 38 39 40 0.85 _o 0.75 —b- V 0.65 ...,...b,.... b 0.55 0.45 E 0.35 ro 0.25 T3 0.15 ...a 4. ...j L. V control low medium high control low medium high control low medium high T3 125X59 36X98 61 X67 ro 0.85 0) 0.75 0.65 :::ii::4:::,::±:&:±*:: _[_ ±1.96*SEof meaf 0.55 - y 0.45 • ±1.00*SEof mea| 0.35 0.25 ....a.—; 4- i •• • Mean 0.15 control low medium high control low medium high 92X118 658 Family and Fertilizer treatment
Figure 4. Leader diameters of 2 year-old potted Sitka spruce seedlings, from Pissodes strobi susceptible families, fertilized with four levels (0, 7, 16, 25 gr) of NPK fertilizer (n=15 for each group). Within families, treatments with the same letter are not significantly different (Tukey's HSD, p<0.05).
A range of tree sizes was achieved by the different levels of fertilization. Gains in height, leader length and leader diameter, with respect to control trees, varied among families and in most cases increased with higher fertilization dosages. Although height gains were considerable following fertilization, after the second growing season, the range of tree heights was still significantly lower than the range of 1.5 to 9 m, at which trees are generally susceptible to weevil attack in the field (Turnquist and Alfaro 1996).
The range of leader diameters achieved by fertilization contained diameters above and below the minimum diameter of 4 mm, acceptable to the weevil, as determined by
Sullivan (1961) on white pine. Although on occasion, very small seedlings are attacked by weevils in the field (Rene Alfaro pers. comm.), the results of this trial should be interpreted with caution, since the trees used in this study were younger and generally
39 smaller than those that would usually be attacked by the weevil in the field. Host trees of sizes more likely to be selected by the weevil, might respond differently to fertilizer treatments, influencing the host/weevil interaction.
Constitutive resin canal defenses
There were significant effects of fertilization on most of the constitutive resin canal variables measured. A summary of ANOVA results is presented in Table 5.
Variable means for each fertilizer group and the results of Tukey's HSD tests are presented in Table 6.
Fertilizer treatment had a significant effect on bark thickness (Table 5), which ranged from an average of 0.48 mm in control trees to 0.98 mm in medium fertilizer trees (Table 6). Trees from all fertilized groups had significantly thicker bark than control trees. Trees in medium and high fertilizer treatments had significantly thicker bark than trees in the low fertilizer treatment.
The number of inner resin canals per quarter cross section ranged from three to six but did not vary significantly with fertilizer treatment (Table 5). The number of outer resin canals however, differed with fertilizer treatment (Kruskal Wallis ANOVA,
2 5C (3)=33.94, p<.0001), ranging from zero in control groups to five in high fertilizer groups
(Table 6). NOUT for medium and high fertilizer trees were significantly greater than those of low and control trees (Table 6). Although no outer resin canals were observed in any of the control trees, the number of outer resin canals in the control group was not significantly different from that of the low fertilizer group.
Inner resin canal size varied significantly among fertilizer treatments (Table 5).
Control trees had the smallest inner resin canals, while medium fertilizer trees had the
40 largest (Table 6). Although no statistical test was conducted to determine differences in size of outer resin canals, the biggest (.00815 mm2) occurred in the high fertilizer group while the smallest (.000416 mm2) occurred in the low fertilizer group.
Table 5. Results of analysis of variance to determine the effects of four levels (0, 7, 16, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) on constitutive resin canal variables of two year-old potted Sitka spruce seedlings.
Variable* MS Effect MS Error F(df 3,60) p>F NIN 0.14 0.54 0.26 0.855 SZIN 32.64 1.02 32.15 0.000 NMMS 7.37 0.09 85.20 0.000 AOCC 1.72 0.47 3.68 0.017 BTHK 0.3205 0.01 55.00 0.000 GAP 3.0216 032 9.47 0.000 * for variable names, see Table 1.
Resin canal density, expressed in terms of bark area occupied by resin canals
(AOCC), increased significantly with fertilizer treatment (Table 5). However, the only group that was statistically different from the control group were trees from the medium fertilization treatment which had the highest density (4.4 %)in terms of area (Table 6).
The effects of fertilization on resin canal density expressed as the number of resin canals per square millimeter of bark (NMMS) were significant but opposite from the effects on AOCC. Fertilized trees had significantly lower NMMS than control trees
(Table 6).
Fertilization did not affect the depth of the ring of inner resin canals (Kruskal
2 Wallis ANOVA, 5C (3)=4.5, p<.2), but did affect the distance between inner resin canals
(GAP). The average GAP of fertilized trees (0.53 mm) was significantly greater than the gap size of control trees (0.21 mm) and was larger than the average snout diameter of a
41 weevil (0.4 mm) (Table 6).
For a number of constitutive resin canal variables, the observed effects of fertilization were contrary to what was expected. Although increased nutrient availability resulted in an increase in growth, there did not appear to be a trade-off between growth and differentiation since fertilization also caused an increase in the number of outer resin canals and the size of both inner and outer resin canals. The variation in resin canal characteristics also appeared to be positively related to fertilizer effects on tree size.
Table 6. Average constitutive resin canal characteristics observed in quarter cross sections of leaders from two year-old potted Sitka spruce maintained under four levels (0, 7, 16, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation).
fertilizer treatment Variable1 control (Og) low (7g) medium (16g) high (25g) NIN 3.9 a 3.7 a 3.9 a 3.7 a NOUT* 0.0 a 0.3 a 0.9 b 2.0 b SZIN (mm2) .002489 a .013019 ab .038353 c .030575 bc SZOUT (mm2) - .000647 .001193 .002019 NMMS 7.2 c 2.3 b 1.4a 1.9 ab AOCC 1.8 a 2.6 ab 4.4 b 3.4 ab DEP (mm) 0.187a 0.230 3 0.221 a 0.252 a GAP* (mm) 0.208 a 0.463 b 0.592 b 0.533 b BTHK (mm) 0.474 a 0.716 b 0.980 c 0.973 c 1for variable names, see Table 1. Values with the same letter are not significantly different within rows, Tukey HSD (p<0.05). * these variables were tested using Kolmogorov-Smirnov two-sample tests (p<0.05)
The consistency in number of inner resin canals (NIN) regardless of fertilizer treatments implies that a predetermined, fixed number of inner resin canals are produced by the apical meristem and that this number is not influenced by nutrient availability. The lack of outer resin canals (NOUT) in the nutrient-stressed control trees
42 may indicate that inadequate resources were available for the formation of resin canals.
If this was the case, these observations are contrary to what might be expected according to Bryant et al. (1983) who proposed that stressed plants would allocate more resources to defense. The increase in NOUT, with fertilization is likely the result of an increase in the frequency of branching of the inner resin canals as the bark becomes thicker. I hypothesize that in small trees, with relatively thin bark, the inner resin canals alone provide an adequate supply of resin protection to the tissues. This is supported by the observation that resin canal density expressed by number (NMMS) is much greater in control trees that in fertilized trees (Table 6).
The observations also indicate that resin canal size increases with increasing stem diameter and bark thickness. In trees with thicker bark, additional resin defense is provided by branching of the inner resin canals into outer resin canals, and increased resin canal size. These hypotheses also explain the observation of higher resin canal density expressed by area (AOCC) in fertilized trees (Table 6). The decrease in NMMS with increasing fertilization may be explained by the dilution of resin canals as a result of increased radial growth, as observed by Alfaro et al. (1997a) and Brescia (2000).
Although the number of outer resin canals increased with fertilization, the increase must not have been great enough to negate the effects of increasing bark area.
Traumatic resin canal defenses
Wounding was highly effective in triggering the formation of traumatic resin canals in the xylem of drilled leaders. The wounding treatment produced higher levels of longitudinal resin duct densities relative to unwounded controls. The initiation of these traumatic resin ducts coincided with the timing of drilling, and thus was a direct result of
43 mechanical wounding (Figure 5). Traumatic response ratings in wounded trees ranged from 1 (dark ring) to 4 (complete ring of traumatic resin canals) (Figure 6). In the unwounded control trees, the average traumatic response rating was less than 1. The weak traumatic response observed in unwounded control trees was likely elicited by feeding and oviposition by rogue weevils present in the outdoor compound.
Fertilization had a significant effect on traumatic response rating, with fertilized trees having considerably stronger traumatic responses to wounding relative to wounded but unfertilized control trees (Table 7). However, there was no significant difference between low and high fertilizer groups (Table 8).
Figure 5. Stem cross section of a Sitka spruce internode wounded with a 1 mm drill bit to simulate weevil feeding, in the spring of the second year of growth.
44 Figure 6. Examples of traumatic responses ratings used in the assessment of the traumatic response intensity in cross sections of internodes of Sitka spruce fertilized with three levels of NPK fertilizer, and mechanically wounded during the second year of growth.
Although statistical analysis indicated a significant family effect on traumatic response rating (Table 7), only two of the families were significantly different from each other. Family 658 had an average response rating of 2.14 which was significantly lower than 2.92, the average response rating of family 1123.
45 Table 7. Summary of ANOVA to detect the effects of fertilization (FERT) and family (FAM) on traumatic response rating of 3 year-old potted Sitka spruce mechanically wounded to simulate weevil feeding and fertilized with three levels (0, 7, 25 gr) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation).
Variable and effects df MS Effect MS Error F p>F PERT 2 32.40 0.449 72.09 <0.001 FAM 5 1.21 0.449 2.68 0.028
Table 8. Means and standard deviations (SD) of traumatic response rating, on a scale from 0-6*, in 3 year-old potted Sitka spruce fertilized with three levels (0, 7, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation) and subjected to mechanical wounding. Values with the same letter are not significantly different according to Tukey's HSD (p<.05).
Fertilization Treatment Mean SD Valid n control 1.23 a 0.43 30 low 3.04 b 0.81 27 high 3.07 b 0.83 27 * For description of scale see Appendix 4.
The lack of a significant traumatic response in wounded control trees, while fertilized trees elicited a strong response, supported the hypothesis that trees in a resource limited environment may be less capable of producing an effective traumatic response. Again, the unfertilized seedlings may not be suitable for attack due to their thin bark, therefore resource allocation to traumatic resinosis may not be necessary.
