Mycorrhizal and Other Root Endophytic Fungi of Lupines in the Pacific Northwest Redacted for Privacy
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AN ABSTRACT OF THE THESIS OF Thomas E. O'Dell for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on May 12. 1992. Title: Mycorrhizal and other Root Endophytic Fungi of Lupines in the Pacific Northwest Redacted for Privacy Abstract approved: Jaws M. Trappe We investigated the root endophytic fungi of lupine using four approaches: (1) occurrence of fungal colonization in field- collected roots; (2) growth response of L. latifolius toinoculation with two types of fungi; (3) structure of root colonizations of Pinus and Lupinus by Phialocephala fortinii, a septate endophytic fungus of lupine; and (4) comparison root morphology, mycorrhizal colonization and natural 15N-abundance N2 fixation of three legumes.In part 1, three species of Lupinus were never observed to have fungal colonization; nine species were colonized by VA mycorrhizal fungi; seven species were colonized by fungi with septate hyphae which often formed intracellular sclerotia, here called septate endophytes.In part 2, shoot weight of 16 week old L.latifolius seedlings in the greenhouse was significantly reduced by Glomus spp. in one experiment; P. fortinii significantly increased nodule weight in one experiment and reduced itin the other.In part 3, P. fortiniicolonized root epidermal and cortical cells in the root hair zone on ultimate lateral pine roots, as well as cortical and epidermal cells of primary roots of Pinus and Lupinus.Fungal colonization was inter- and intracellular with sclerotia forming in cells of both hosts.Labyrinthine tissue, a type of fungal differentiation which occurs in the Hartig net of ectomycorrhizae, formed sporadically on pine roots.In part four, Roots of Lupinus albicaulis cv. hederma had a significantly larger proportion of coarse roots (> 1mm diameter) and significantly less mycorrhizal colonization than two other legumes, Medicago Iupinula and Trifolium hybridum.Estimated aboveground N derived from fixation ranged from 6.1 to 39.9 kg per hectare (average = 22.0 kg/ha) and did not vary significantly among species. Mycorrhizal and other Root Endophytic Fungi of Lupines in the Pacific Northwest by Thomas E. O'Dell A THESIS submitted to Oregon State University inpartial fulfillment of the requirements for the degree of Doctor of Philosophy Completed May 12, 1992 Commencement June 1992 APPROVED: Redacted for Privacy Professoof Botany and Plant Pathology in charge of major Redacted for Privacy ) Head of department of Botany and Plant Path y Redacted for Privacy Dea GraduatecSchool Date thesis is presented 12 May. 1992 Typed by Thomas O'Dell for Thomas E. O'Dell "The most important learning, that of experience, can neither be summoned nor sought out... It comes in its own good time and in its own way to the man who will go where it lives, and wait, and be ready, and watch." - Wendell Berry Table of Contents Introduction 1 Chapter 1 Root Endophytes of Lupine I Colonization in the Field 3 Summary 3 Introduction 3 Methods 6 Results 8 Discussion 9 Chapter 2.Growth Response of Lupinus latifolius to Phialocephala fortinii and Glomus spp. 20 Summary 20 Introduction 20 Methods 23 Plants 23 Fungi 23 Growing conditions and inoculations 24 Fungal Colonization 26 Statisticalanalyses 27 Results 27 Discussion 28 Chapter 3. Root Colonization of Lupinus latifolius and Pinus contorta by Phialocephala fortinii 34 Summary 34 Introduction 35 Methods 37 Plant Material 37 Fungi 38 Bacteria 39 Inoculation & Growing Conditions 39 External Morphology, Light Microscopy & Clearing of Roots .40 Photography 41 Results 41 Isolations of fungi 41 Growth pouch 42 Discussion 43 Chapter 4. Natural 15N Abundance Estimates of Nitrogen Fixation by Three Legume Species in Conifer Plantations of the Wenatchee National Forest 57 Summary 57 Introduction 57 Methods 59 Sites 59 Collecting and processing plants 60 Tissue analyses 61 15N 61 Fungal colonization 62 Statisticalanalyses 62 Results 63 Root morphology 63 Nitrogen and phosphorus content 63 VAM & SE colonization 64 Discussion 64 Chapter 5.General Summary 70 What was Previously Known 70 Vesicular-arbuscular mycorrhizae (VAM) and Legumes 70 Septate Endophytic Fungi of Roots 70 Legume N fixation in Forestry Applications 71 What was Discovered by this Research 71 VAM in Lupines 71 SE in Lupines 71 N-Fixation 72 What it All Means (Conclusions and Speculations) 72 VAM and Lupines 72 Phialocephala fortinii and Lupines 72 Bibliography 74 List of Figures Figure 1.1.Approximate locations of collecting sites 13 Figure 3.1.Lupinus latifolius seedlings in growth pouch with agar plug (arrow) of Phialocephala fortinii 51 Figure 3.2.Conidiophore of P. fortinii in pure culture. 51 Figure 3.3.Root of L.latifolius colonized by P. fortinii. 51 Figure 3.4.Pinus contorta seedlings in growth pouch with agar plug (arrow) of P.fortinii. 