The observation that response rating was higher in fertilized trees but did not differ significantly between high and low fertilizer trees, suggests that there may be a threshold level of energy required to produce a traumatic response, below this level, the tree is unable to produce an effective response.
46 Incidence of weevil attack
The response of weevils to the fertilizer treatments was similar in each of the cages, therefore the results from each cage were pooled together for the purpose of discussion.
Careful inspection revealed no oviposition or feeding punctures present on any of the control trees. Oviposition was greatest on the high fertilizer trees, and low fertilizer trees had about half as many punctures as those in the high fertilizer group (Table 9).
All fertilized trees but one in the high fertilizer group, sustained oviposition. The high fertilizer tree that was not selected by weevils for oviposition was from family 36 x 98 and had a significantly shorter (36.5 cm), but thicker (0.72 cm) leader than the average high fertilizer tree (mean leader length 46.8 cm and leader diameter 0.68 cm).
Table 9. Mean number and range of Pissodes strobi oviposition punctures and number of Sitka spruce trees with oviposition, failed and successful attacks and brood emergence, in three NPK fertilizer treatments (FERT) (Osmocote®, 8-9 month slow release formulation, 0, 7, 25 g) (n=18 trees for each group).
oviposition punctures failed killed brood FERT mean min max oviposition attacks leaders emergence control 0 0 0 0 0 0 0 low 30.1 5 58 18 16 2 0 high 56.8 0 112 17 12 5 2
The number of trees that sustained failed attacks (eggs were deposited, but the larvae failed to kill the leader) was greatest in the low fertilizer group, while the high fertilizer group held the greatest number of trees with killed leaders. The only trees from
47 which brood weevils emerged were two trees from the high fertilizer group. These trees both had longer and thicker leaders than the average in the high fertilizer treatment and also had a higher than average number of oviposition punctures.
Since it has been previously shown that trees from the high fertilizer group had significantly longer and thicker leaders than those from the low fertilizer group, the observed trends in oviposition are in accordance with studies by Silver (1968), Gara et al. (1971) and Alfaro et al. (1993) which have shown that weevils generally prefer trees with longer, thicker leaders.
The absence of feeding and oviposition punctures on the control trees indicates that these trees were not suitable hosts for the weevil. It is possible that constitutive resin canal defenses may have contributed to the unattractiveness of the control trees by controlling resin odours that influence host acceptance. However, is not likely that cortical resin canal distribution played a significant role in deterring weevils since the weevils did not feed on the bark of these trees. The rejection of the control trees as weevil hosts is consistent with the literature on host selection by weevils feeding on white pine. The average leader diameter of the control trees was 0.22 cm which is less than the minimum shoot size (0.4 cm) required for weevil selection, as observed by
Sullivan (1961) studying white pine. The average bark thickness measured for control trees was 0.47 mm (see Table 6) which Sullivan (1961) suggests is less than the minimum bark thickness (0.8 mm) required for egg deposition, since weevil eggs are around 0.8 x 0.5 mm in size. However, since the weevils did not feed on the control trees, it is unlikely that bark thickness was a factor in the rejection of these trees as hosts, unless weevils are capable of assessing bark thickness without penetrating the bark. It is possible that the lack of feeding and oviposition on the control trees was due
48 to chemical or physical deterrents.
The differences in the ratio of failed attacks to killed leaders between high and low fertilizer groups may be explained by a number of factors. More attacks may have failed in the low fertilizer group because, on average, there was less oviposition. It has been suggested that, in order to overcome the tree's resin canal defenses, larvae must feed gregariously in large numbers (Alfaro 1996b). Studying interior spruce, Alfaro et al.
(1996a) found that little emergence occurred if fewer than 60 oviposition punctures were excavated (or approximately 100 eggs were deposited), which is higher than the highest number of oviposition plugs excavated in low fertilizer trees. Lower oviposition rates in low fertilizer trees may have been due to bark thicknesses that were less than the minimum acceptable by the weevil, as suggested by Sullivan (1961).
Parasitoids were able to fit through the screen of the cages, and may have also contributed to larval mortality. Leader dissections revealed chip cocoons in only one low fertilizer tree, and parasitoid larvae (tentatively identified as the hymenopteran Eurytoma pissodes Girault) were observed in three low fertilizer trees. Because the proportion of viable eggs was not estimated, poor brood survival may have also been a result of a low egg viability in the laboratory colony weevils.
Constitutive and traumatic resin canal defenses likely played only minor roles in influencing the variation in susceptibility to weevil attack between low and high fertilizer trees, since only SZIN and NOUT were significantly different between the two groups and both values were larger in the high fertilizer group. The greater proportion of high fertilizer trees that sustained killed leaders is likely due to higher oviposition rates influenced by the effects of fertilization on tree size and bark thickness.
49 CONCLUSIONS
It is clear that fertilizer treatments influence the growth of young Sitka spruce trees as well as the incidence of weevil attack. The results of this trial indicate that the incidence of weevil attack on susceptible trees may be decreased by limiting nutrient availability. The positive effects of fertilization on tree size have a strong influence on the incidence of weevil attack, by providing more attractive hosts. Although constitutive and traumatic resin canal defenses varied between fertilized and unfertilized groups, resulting in more intense traumatic responses and a greater proportion of bark area occupied by resin canals in fertilized trees, it is not likely that these defenses influenced the observed variation in weevil susceptibility between fertilizer groups. It is possible that the application of fertilizer to trees that are comparable in size to 'normal' host trees, may induce enough variation in these defenses to affect host suitability. It would be beneficial to conduct the resin canal studies on trees in a field setting, under conditions where weevil attack would usually occur. Such a study is described in Trial 3.
50 TRIAL 2. Fertilizer effects on constitutive resin canal defenses in resistant and susceptible spruce genotypes
METHODS
Tree material and fertilization
In Trial 2, both resistant and susceptible Sitka spruce genotypes3 were utilized to test whether spruce responds differently to fertilizer treatments depending on genotype and resistance status, and to confirm the findings of Trial 1. The genotypes used in this trial were designated resistant or susceptible using the same criteria as described for
Trial 1. Four putatively weevil resistant and four susceptible genotypes of 1 year-old
Sitka spruce somatic seedlings4 were provided by the BC MoF in March 1999. Parent tree origins are given in Table 10. All of the genotypes used in this study were from the
Big Qualicum provenance.
Table 10. Identification, origin and resistance status of parent trees of the Sitka spruce somatic seedlings used in Trial 2.
Family Genotype Location status 1017 247 Fanny Bay2 resistant 1018 59 Fanny Bay3 resistant 1024 338 Qualicum2 resistant 1024 356 Qualicum2 resistant 1025 763 Qualicum3 susceptible 1034 8548 Gilles Bay2 susceptible 1106 Coombs susceptible 1123 Coombs susceptible
Twenty trees from each genotype (only 15 seedlings were available for genotypes 1018 59, 1018 435 and 1034 8548) were planted into 1 gallon pots and
3 A genotype is a clonal line, all trees belonging to the same genotype have the same genetic make-up. 4 Somatic seedlings are produced by somatic embryogenesis, resulting in clones or a series of seedlings of identical genotype.
51 maintained as described for Trial 1.
On May 4th 1999, somatic seedlings in each genotype were randomly divided into four groups of five plants each. Each group was fertilized with a different level of NPK fertilizer. Fertilizer rates for each treatments are given in Table 11. The control group was omitted for genotypes that had only 15 somatic seedlings. The same fertilizer and application methods were used as for Trial 1.
Fertilizer effects on height, leader length, leader diameter and constitutive resin canal characteristics were measured following one season of growth under the fertilizer treatments.
Table 11. NPK fertilizer (Osmocote®, 8-9 month slow release formulation) applied to 1 year-old Sitka spruce somatic seedlings in 1 gallon pots, in each of four treatments, in a study of fertilization effects on spruce defenses. Treatments were applied in May 1999.
Fertilizer treatment Amount applied(g) Control 0 Low 3 Medium 8 High 25
Data collection and analysis
Tree size
On May 4th 1999, prior to fertilization, height and diameter measurements were taken (using the same methods as for Fertilizer Trial 1) for all seedlings. Trees were measured again on April 7th 2000, after one growing season under the various fertilizer regimes. ANOVA were performed to determine the effects of fertilization, resistance status and genotype on tree size variables. For model see Appendix 5. Type III sums of
52 squares were calculated using the general linear model (GLM) in SAS from the SAS
Institute Inc. (SAS Circle, Box 8000, Cary, NC, 27512-8000). Fertilization was treated as a fixed effect. Tukey's HSD was performed to detect significant differences (p<0.05) between groups.
Constitutive resin canal defenses
On April 7th 2000, four seedlings were randomly selected from each of the four resistant and four susceptible genotypes in each fertilizer treatment, and sampled for constitutive resin canal analysis. Thus, there were a total of 16 resistant and 16 susceptible somatic seedlings for each fertilizer treatment, except in the control treatment, in which there were only 12. Using the same methods as for Trial 1, slides of leader cross sections were prepared, scanned and measured.
ANOVA were conducted to detect the effects of fertilization, resistance status and genotype on constitutive resin canal defenses (for model see Appendix 5). Type III sums of squares were calculated using SAS® GLM procedures. The following variables were transformed using log transformations in order to meet the assumptions of normality and homogeneous variances: NOUT, SZIN, BTHK, AOCC, NMMS and GAP.
SZOUT was not examined statistically as there were an inadequate number of outer resin canals in samples from each fertilizer group. Fertilization was treated as a fixed effect.
RESULTS AND DISCUSSION
Tree size
Prior to fertilizer treatment, average height varied among genotypes, independently of resistance status and ranged from 23.0 cm to 37.4 cm (Table 12). The
53 effects of fertilization varied with genotype, but not with resistance status, for all tree size variables measured (Table 13). Fertilizer treatments produced gradients of percent gains in heights, leader lengths and diameters within each genotype (Figures 7-9). For each genotype, all measured variables of tree size increased with subsequent levels of fertilizer although not all treatments were significantly different within each genotype
(Figures 7-9).
Table 12. Height (cm) prior to fertilization, of 1 year-old Sitka spruce somatic seedling from weevil resistant (R) and susceptible (S) genotypes.
Genotype Status Height Standard Deviation n 1018 59 R 33.3 2.5 12 1017 247 R 23.0 3.6 16 1024 338 R 33.8 4.6 16 1024 356 R 37.4 2.2 16 1106 S 29.8 2.1 16 1123 S 25.7 3.8 16 1025 763 S 29.9 2.5 16 1034 8548 S 33.8 5.8 12
As in Trial 1, a range of tree sizes was achieved by the fertilizer treatments.
However, on average, Trial 2 trees were shorter and had shorter, thinner leaders than
Trial 1 trees. Therefore, Trial 2 trees were also smaller than the average spruce that would be attacked by the weevil in the field. In most of the genotypes, only the high fertilizer trees had leaders that were thick enough to be accepted by the weevil, according to Sullivan's (1961) study on white pine. It is suggested that, as with the results of Trial 1, the results of this trial may not represent the effects of fertilizer
54 treatments on trees of an adequate size to host the weevil. However, they serve as both a model to provide information on the influence of fertilization on tree defenses and a basis for future studies.
Table 13. Results of ANOVA to detect the effects of fertilization, resistance status and genotype on height, leader length and leader diameter of 2 year-old Sitka spruce somatic seedlings fertilized with four levels of NPK fertilizer (Osmocote®, 8-9 month slow release formulation).
variable and effects df MS Effect MS Error F p level Height RES 1 147.407 584.401 0.25 0.6334 GENO(RES) 6 584.401 26.360 22.17 <0.0001 FERT 3 4571.190 32.025 142.74 0.0010 RES*FERT 3 32.025 99.453 0.32 0.8094 GENO(RES)*FERT 16 99.453 26.360 3.77 <0.0001
Leader length RES 1 573.674 169.222 3.39 0.1152 GENO(RES) 6 169.222 18.324 9.24 <0.0001 FERT 3 4762.162 16.915 281.54 0.0004 RES*FERT 3 16.915 80.848 0.21 0.8885 GENO(RES)*FERT 16 80.848 18.324 4.41 <0.0001
Leader diameter RES 1 0.0041 0.0129 0.32 0.5941 GENO(RES) 6 0.0129 0.0015 8.41 <0.0001 FERT 3 0.2805 0.0008 328.09 0.0003 RES*FERT 3 0.0008 0.0029 0.29 0.8326 GENO(RES)*FERT 16 0.0029 0.0015 1.92 0.0285
55 100 80 b c c ; C • 60 ab b «s a -o- 40' ... o 20 control low medium high 1106 S 1123 S 1025 763 S 100 r
E 80 C 60 ...a '•...*>... i b i a £ 40 a i ; CP i '55 20 O ; I control low medium high 1034 8548 S 1018 59 R 1017 247 R 100 r 80 c "T" Mean±1.96*SE of mean 60 ..St.. •| MeaniSE of mean 40 • Mean 20 control low medium high control low medium high 1024 338 R 1024 356 R
Genotype and Fertilizer Treatment
Figure 7. Height of 2 year-old potted Sitka spruce somatic seedlings from putatively resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
60 50 d 40 b C : C 30 b ; o •; .£> 20 b L.J&. a 4 10 ^- ;. 0 control low medium high 1106 S 1123 S 1025 763 S
c b a a o -O- : control low medium high
"T~ Mean±1.96*SEof mean •| MeaniSE of mean • Mean
control low medium high control low medium high 1024 338 R 1024 356 R
Genotype and Fertilizer Treatnant
Figure 8. Leader length of 2 year-old potted Sitka spruce somatic seedlings from putatively resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
56 0.60 b
0.45 ;--a~ 1 b i-
0.30 a A a ; ©IT: 0.15 control low medium high 1106 S 1123 S 1025 763 S 0.60 b 0.45 ...!.. ! . h 3> a 0.30 » La! ..IS.
0.15 control low medium high 1034 8548 S 1018 59 R 0.60 b ! b ! 0.45 be "T~ Mean+196*SE of mean \ ab ^| MeantSE of mean 0.30 • Mean a : 0.15 o : control low medium high control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatrent
Figure 9. Leader diameter of 2 year-old potted Sitka spruce somatic seedlings from putatively resistant (R) and susceptible (S) genotypes, fertilized with four levels (0, 3, 8, 25 gr) of NPK fertilizer (n=4 for each group). Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
Constitutive resin canal defenses
For all of the variables measured except depth of inner resin canals (DEP), there was a significant interaction between the effects of fertilization and genotype (Table 14).
DEP was however, significantly affected by fertilization independently of genotype effects, which were also significant. Independently, resistance status did not significantly affect any of the variables. However, resistance status and genotype influenced the effects of fertilizer treatment on one variable, the number of inner resin canals (NIN). A summary of ANOVA for detecting the effects of fertilization, resistance status and genotype effects on constitutive resin canal characteristics, is presented in Table 14.
Within each genotype, increased fertilization resulted in increased bark thickness
(Figure 10). Although not all treatments were significantly different within each
57 genotype, the thickest bark was always found in the highest fertilization treatment and the thinnest bark in the control treatment (or low treatment if there was no control group for that genotype).
c b b c C i b A b d a o ~ a a O o control low medium high control low medium high control low medium high E 1106 S 1123 S 1025 763 S
; ab c ibV b $ a . be c ' «* • a a bo-. CS a O! control low medium nigh control low medium high control low medium high 1034 8548 S 1018 59 R 1017 247 R
d d C cd ' ~T~ Mean±1.96*SE of mean be MeantSE of mean b i a • Mean a •Efr : a • ..-0-...1 control low medium high control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatment
Figure 10. Bark thickness of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
The effects of fertilization on NIN varied with genotype depending on resistance status (Figure 11). In two of the susceptible genotypes, NIN was not significantly different among fertilizer groups. In the other two susceptible genotypes, although not all groups were statistically different, NIN was highest in the control groups and lowest in either the low or medium groups, while the high fertilizer group had intermediate values. In the resistant class, there was no significant difference in two genotypes while in the other two, NIN decreased with increasing fertilizer treatment, although not all treatments were significantly different.
58 Table 14. Summary of ANOVA to determine the effects of fertilization (FERT), resistance status (RES), genotype (GENO) and their interactions, on characteristics of the cortical resin canal system in two year-old Sitka spruce somatic seedlings.
variable and effects df MS Effect MS Error F p level Bark thickness (BTHK) RES 1 0.0661 0.1911 0.35 0.5778 GENO(RES) 6 0.1911 0.0211 9.05 O.0001 FERT 3 5.4656 0.0085 639.40 0.0001 RES*FERT 3 0.0085 0.0379 0.23 0.8774 GENO(RES)*FERT 16 0.0379 0.0211 1.80 0.0436 Number of inner resin canals (NIN) RES 1 0.6787 5.0370 0.13 0.7262 GENO(RES) 6 5.0370 0.4389 11.48 O.0001 FERT 3 7.9896 3.6609 2.18 0.2690 RES*FERT 3 3.6609 0.8741 4.19 0.0229 GENO(RES)*FERT 16 0.8741 0.4389 1.99 0.0219 Number of outer resin canals (NOUT) RES 1 0.2041 0.2565 0.80 0.4067 GENO(RES) 6 0.2565 0.0584 4.39 0.0006 FERT 3 2.1568 0.0378 57.10 0.0038 RES*FERT 3 0.0378 0.1790 0.21 0.8873 GENO(RES)*FERT 16 0.1790 0.0584 3.06 0.0004 Size of inner resin canals (SZIN) RES 1 0.1393 1.1861 0.12 • 0.7435 GENO(RES) 6 1.1861 0.1969 6.02 O.0001 FERT 3 23.1642 0.2023 114.51 0.0014 RES*FERT 3 0.2023 0.5866 0.34 0.7933 GENO(RES)*FERT 16 0.5866 0.1969 2.98 0.0005 Bark area occupied by resin canals (AOCC) RES 1 0.0269 0.0578 0.47 0.5204 GENO(RES) 6 0.0578 0.0222 2.61 0.0225 FERT 3 0.2179 0.0049 44.76 0.0054 RES*FERT 3 0.0049 0.0418 0.12 0.9492 GENO(RES)*FERT 16 0.0418 0.0222 1.89 0.0319 Number of resin canals per mm2 bark (NMMS) RES 1 18.7124 9.8761 1.89 0.2178 GENO(RES) 6 9.8761 1.7117 5.77 O.0001 FERT 3 397.6731 0.4682 849.26 O.0001 RES*FERT 3 0.4682 4.5147 0.10 0.9567 GENO(RES)*FERT 16 4.5147 1.7117 2.64 0.0020 Average distance between inner resin canals (GAP) RES 1 0.1801 0.2986 0.60 0.4669 GENO(RES) 6 0.2986 0.0431 6.93 <0.0001 FERT 3 3.6431 0.1309 27.84 0.0108 RES*FERT 3 0.1309 0.0815 1.61 0.2272 GENO(RES)*FERT 16 0.0815 0.0431 1.89 0.0315 Average depth of inner resin canals (DEP) RES 1 0.0101 0.0169 0.60 0.4689 GENO(RES) 6 0.0169 0.0071 2.38 0.0352 FERT 3 0.8272 0.0040 206.23 0.0006 RES*FERT 3 0.0040 0.0110 0.37 0.7791 GENO(RES)*FERT 16 0.0110 0.0071 1.54 0.1024
59 8.0 a a i ! ab a • : a 6.5 — [ a
-a- i b : b | ! 5.0 A * _,...] b : b •& ^ ^ « 4 . 3.5 ^ & & 2.0 TO tow medium high control low medium high control low medium high C TO U 1106 S 1123 S 1025 763 S 8.0 3 6.5 a a i a i a I a a i a • 5.0 \ J. i ^ j & i _i g. ! # . i 4 ! ^ ! b 3.5 ¥ ...•P.; |S 2.0 low medium high control low medium high control low medium high .a 1034 8548 S 1018 59 R 1017 247 R E 8.0 6.5 ab z ~T~ Mean±1.96*SE of mean ab 5.0 •• MeaniSE of mean 344 :S : . 3.5 • Mean 4..^.. 2.0 control low medium high control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatment
Figure 11. Number of inner resin canals per quarter cross section of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
In three of the eight genotypes (1106, 1123 and 1024 338), independent of resistance status, NOUT increased with increasing fertilization, although not all treatments were significantly different. In the other five genotypes (1018 59, 1017 247,
1024 356, 1025 763 and 1034 8548) NOUT did not vary significantly among fertilizer groups (Figure 12). No outer resin canals were observed in trees from the control groups and only three genotypes (1106, 1025 763 and 1017 247) had trees with outer resin canals in the low fertilizer group. Genotype 1025 763 had no outer resin canals in control or medium fertilizer group trees.
In the majority of the genotypes, SZIN was greater in fertilized groups than in unfertilized controls, and in all cases, either the medium or high fertilizer treatment had
60 the biggest inner resin canals (Figure 13). The lack of outer resin canals in a number of the groups made it impossible to detect trends in SZOUT.
ab ! ab a a a
(0 control low medium high control low medium high control low medium high c ro 1106 S 1123 S 1025 763 S u (A
3 a O control low medium high control low medium high control low medium high 0> 1034 8548 S 1018 59 R 1017 247 R E 3 "T~ Mean±1.96*SE of mean ab b •i Mean±SE of mean a a ab • Mean w -o- -: -o- control low medium high control low medium high 1024 338 R 1024 356 R
Genotype and Fertilizer Treatment
Figure 12. Number of outer resin canals per quarter cross section of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S), fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
Fertilizer effects on AOCC were significant in only two genotypes, both of which were resistant (Figure 14). In these two genotypes, AOCC was highest in the low or medium treatment and lowest in the control groups, however, not all groups were significantly different. Although there were no significant differences between treatments in any of the other genotypes, the greatest average AOCC was always observed in either the low or medium fertilizer group.
61 .060
.040 L.JL j be c be .020 b ; " y ab b ; TT ; -r- .... a a & c & 0 •n- w .... =Qr.. ± ; E control low medium high control low medium high 1106 S 1123 S 1025 763 S ro c .060 ro o .040 : c a a i .020 b ! b be A in r«a ^ a O ° 0 -o- 0) c control low medium high control low medium high c 1034 8548 S 1018 59 R 1017 247 R
0) N ]tc T~ Mean±1.96*SE of mean w Mean±SE of mean ! c A a Mean a [ b •» W 4- *> i control low medium high control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatment
Figure 13. Size of inner resin canals in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
a T a a; a ' a i O -o & i a control low medium high control low medium high 1123 S 1025 763 S
T a ; f • J. ; ab "'"a a o . ;|j §j .11
control low medium high control low medium high 1034 8548 S 1018 59 R ro A T~ Mean±1.96*SE of mean •*-< i b i b ^| MeantSE of mean oC o a ab A • Mean L. t CD '** Q. control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatment
Figure 14. Percent of cross sectional bark area occupied by resin canals (AOCC) in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05).
62 Within each genotype, NMMS (the number of resin canals per square millimeter of bark) decreased with increasing fertilizer treatment (Figure 15). The control groups had higher NMMS than the other groups, while the medium or high groups had the lowest.
a i ; a ; i 6 :mr: • 1 ab *c b a o I -«"T C ::T -|i Ti;~b:t b ^ c .,.d C3• "' o .i...... y ra | J —a-"j c n • A ; b C|i d -G- control low medium high control low medium high 1034 8548 S 1018 59 R 1017 247 R xi E "T~ Mean±1.96*SE of mean •i MeaniSE of mean 3 i .. c ....c ... -• • Mean control low medium high control low medium high 1024 338 R 1024 356 R Genotype and Fertilizer Treatment Figure 15. Number of resin canals per square millimeter of bark (NMMS) in cross sections of leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05). In all genotypes, with two exceptions, there was a general increase in GAP with increasing fertilization, however, not all groups were significantly different. In two genotypes, one resistant and one susceptible, there was no significant difference in the average distance between inner resin canals (GAP) (Figure 16). Fertilization and genotype each affected the depth of inner resin canals (DEP) independently. The largest average DEP occurred in genotypes 1123 S and 1018 59 R 63 while the smallest occurred in genotypes 1025 763 S and 1017 247 R. The rest of the genotypes had intermediate DEP values which were not significantly different from the highest and lowest values (Figure 17). DEP increased significantly with corresponding increases in fertilization (Figure 18). E 0.7 0.6 b E, 0.5 i b b tn 0.4 D ab ° b • =: ab r co 0.3 a • c 0.2 a .Q. -O- " 0.1 -D- • ^ T •8 c control low medium high control low medium high control low medium high in 1106 S 1123 S 1025 763 S ia> i CD 0.7 c 0.6 c a tz 0.5 a ; a 0.4 • •a 4 c a D Ir? b 1, 0J 0.3 •£> CD 0.2 a -a- Q 0.1 * CD XI control low medium high control low medium high control low medium high o0) 1034 8548 S 1018 59 R 1017 247 R c CO 0.7 • •••c —1 0.6 h c (0 0.5 b ~T~ Mean+1.96*SE of mean T3 0.4 b $ •1 MeaniSE of mean a CU 0.3 a a o • Mean •> 0.2 -o- CO 0.1 > control low medium high control low medium high < 1024 338 R 1024 356 R Genotype and Fertilizer Treatment Figure 16. Average distance between inner resin canals (GAP) in leaders from 2 year- old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05). The average values of several constitutive resin canal variables were noticeably different from Trial 1 values (Tables 6 and 15). For each fertilizer treatment, NIN was larger in Trial 2, however, SZIN was smaller. NMMS was considerably larger in Trial 2, due to higher NIN while BTHK was similar to that of Trial 1. DEP was noticeably greater in Trial 2 for each fertilizer treatment, while GAP was smaller due to relatively higher NIN. 64 0.52 , •C Mean±1.96*SEofmean I I Mean±SE of mean n Mean 0.46 ab ab ab ab « 0.40 Z 0.34 • 0.28 0.22 1106 S 1025 763 S 1018 59 R 1024 338 R 1123 S 1035 8548 S 1017 247 R 1024 356 R Genotype Figure 17. Average depth of inner resin canals (DEP) in leaders from 2 year-old Sitka spruce somatic seedlings from putatively weevil resistant (R) and susceptible (S) genotypes, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different within each genotype (Tukey's HSD, p>0.05). 0.60 ~T~ Mean±1.96*SE of mear) 0.55 I I Mean±SE of mean • Mean 0.50 0.45 0.40 • 0.35 0.30 0.25 0.20 0.15 control low medium high Fertilizer treatment Figure 18. Average depth of inner resin canals (DEP) in leaders of 2 year-old Sitka spruce somatic seedlings, fertilized with four levels of NPK fertilizer. Treatments with the same letter are not significantly different (Tukey's HSD, p>0.05). 65 Table 15. Means of constitutive resin canal variables in two-year-old Sitka spruce somatic seedlings fertilized with four levels (0, 3, 8, 25 g) of NPK fertilizer (Osmocote®, 8-9 month slow release formulation). Fertilizer treatment Variable1 control (Ogr) low (3gr) medium (8gr) high (25g) NIN 5.8 .5.0 4.8 4.7 NOUT 0.0 0.3 1.0 2.1 SZIN (mm2) 0.001941 0.007318 0.016056 0.015467 SZOUT (mm2) — 0.000438 0.002049 0.001222 AOCC(%) 2.2 3.2 3.7 2.6 NMMS 11.3 5.5 3.4 2.5 DEP (mm) 0.212 0.276 0.395 0.535 GAP* (mm) 0.177 0.255 0.333 0.415 BTHK (mm) 0.407 0.625 0.905 1.151 For variable descriptions see Table The trends observed in Trial 2 were comparable to those of Trial 1 with a few distinctions. In Trial 1, small sample sizes necessitated pooling families together, making it impossible to detect family effects, however in Trial 2, I was able to demonstrate significant genotype effects for most variables. For each variable measured, some of the genotypes, regardless of resistance status, responded to fertilizer treatment in the same way as trees in Trial 1. Fertilizer effects on some of the variables were not significant for all genotypes, whereas they were significant in Trial 1. However, there were no genotypes for which fertilizer effects were the opposite (ie. negative as opposed to positive) of those observed in Trial 1. Similar to the results of Trial 1, the variation in some resin canal characteristics appeared to be related to fertilizer effects on tree size. As in Trial 1, on average, increases in NOUT, SZIN, DEP and GAP in Trial 2 corresponded with increases in leader diameter and bark thickness, while NMMS decreased with increasing tree size (Table 15 and Figure 15). 66 Unlike the results of Trial 1 which indicated no significant effects of fertilization on NIN, in Trial 2 there were significant fertilizer effects on NIN depending on genotype and resistance status. These findings suggest that in some genotypes the number of inner resin canals produced by the apical meristem may be fixed, while in others nutrient availability may influence inner resin canal formation. However, these findings were based on significant results in only two resistant and two susceptible genotypes. The lack of significant differences in constitutive resin canal characteristics between resistant and susceptible trees has been reported in the past, by researchers who also studied trees from the Big Qualicum provenance (Tomlin 1996; Brescia 2000; Grau et al. 2001). Tomlin (1996) concluded that the high degree of resistance exhibited by the resistant Big Qualicum trees must be attributed to other traits than cortical resin canals. It is recommended that further studies be conducted on trees from different provenances to determine whether resistance status does indeed influence fertilizer effects on constitutive resin canal characteristics. The between-trial variation observed in many of the constitutive resin canal variables may have been due to differences in tree material. The parents of the seedlings used in Trial 1 represented a different, and broader, range of geographic locations, from northern Washington to northwestern Vancouver Island, while the Trial 2 parents were all from a relatively small area on east coast of Vancouver Island (near Qualicum). Differences in constitutive resin canal characteristics among trees from different provenances has been reported by Tomlin (1996) and Grau et al. (2001). It is also possible that differences in the nature of the tree material (seedling vs somatic seedling) may have influenced fertilizer effects on constitutive defenses. 67 CONCLUSIONS The results of Trial 2 confirmed the findings of Trial 1, emphasizing the importance of recognizing the variation in fertilizer effects due to Stika spruce genotype. Resistance status influenced fertilizer effects on only one variable (NIN) which was also influenced by genotype. It is recommended that further studies be conducted to confirm the influence of resistance status. These findings indicate that while constitutive resin canal defenses may be genetically based, for some genotypes the phenotypic expression of these defenses can be altered by fertilization. The effects of fertilization may act either indirectly, by influencing tree size, or possibly directly, by influencing allocation of resources to constitutive defenses, as suggested by Loomis (1953) and Bryant et a/.(1983). 68 TRIAL 3. Fertilizer effects on incidence of weevil attack and resin canal defenses of interior spruce of unknown resistance status METHODS Tree material and fertilization Trial 3 was conducted in an operational interior spruce (P. englemanni x P. glauca) plantation. A number of fertilizer treatments have been and are still being tested at this site, which has also been naturally infested by Pissodes strobi. Trees in this trial can be identified only by seedlot and have not been tested for weevil resistance. The goal of this trial was to determine the effects of fertilization on the resin canal defenses and incidence of natural weevil attack in interior spruce in a plantation setting and to compare the results of this trial to results from the previous two tests. In 1995, Dr. Robert Brockley of the BC Ministry of Forests, established a maximum productivity research trial in the SBSwk1/01 biogeoclimatic zone east of Hixon, BC, in Dunkley Lumber Ltd. Tree Farm License 53, near Lodi Lake. The goal of the trial was to study the effects of intensive, repeated fertilization on the growth and yield of interior spruce and to document the effects of repeated nutrient additions on various ecosystem processes. The site was harvested in 1985, broadcast burned in 1986 and planted in 1987 with interior spruce, at a stand density of 1100 stems per hectare. In 1995, eighteen 36.2m x 45.3m fertilizer treatment plots were established, each including 64 trees. Each plot is surrounded on three sides by two rows of treated buffer trees and on the fourth side by five rows of treated buffer trees. There are three replications, or blocks, each consisting of one plot for each of six treatments (Table 16). Plots were randomly assigned fertilizer treatments. In the spring, commencing in 1996, each treatment plot was divided into 16 equally sized segments with string tied to 69 aluminum posts. Pre-weighed amounts of specified fertilizer were then hand spread within each sector. Table 16. Descriptions of fertilizer treatments employed in the BC Ministry of Forests Maximum Productivity research trial at Lodi Lake, BC. Treatment code Treatment (quantities in kg/ha) ON2 fertilized yearly to maintain foliar N levels at 1.6%, other nutrient levels maintained at optimal levels* ON1 fertilized yearly to maintain foliar N levels at 1.3%, other nutrient levels maintained at optimal levels Complete fertilized every six years with: 200N, 100P, 100K, 50S, 25Mg, 1.5B NSB fertilized every six years with: 200N, 50S, 1.5B NB fertilized every six years with: 200N, 1.5B Control not fertilized "typically, ON1 and ON2 treatments receive 50-75 and 100-150 kg N/ha, respectively each year. In addition, other nutrients are added about every 2nd or 3rd year. The formulations for the fertilizations used in the ON1 and ON2 treatments were based on the results of foliar analyses undertaken the previous fall by Dr. R. Brockley of the BC Ministry of Forests. The nutrient sources used for the fertilizer treatments are given in Table 17. Table 17. Nutrient sources for fertilization treatments applied at the BC Ministry of Forests Maximum Productivity research trial at Lodi Lake, BC. Nutrient Source NPK Ratio Nitrogen urea 46-0-0 ammonium nitrate 34-0-0 Nitrogen and phosphate monoammonium phosphate 11-52-0 Potassium, magnesium and sulfur sulphate potash magnesia 0-0-22-11Mg-22S Potassium potassium chloride 0-0-60 Magnesium Tiger 36 36% Mg and 6% S Boron granular borate 15% B 70 Fertilizer effects on height, constitutive and traumatic resin canal defenses and incidence of weevil attack were assessed for these trees. Data collection and analysis Tree size All of the trees in each plot were measured in the fall of 1998 and again in the fall of 2001, using a telescoping height pole. The heights of trees with top kills due to weevil attack, were measured to the tallest live growth and were included in the means. Three- year height increment was calculated for each tree and divided by three to give the average leader length for the last three years. Leader diameter was measured using the same methods as in Trial 1, in the spring of 2001, on 10 trees per plot, the same trees that were sampled for analysis of constitutive resin canal defenses. Constitutive resin canal defenses In late May 2001, leader samples were collected for analysis of constitutive resin canals. Samples were collected from ten trees randomly chosen from the treated buffer of each plot. Prior to sampling, height, leader length and apical bud phenology stage were measured. Four centimeter sections of leader were collected starting approximately two centimeters below the apical bud, in the area where weevil oviposition would usually occur. The samples were fixed, and slides were prepared, scanned and measured using the same methodology as described for Trial 1. Analysis of variance (ANOVA) was performed using STATISTICA@to detect the effects of replicate and fertilizer treatment on constitutive resin canal variables (for model see Appendix 6). Type III sums of squares were calculated using the general 71 linear model. Fertilization was treated as a fixed effect. A log transformation was applied to SZOUT in order to meet the assumptions of normality within treatment groups and homogeneous variances among groups. Tukey's HSD was used to detect differences between groups. Traumatic resin canal defenses In late May 2001, ten trees from each of the fertilized buffer areas of plots 1-12 were randomly selected for artificial wounding. Artificial wounding was conducted using the same methodology as used in Trial 1. A total of 18 punctures, in three vertical rows of six, were made. At the time of wounding, weevils were observed feeding and mating on the leaders; however, no egg plugs were observed. In September 2001, using the same methodology as in Trial 1, stem samples were collected from wounded trees, and microscopy slides were prepared and scanned. ANOVA were conducted to detect the effects of fertilization and replicate on traumatic response rating (TRAU). Preliminary analyses indicated negligible effects of replicate, consequently this variable was excluded from the analysis. Type III sums of squares were calculated using GLM (STATISTICA®) procedures. Examination of the residuals indicated normally distributed errors for TRAU. Fertilization was treated as a fixed effect. Effects were considered significant at p< 0.05. Incidence of weevil attack In May 2001, all 64 trees in each plot were surveyed for weevil damage from the ground. Damage was evidenced by the presence of crooks and forks in the bole accompanied by an old, dead leader or leader stub if the dead leader had broken off, at 72 the site of the deformity. Each tree was scored either 1, for attacked or 0, for not attacked. The proportions of attacked and unattacked trees were calculated for each fertilizer treatment within each plot. ANOVA was performed using replicates as experimental units. No transformations were required to meet the assumptions of ANOVA. Tukey's HSD was performed to detect differences between groups. RESULTS AND DISCUSSION Tree size In each replicate, control trees were the shortest and trees from either the ON2 or ON1 treatments were the tallest (Figure 19). In replicate 1, ON1 trees were shorter than NSB and complete trees, although this difference was not significant. In replicate 2, complete trees were shorter than all but control trees, but not significantly shorter than NB trees. Fertilizer effects on leader length mirrored the effects on height with the longest average leader lengths in the ON1 or ON2 treatments within each replicate (Figure 20). In replicate 2, ON2 leaders were 51 percent longer than control leaders, whereas in replicates 1 and 3 the longest leaders (ON2 and ON1 respectively) were only 36 and 15 percent longer than the control groups. Fertilizer effects on leader diameter appeared to vary among replicates, however, trees in the NB treatment consistently had leader diameters that were smaller than those of the control trees (Figure 21). No fertilizer treatment consistently produced the largest leaders in each replicate. 73 d ' d be C n # a I? ; control NB NSB complete ON1 ON2 REP 2 ~T~ ±1.96*SE of mean ±1.00*SE of mean • Mean control NB NSB complete ON1 ON2 REP 3 Block and Fertilizer treatment Figure 19. Average height of 16 year-old interior spruce trees fertilized with six fertilizer treatments in three replicates (n=64 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05). ' ' ' ' c ; o T ..a i a. b ab a a n a _i_ 9 i-i; control NB NSB complete ON1 ON2 REP 1 REP 2 ±1.96*SE of mean ±1.00*SE of mean • Mean control NB NSB complete ON1 ON2 REP 3 Block and Fertilizer treatment Figure 20. Average leader length of 16 year-old interior spruce trees fertilized with six fertilizer treatments in three replicates (n=64 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05). 74 11.5 a a 10.5 ab 9.5 a a ^ t E 8.5 b . E i f 7.5 o V E 6.5 .2 control NB NSB complete ON1 ON2 control NB NSB complete ON1 ON2 •a REP 1 REP 2 k. CD TJ rs © o 2 > < 6.5 control NB NSB complete ON1 ON2 REP 3 Block and Fertilizer treatment Figure 21. Average leader diameter of 16 year-old interior spruce trees fertilized with six different fertilizer treatments in three replicates (n=10 for each group). Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05). The observed variation in fertilizer effects between replicates may be explained by a number of factors. Since the topography of the site was not uniform, the replicates, and plots within replicates, sometimes differed in slope and aspect which likely affected soil drainage and heat accumulation. Natural soil fertility levels may also have been different among the plots. Any of these factors may have influenced tree height. The trees in this trial were considerably larger (average size of 66 cm in Trial 1, 54 cm in Trial 2 and 473 cm in Trial 3) than in the other two trials. Unlike the trees used for the previous two trials, the trees in Trial 3 were tall enough (all trees were taller than 1.5 m) and had thick enough leaders (all leaders were greater than 4 mm thick), to be considered acceptable hosts to the white pine weevil (Sullivan 1961; Turnquist and 75 Alfaro 1996). As a result, weevil attacks at this site have also likely influenced tree height, since there was evidence of weevil damage that dated back to 1996. The formation of multiple leaders as a result of weevil damage, likely influenced leader diameter, resulting in thinner leaders on attacked trees, as resources would have been partitioned amongst them. Weevil damage likely also influenced leader length in the same way, however since leader length was averaged over three years, the influence of weevil damage on leader length was less severe. Constitutive resin canal defenses There was a significant effect of fertilization on most of the constitutive resin canal variables measured (Table 18). The only variable for which there was a significant interaction between replicate and fertilizer effects was the number of outer resin canals (NOUT). Average number of inner resin canals (NIN) varied significantly between replicates, but the effects of replicate did not interact with the effects of fertilization. As in Trials 1 and 2, bark thickness (BTHK) was significantly affected by fertilizer treatment. The two optimal nitrogen treatments (ON1 and ON2) yielded the thickest bark whereas the thinnest bark was found in nitrogen/boron (NB) treated trees. The other two treatments (NSB and complete) had intermediate BTHK but were not significantly different from the control trees (Table 19). NIN varied with both fertilizer treatment and replicate. Average NIN was significantly smaller in replicate 3 than in the other two replicates. Pooling the replicates together, only two fertilizer treatments had significantly different NIN (Table 19). ON2 trees had the greatest NIN while NB trees had the least. 76 Table 18. Summary of ANOVA for analysis of the effects of fertilization (FERT) and replicate (REP) and their interactions, on characteristics of the cortical resin canal system of interior spruce maintained under six fertilizer regimes*. Variable and effects df MS Effect MS Error F p>F Number of inner resin canals (NIN) FERT 5 6.3867 1.5067 4 24 0 0249 REP 2 10.4000 2.3975 4 34 0 0146 FERTxREP 10 1.5067 2.3975 0 63 0 7880 umber of outer resin canals (NOUT) FERT 5 9.4222 10.1656 0 93 0 5027 REP 2 9.7056 3.9469 2 46 0 0887 FERTxREP 10 10.1656 3.9469 2 58 0 0064 Size of inner resin canals (SZIN) FERT 5 0.0008163 0.0001533 5.33 0.0121 REP 2 0.0001405 0.0002010 0.70 0.4986 FERTxREP 10 0.0001533 0.0002010 0.76 0.6645 Size of outer resin canals (SZOUT) FERT 5 1.2020 0.3354 3.58 0.0407 REP 2 0.3964 0.3034 1.31 0.2737 FERTxREP 10 0.3354 0.3034 1.11 0.3612 Bark thickness (BTHK) FERT 5 0.7172 0.1067 6.72 0.0054 REP 2 0.0871 0.0839 1.04 0.3566 FERTxREP 10 0.1067 0.0839 1.27 0.2511 Bark area occupied by resin canals (AOCC) FERT 5 2.1368 1.8149 1.18 0.3848 REP 2 1.1515 1.9111 0.60 0.5486 FERTxREP 10 1.8149 1.9111 0.95 0.4896 Number of resin canals per mm2 bark (NMMS) FERT 5 1.6613 0.3351 4.96 0.0153 REP 2 0.1055 0.2851 0.37 0.6914 FERTxREP 10 0.3351 0.2851 1.18 0.3112 Average distance between resin canals (GAP) FERT 5 0.0070 0.0153 0.46 0.7990 REP 2 0.0259 0.0106 2.45 0.0893 FERTxREP 10 0.0153 0.0106 1.45 0.1638 Average depth of inner resin canals (DEP) FERT 5 0.2379 0.0657 3.62 0.0396 REP 2 0.2054 0.0743 2.76 0.0661 FERTxREP 10 0.0657 0.0743 0.88 0.5493 * For a description of fertilizer regimes, see Table 16. There was a significant interaction between the effects of fertilization and replicate on NOUT (Table 18). However, the effects were only significant in replicate 1 77 and there was no apparent trend in the response of NOUT to fertilizer treatments within each replicate (Figure 22). Table 19. Average constitutive resin canal characteristics* observed in quarter cross sections of interior spruce leaders maintained under six fertilizer regimes**. Values with the same letter are not significantly different within columns, Tukey HSD (p<0.05). treatment BTHK NIN SZIN SZOUT AOCC NMMS GAP DEP mm mm2 mm2 % mm mm control 1.666 a 7.5 aD 0.024476 aD 0.013998 ab 3.595 a 1.979 aD 0.388 a 1.622 ab NB 1.529 a 7.0 a 0.024581 ab 0.010967 a 3.869 a 2.302 b 0.380 a 1.565 a NSB 1.752 ab 7.5 ab 0.023581 a 0.014834 ab 3.526 3 1.994 ab 0.396 a 1.743 ab ab abc b a ab complete 1.767 7.5 ab 0.027434 0.017352 3.752 a 1.793 a 0.401 1.755 ON1 1.931 b 7.5 ab 0.035115 c 0.017579 b 4.028 3 1.664 a 0.419 a 1.800 b ON2 1.927 b 8.4 b 0.034475 be 0.021726 b 4.232 a 1.716 a 0.416 a 1.671 ab * For variable definitions, see Table 1. **For a description of fertilizer regimes, see Table 16. 12 11 10 • —4 a g :::?:i:::::±::::::i:j[': 8 rs 7 • c I I rs 6 u 5 10 4 Q) control NB NSB complete ON1 ON2 REP 1 REP 2 I o t L. L.x..i <*- o - t- —-a—r 0) XI Mean±1.96*SE of mean MeantSE of mean E D Mean 3 Z r j ...j .„..;.....a... control NB NSB complete ON1 0N2 REP 3 Fertilizer teatment Figure 22. Effects of fertilization and replicate on the number of outer resin canals in quarter cross sections of interior spruce leaders grown under six fertilizer regimes. Treatments with the same letter are not significantly different within each replicate (Tukey's HSD, p>0.05). 78 There were significant differences in the size of inner (SZIN) and outer (SZOUT) resin canals between fertilizer groups (Table 18). Although they were not significantly different from all other treatments, ON1 and ON2 treatments had the largest inner resin canals while the NSB treatment had the smallest. ON1, ON2 and complete treatments also had significantly larger outer resin canals than NB trees. SZOUT of control and NSB trees were not significantly different from any of the other treatments. Fertilization did not affect resin canal density when expressed by area (AOCC), but significantly affected resin canal density when expressed by number (NMMS). The highest NMMS occurred in NB trees while the lowest density occurred in the ON1, ON2 and complete groups. NMMS of the control and NSB groups were not significantly different from that of any of the other groups. There was no significant effect of fertilization on the distance between inner resin canals (GAP), however, depth of inner resin canals (DEP) was affected by fertilization. ON1 trees had the deepest inner resin canals while NB trees had the shallowest inner resin canals. DEP was not significantly different in any of the other treatments. Compared to the first two trials, average bark thickness and size, number and depth of resin canals were larger in Trial 3. This is likely the result of a number of factors, including tree age, size and species of spruce. The intensity and composition of the fertilizer regimes may also have influenced the observed variation in cortical resin canal characteristics between the trials. Regardless of the between-trial differences in average values of resin canal variables, the effects of fertilization on the cortical resin canal system of interior spruce in a plantation setting were similar to the effects observed in the previous two trials using potted Sitka spruce, with a few notable differences. As in Trial 2, the number of 79 inner resin canals (NIN) was significantly influenced by fertilization. However, unlike the results of Trials 1 and 2 which respectively showed no significant fertilizer effects on NIN and fertilizer effects depending on resistance status, in Trial 3, the greatest NIN was found in the most intense fertilization treatment (ON2) and the smallest NIN was found in the least intense fertilization treatment (NB). Fertilizer effects on NOUT and GAP were not significant in Trial 3 whereas they were significant in the first two trials. The observation of no significant fertilizer effects on AOCC in Trial 3 is similar to the results of Trial 2 which indicated significant fertilizer effects on AOCC in only two of the eight genotypes. It is possible that discrepancies between the Trial 3 and the first two trials may be due to varying responses to fertilization depending on spruce species. Similar to the first two trials, the variation in all of the resin canal characteristics, appeared to be related to fertilizer effects on tree size except the number of outer resin canals (NOUT). Although it was not tested statistically, as bark thickness increased, constitutive resin canal variables generally increased, except NMMS, which decreased with increasing bark thickness. While the first two trials suggest that fertilizer effects on tree size influence NOUT in the cortex, the results of Trial 3 suggest that once the leaders reach a certain size and diameter, fertilization may no longer affect NOUT. Similarly, fertilizer effects on GAP may also diminish since Trial 3 was the only trial in which GAP was not significantly affected by fertilizer treatment. In summary, the effects of fertilization on the constitutive resin canal defenses of interior spruce were as follows. There was a positive relationship between fertilization and these variables: bark thickness (BTHK), number of inner resin canals (NIN), size of inner and outer resin canals (SZIN and SZOUT) and the distance between inner resin 80 canals (GAP). Increasing fertilization had no effect on the number of outer resin canals (NOUT) and the percent of bark area occupied by resin canals (AOCC). There was a negative relationship between increasing fertilization and resin canal density (NMMS) while the effects of increasing fertilization on inner resin canal depth were variable, depending on the fertilization treatment. Traumatic resin canal defenses As in trial 1, mechanical wounding induced a clear traumatic response, which ranged from 0 (no response) to 4 (complete ring of traumatic resin canals). ANOVA indicated significant effects of fertilization on the traumatic response rating (F(5,114)= 2.70, p<0.03), however post hoc comparisons revealed differences only between the NB treatment which had the strongest response, and the NSB treatment which had the weakest response (Table 20). Table 20. Means and standard deviations (SD) of traumatic response rating in interior spruce treated with six fertilization regimes* (n=20 for each group). Treatment Mean SD control 2.9aD 07 NB 3.2b 1.0 NSB 2.3a 1.5 complete 3.1 ab 0.8 ON1 2.9ab 1.0 ON2 2.4ab 1.0 *For a description of fertilizer regimes, see Table 16. 81 The response ratings observed in Trial 3 were similar in magnitude to the ratings observed in Trial 1. However, the lack of significant differences in response ratings between the fertilized groups and the control group indicate that the significant difference between the NB and NSB groups may have been due to something other than fertilizer effects. Response intensity has been shown to vary with wound intensity, tree phenology and genotype (Tomlin et al. 1998; Brescia 2000). While wound intensity was held as constant as possible, any of these factors may have influenced the results of this study. In summary, fertilization had no dramatic influence on the ability of trees to produce traumatic resin canals in response to artificial wounding in this experiment. This may indicate that all treatments including the controls, had adequate energy and nutrient stores to produce a traumatic resin response. Incidence of weevil attack Fertilizer treatment significantly influenced weevil attack rates (F(5,12)= 5.1571, p< 0.01). There were more than twice as many weevil-damaged trees in the ON2 and ON1 treatments than in the control treatment (Figure 23). The other three treatments were not significantly different from each other or the rest of the treatments. It is apparent that the observed distribution of weevil attacks was due to the preference of weevils for tall, vigorously growing trees, with the thickest bark. These characteristics, in combination with longer leaders, would have provided weevils with more resources for feeding and oviposition than trees in the other treatments. It should also be noted, that although trees in the ON2 and ON1 treatments sustained the highest frequencies of weevil attacks, on average, trees in these groups remained the tallest. 82 60 ~r~ ±1.96*SE of mean CD ±1.00*SE of mean D) •CO • Mean 50 £ ro > CD CD 40 x: CD 30 CD CD 20 o s 0 CL 10 Control NSB Complete Fertilizer treatment Figure 23. Percent of interior spruce trees with weevil damage in each of six fertilizer treatments. Treatments with the same letter are not significantly different (Tukey's HSD, p>0.05). Resin canal defenses may also have played a role in influencing incidence of weevil attack. The treatments with the highest frequencies of attack (ON2 and ON1) also had the lowest cortical resin canal densities (NMMS). These findings are in accordance with observations by Tomlin and Borden (1997a) who reported that Sitka spruce with higher densities of resin canals in combination with thinner bark sustained fewer weevil attacks. It is unlikely that the strength of the traumatic response, as measured by the relative number of traumatic resin canals, influenced tree susceptibility to weevil attack since response ratings between the majority of the treatments were not significantly different. 83 CONCLUSIONS The results of this trial indicate that the incidence of weevil attacks in interior spruce increases with fertilizer treatment intensity. This is likely due to the preference of weevils for the larger, more vigorous leaders produced by the higher intensity fertilization treatments. Increased area for feeding and oviposition in the cortex, due to fertilization, likely improved weevil brood survival. Although the size and number of cortical resin canals increased with increasing fertilizer intensity, the density of the resin canals as expressed by NMMS decreased due to thicker bark in higher fertilization treatments. This decrease may have resulted in weevils encountering resin canals less frequently, which resulted in the trees from the more intense fertilizer groups being more susceptible to the weevil. It is not likely that the traumatic resin response contributed to the variation in the incidence of weevil attacks among fertilizer groups. 84 SUMMARY AND DISCUSSION Constitutive resin canal defenses The effects of fertilization on constitutive resin canal defenses were explored in each of the three trials. The results suggest that fertilization influences constitutive resin canal systems in both Sitka and interior spruce by affecting tree growth rate. Although the effects of fertilization on some of the constitutive resin canal variables depended on genotype, in general the results of these studies show that fertilization causes an increase in cortical resin canal size and a decrease in the density of resin canals (as expressed by NMMS). A summary of my findings of the effects of fertilization on constitutive and traumatic resin canal characteristics of Sitka and interior spruce is given in Table 21. The observed effects of fertilization on resin canal size, and number (of inner resin canals in interior spruce and some Sitka spruce genotypes, and outer resin canals in Sitka spruce), are contrary to what was expected according to theories of resource allocation to defense (Loomis 1953; Bryant et al. 1983). Loomis (1953) and Bryant et al. (1983) proposed that resources tend to be allocated to growth when conditions are favorable. Conditions that limit growth, but not photosynthesis, promote increases in differentiation and secondary metabolism, processes which are responsible for the formation of resin and resin canals. Following this theory, one would expect that fertilization would have either no effect or negative effects on resin canal size and number. However, contrary to this theory, increases in the size or number of resin ducts in response to fertilization and other environmental factors have been reported in the past, in the woody tissues and needles of conifers (Smith et al. 1977; DeAngelis et al. 85 1986; Kainulainen etal. 1996; Kyto etal. 1996; Wainhouse etal. 1998). The observed increase in resin canal size may be explained by the tendency of resin canals to enlarge with increasing stem diameter and bark thickness in response to fertilization, or as a result of seasonal growth. In this study I measured cortical resin canal attributes at the beginning of the growing season. Brescia (2000) studied the changes in resin canal characteristics over a growing season and observed an increase in both inner and outer resin canal size in Sitka spruce. Smith et al. (1977) also observed that resin canal diameter increased as ring width increased in the wood of Corsican pine. According to Jou (1971), there is also an increase in the size and branching of cortical resin canals as diameter increases progressively down a terminal internode. The trend of increasing branching of cortical resin canals with increasing stem diameter, might explain the observed increase in the number resin canals in interior spruce and some of the Sitka spruce genotypes, as stem diameter increases in response to fertilization. Table 21. Summary of the significant effects of fertilization treatments varying in intensity, on constitutive resin canal systems of 2 year-old Sitka and 16 year-old interior spruce. Effect of fertilization treatments increasing in intensity Variable Sitka spruce Interior spruce NIN varied with genotype and resistance status increased NOUT increased no effect SZIN increased in some genotypes increased SZOUT increased increased NMMS decreased decreased AOCC varied with genotype and resistance status no effect DEP increased varied with treatment GAP increased increased* *this increase was not statistically significant, but consistent and therefore noteworthy 86 It is possible that fertilization also affects the rate of increase in resin canal size and frequency of branching (thereby influencing the number of resin canals in cross section), over a growing season. Fertilization affects physiological processes, such as bud phenology and developmental rate (personal observations); these processes have been linked to weevil resistance (Alfaro et al. 2000) and are known to be important regulators of host/insect synchrony (Quiring 1992; Hulme 1995). Therefore, fertilization may affect the window of susceptibility available for weevils to attack the tree, by influencing seasonal changes in cortical resin canal defenses (Brescia 2000). While the observed effects of fertilization on resin canal size and number were contrary to what was expected, the observed decrease in resin canal density in response to increasing fertilization agreed with hypotheses of resource allocation (Loomis 1953; Bryant et al. 1983). An inverse relationship between fertilization intensity and cortical resin canal density (NMMS) indicates that allocation of additional resources in fertilized trees is mainly to growth. Although the number of resin canals increases with fertilization in interior spruce, this increase may not be substantial enough to maintain the levels of resin canal densities observed in the lower fertilization treatments as the cortex thickens due to fertilization. It is likely however, that the decrease in resin canal density (NMMS) is compensated for by an increase in resin canal size as the bark thickens. This may explain the observed stability in the percent of bark area occupied by resin canals (AOCC) with increasing fertilization. Although resin canal size increases, the resulting AOCC, regardless of its stability, apparently does not provide adequate protection against the weevil. Fertilization also affected the distribution of inner resin canals within the cortex. The observed increase with fertilization, in the average depth of the inner resin canal 87 ring (DEP) and the average distance between inner resin canals (GAP), may influence the rate of oviposition and successful feeding behavior of adult weevils as well as brood survival. Stroh and Gerhold (1965) found that the amount of weevil feeding and feeding cavity dimensions, were positively correlated with resin canal depth. They observed that weevil feeding cavities often contacted the epithelial cells of the resin canals. They proposed that when weevils encounter these cells, the direction of feeding is reoriented, continuing around the resin canal, as long as there is adequate space between the inner and outer resin canals. Sullivan (1961) suggested that the average bark thickness required for weevil oviposition is 0.8 mm since that is the average length of a weevil egg. However, if a weevil can avoid encountering outer resin canals, it is less likely to encounter inner resin canals if it can achieve this depth before reaching the inner resin canal ring. This could have been achieved in all of the fertilizer treatments in Trial 3. However, none of the DEP values in Trials 1 and 2 were large enough for this to be achieved. As a result, the depth of weevil feeding and oviposition punctures in these trees would likely be dictated by the ability of the weevil to successfully avoid, inner resin canals (Stroh and Gerhold 1965) or ultimately deactivate them by severing their connection to the vascular bundles (Alfaro et al. 1999). This may depend in part, on the distance between inner resin canals (GAP). Exploring a variable similar to GAP, Tomlin and Borden (1997a) correlated weevil resistance with a combination of thin bark and low outer resin canal density (which was expressed by the number of resin canals per centimeter of circumference). They proposed that a combination of thin bark and a high density of outer resin ducts would increase the chance that weevils would encounter resin ducts, and in some genotypes, 88 would prevent the acceptance of the tree by a weevil. The GAP values measured in Trials 1-3 ranged from 0.208 mm to 0.380 mm in the control groups, and in higher intensity fertilizer groups, ranged from 0.415 to 0.592 mm. Since average weevil snout diameter is 0.4 ± 0.02 mm (Manville et al. 2002) weevil-feeding early in the season on control trees would likely be limited to the area of the bark outside the inner resin canal ring. Weevils feeding on fertilized trees would be more able to feed between inner resin canals with each successive fertilizer treatment. Greater distances between inner resin canals also decreases the likelihood that a weevil larva will contact an inner resin canal while feeding. The average width of the head capsule of a first instar weevil larva ranges from 0.237 to 0.451 mm (Silver 1968). These values indicate that some first instar larvae might fit between the inner resin canals of some trees but not others. However, there is less of a likelihood that larvae feeding on a tree from a more intense fertilizer treatment would encounter a GAP too small to fit through. Because the GAP values in these trials were measured at the beginning of the growing season, when weevils were just starting to oviposit, it is possible that these values do not represent the GAP sizes encountered by newly hatched weevils, since GAP may increase with increasing stem diameter during the growing season. It would be beneficial to conduct a study that investigates the effects of fertilization on tree development and the changes in the constitutive resin canal system through the season as described by Brescia (2000). The results of Trial 2 indicate that there are genotype effects that influence the effects of fertilization on constitutive resin canals variables in young Sitka spruce. The finding that the phenotypic expression of these defenses can be altered by fertilization will be important to consider while employing resistant genotypes in attempt to manage 89 weevil damage. Due to constraints in available resources, I was not able to study the interaction of genotype and fertilizer effects on incidence of weevil attack. It would be useful to conduct such a study, in effort to relate the combined effects of fertilization and genotype on constitutive resin canal defenses, to their effects on incidence of weevil attack. From the results of this study, considering only constitutive resin canal defenses, it can be expected that trees from more intense fertilization treatments are more favorable hosts for the weevil. Traumatic resin canal defenses The effects of fertilization on traumatic resin canal defenses were studied in Trial 1, using young, potted Sitka spruce, and again in Trial 3, utilizing interior spruce in a plantation setting. The results of these studies indicate that fertilization influences the traumatic resin response in young, severely nutrient-stressed Sitka spruce. However, the lack of a significant difference in response rating between the fertilized treatments in Trial 1, and between all of the treatments in Trial 3, indicate that only severe nutrient deficiencies influence the ability of a tree to produce an energy-expensive traumatic response. The control trees in Trial 1 were indeed stunted and very chlorotic whereas the control trees in Trial 3 looked fairly healthy but were smaller than the fertilized trees. It is also possible that since the control trees in Trial 1 had invested in a strong constitutive defense system (ie. very dense cortical resin canals compared to the other treatments), it may have been inefficient or energetically impossible for these trees to also produce a strong traumatic response. 90 Incidence of weevil attack It is evident from the studies described herein, that fertilization to improve growth rates influences incidence of weevil attack. In both studies (Trials 1 and 3) the weevil indicated a preference for vigorously growing hosts with thicker bark and longer leaders. This finding is in accordance with a number of studies (Kriebel 1954; Silver 1968; VanderSar and Borden 1977; Weetman etal. 1989; Alfaro etal. 1993 ; King etal. 1997). These results were also predicted by Price (1991) who stated that herbivore species that are intimately involved in plant growth processes, such as stem borers like Pissodes strobi, will prefer vigorous plants. The preference of weevils for well fertilized trees may be the result of fertilizer effects on a number of variables measured in the preceding trials. Fertilizer effects on tree size may influence host recognition. Studying the visual orientation of weevils, VanderSar and Borden (1977) found that weevils are predisposed to attack longer, thicker leaders. Brood survival may be affected since vigorous leaders with thicker bark can support a greater number of oviposition punctures and more larval feeding. Deposition of a large number of eggs in a concentrated area is thought to enable the weevil brood to overcome the tree's constitutive defenses by collapsing the resin canal system (Alfaro et al. 1999). Increased resource availability could also decrease weevil mortality due to larval competition. Although the traumatic response did not play a role in influencing incidence of weevil attack, it was found that the distribution of constitutive resin canals in the cortex is more favorable for weevil survival in trees from more intense fertilizer treatments. A number of other tree characteristics that have been shown to influence incidence of weevil attack, but were not studied in this thesis, may have also been 91 influenced by fertilization. The chemical composition of resin and bark constituents as well as resin flow, have been shown to be influenced by fertilization (Holopainen et al. 1995; Kainulainen etal. 1996; Kyto etal. 1996, 1998; Wainhouse etal. 1998; Warren et al. 1999; Viiri et al. 1999). Although it has not been studied to date, it is possible that the nutritive value of the cortex may also be affected by fertilization. 92 CONCLUSIONS The use of fertilizers to improve growth in young interior spruce can cause an increase in the incidence of weevil attack of up to 30 percent (50% of trees attacked in ON2 group compared to 20% in the control group, in Trial 3). Incidence of attack in young Sitka spruce is also increased by fertilization. This study has identified a number of characteristics of Sitka and interior spruce that are affected by fertilization and may be the cause of the observed increase in incidence of weevil attack. Fertilizer causes enhanced leader length, diameter and bark thickness, essentially increasing the resources available for weevil feeding and oviposition. Fertilization also causes increased diameter growth that leads to a dilution of cortical resin canal defenses including an increase in the depth and distance between inner resin canals in the cortex. The observed variation in constitutive resin canal characteristics may be the result of direct effects of fertilization on the ability of the tree to produce cortical resin canals, or it may be indirectly due to the effects of fertilization on tree size. The effects of fertilization on traumatic response were too insufficient to influence incidence of weevil attack. However, in severely nutrient stressed trees, fertilization may improve the ability of the tree to produce a traumatic resin response. The results of Trial 2 demonstrated that fertilization acts on constitutive resin canal defenses of young Sitka spruce independently of the tree's weevil resistance status. However, the effects of fertilization varied among genotypes. The observations of this trial have important implications in the deployment of resistant spruce genotypes for weevil management purposes. In areas where fertilization may be required, special consideration should be given to the selection of genotypes employed for weevil management. Because these studies tested only a few genotypes, it is recommended 93 that a larger scale study be conducted utilizing genotypes likely to be employed in the field. A study similar to that described in Trial 3 but utilizing known, weevil resistant genotypes, would be ideally suited for such a purpose. There are many other aspects of the weevil/spruce interaction that may be influenced by fertilization but were not studied in this thesis. A number of studies have demonstrated significant effects of resource availability on resin composition and flow (Holopainen etal. 1995; Kainulainen etal. 1996; Kyto etal. 1996, 1998; Wainhouse et al. 1998; Warren etal. 1999; Viiri etal. 1999). Resin flow has not been studied with respect to incidence of weevil attack, however it has been correlated with resistance to bark beetles (Hodges et al. 1979). Aspects of resin composition have been correlated with weevil resistance in a number of studies (Tomlin 1996; Nault etal. 1999; Tomlin et al. 2000). Further studies of fertilizer effects on traits such as these may lead to a more successful deployment of weevil resistant spruce in high weevil hazard areas. There is also a need to study the trade-offs between the positive effects of fertilization on growth and the negative effects of fertilization which result from increased weevil damage. Replication of this study in areas of different weevil hazard is recommended. This would enable the development of guidelines to maximize the positive effects of fertilization while minimizing the negative ones. It is anticipated that the results of these studies and future studies of fertilizer effects on weevil resistant genotypes will be utilized in the development of a system aimed at the successful regeneration of spruce species in British Columbia. Such a system should incorporate the use of genetically improved stock and pest management strategies. In addition, decisions regarding silviculture treatments such as fertilization, will be necessary to maintain stand productivity and to fully realize the potential gains 94 from tree improvement and pest management. 95 LITERATURE CITED Alfaro, R.I. 1982. Fifty year-old Sitka spruce plantations with a history of intense weevil attack. J. Entomol. Soc. Brit. Columbia 79: 62-65. Alfaro, R.I. 1989a. Stem defects in Sitka spruce induced by Sitka spruce weevil, Pissodes strobi (Peck). In Alfaro, R.L, Glover, S.G. (Eds.), Insects Affecting Reforestation: Biology and Damage. 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Note 106. 109 APPENDICES 110 Appendix 1. Statistical model for the analysis of fertilizer and family effects on tree size variables and constitutive and traumatic resin canal defenses in Sitka spruce seedlings, Trial 1. Model: y^ = p. + Fk + Fam; + FFamy/< + e,-^; Where y,^ is the observation of seedling i in family j within fertilizer treatment k; u is the overall mean; Fk and Famy are the effects of fertilizer treatment k and family j respectively; FFam7v< is the interaction between fertilizer treatment k and family j; e,^ is the residual composed of the interaction of the individual seedlings with fertilizer treatment and family. Note: To test fertilizer effects on constitutive resin canal variables, observations from all families were pooled together and family effects were not tested. Appendix 2. Recipe for formalin acetic acid (FAA). 90 mL 70% EtOH 50 mL glacial acetic acid 50 mL formaldehyde 5% glycerin Appendix 3. Formulae for the calculation of constitutive resin canal variables: SZIN, SZOUT, AOCC, NMMS, DEP, GAP and BTHK. SZIN (mm2) = total area of inner resin canals (LI2) X 1 number of inner resin canals 106 SZOUT (mm2) = total area of outer resin canals (u2) X 1 number of inner resin canals 106 AOCC = total area of inner and outer resin canals (u2) X 100 bark area (u2) NMMS = number of inner and outer resin canals X 1 bark area (u2) 106 in Appendix 3. continued DEP (mm) = radius* - radius to inner resin canals* (u2) 1000 GAP = (7i2r)/4 - n(2(SZIN/7t) ) where r= radius to inner resin canal ring n n= number of inner resin canals in quarter cross section BTHK (mm) = radius* - radius to cambium* (u2) 1000 Appendix 4. Traumatic response rating system as used by Brescia (2000). 0 No resin canal formations or pre-formations 1 Dark ring (pre-canals) 2 Scattered formatin 3 Incomplete ring with scattered canals 4 Complete ring 5 Complete inner ring and incomplete outer ring 6 Two complete rings Appendix 5. Statistical model for the analysis of fertilizer, resistance class and genotype effects on constitutive resin canal variables in Sitka spruce somatic seedlings, Trial 2. Model: yijkl = u. + R, + Fk + RFjk + G(R)ffj,+ FG(R)fflW+ em Where y fertilizer treatment k; u is the overall mean; R/ and Fk are the effects of resistance class I and fertilizer treatment k respectively; G(R)©/ is the effect of genotype j within resistance class I; RF//< is the interaction between resistance class I and fertilizer treatment k; FG(R)(p/is the interaction between genotype j within resistance class I and fertilizer treatment k; ew is the residual composed of the interaction of the somatic seedlings with resistance class, fertilizer treatment and genotype within resistance class. * The average of several radii was taken to obtain these values. 112 Appendix 6. Statistical model for the analysis of fertilizer and replicate effects on constitutive and traumatic resin canal variables in interior spruce, Trial 3. Model: yijk = LI + Repy + Fk + RepFy* + eiJk Where is the observation of tree i in replicate j and fertilizer treatment k; u is the overall mean; Repy and Fk are the effects of replicate j and fertilizer treatment k respectively; RepFy^ is the interaction between replicate j and fertilizer treatment k; e^ is the residual composed of the interaction of the trees with replicate and fertilizer treatment. Note: To assess fertilizer effects on traumatic response, replicates were pooled and only the effects of fertilization were tested. 113