51 Figure 3.5. P.fortiniiin pure culture with characteristic conidiophore and hyphal coils. 51 Figure 3.6.P. contorta roots on growth pouch with agar plugs colonized by P. fortinii 51 Figure 3.7.Cleared root of L.latifolius showing colonizationby P. fortinii 5 3 Figures 3.8 and 3.9.Cleared root nodule of L.latifolius colonized by P. fortinii 5 3 Figure 3.10.Cleared root of P. contorta colonized by P. fortinii 5 3 Figure 3.11.Cleared primary root of P. contorta showing labyrinthine tissue (arrow) of P.fortinii. 53 Figure 3.12.Transverse section of P. contorta root showing colonization of root hairs by P.fortinii. 53 Figure 3.13.Transverse section of uncolonized L.latifolius root 5 5 Figure 3.14.Transverse section of L.latifolius root colonized by P. fortinii 5 5 Figure 3.15.Longitudinal section of L. latifolius root 55 Figure 3.16.Transverse section of P. contorta short root colonized by P. fortinii 5 5 Figure 3.17.Transverse section of P. contorta short root colonized by P. fortinii 5 5 Figure 3.18.Longitudinal section of P. contorta short root colonized by P. fortinii with basipetal colonization of wound caused by secondary root emergence 5 5 List of Tables Table 1.1.Fungal colonization of roots of native legumes. 14 Table 1.2.Species of papilionoid legumes reported to sometimes occur in nature without mycorrhizae. 16 Table 1.3.Species of papilionoid legumes reported to have septate root endophytes. 19 Table 2.1.P-values from analysis of variance of selected response variables 32 Table 2.2. Nodule and shoot mass of L. latifolius 32 Table 2.3.Average shoot weight of Clover, experiment 2 33 Table 4.1.Average aboveground biomass of legume species 68 Table 4.2.P-values from analysis of variance for selected response variables. 68 Table 4.3.Medians by species of selected response variables69 Mycorrhizal and other Root Endophytic Fungi of Lupines in the Pacific Northwest Introduction Legumes are among the most widely cultivated and economically important crop plants.They are grown for food, fodder, timber and fuel wood, and as cover crops. A major value of many legumes is their ability to host symbiotic nitrogen- fixing bacteria and thereby contribute N to natural and agroecosystems. Lupinus (lupine) is a widespread legume genus in the Pacific Northwest.Franklin and Dyrness (1974) report species of Lupinus as components of more plant communities than any other legume genus.Although cropped commercially elsewhere (e.g. Australia) lupines are not used much in Pacific Northwest agriculture. There is interest in using lupines as N-fixingcover crops in forest plantations in the region and experimentsare underway to assess their value in this regard. Lupines tend to be colonized by vesicular-arbuscular mycorrhizal (VAM) fungi to a lesser degree than other legumes (Trinick, 1977; Bedmar & Ocampo, 1986). VAMare important for P uptake by many legumes, so the observation that lupinesmay be non-mycorrhizal led to discovery of the P-solubilizing capability of L. albus roots (Gardner & Boundy, 1983). Covercropsof 2 lupines can increase P as well as N availability to subsequent Pinus radiata plantations (Smethurst Turvey & Attiwill, 1986). The conclusion that lupines are nonmycorrhizal came from observations of species in cultivation out of their native range. Our investigations of fungi in lupine roots began with a survey of native species to learnif they were colonized by VAM fungi in non-agricultural soils (Chapter 1).Lupine roots were colonized by VAM-type or other fungi in about half of the populations sampled.The non-VAM colonization's were formed by septate fungi (VAM fungi are coenocytic) and resembled fungal colonization's of roots described in reports scattered through decades of mycorrhizal research (Peyronel, 1923; Thomas, 1943; Read & Haselwandter, 1980; Currah & Van Dyk, 1986). These apparently symptomless colonization's variously called "dark- septate" or "rhizoctonia-like" occur on a wide variety of plants, are often intracellular and can occur in roots with typical VAM. Because these phenomena have rarely been investigated, we isolated one of the fungi involved and conducted experiments on its effect on growth and nodulation of Lupinus latifolius (Chapter 2) and studied the structures it formed on L.latifolius and Pin us contorta roots in dual culture (Chapter 3).The final study described herein is a comparison of N-fixation, root morphology and mycorrhizal colonization of a lupine and two other legumes planted in clearcut forest sites. 3 Chapter 1.Root Endophytes of Lupine I: Colonization in the Field By T. E. O'Dell and J. M. Trappe Summary Roots of ten species of Lupinus and three other legumes collected from field soils were cleared, stained, and examined for fungal colonization. Three species were not observed to have fungal colonization. Nine species had colonization's with aseptate hyphae and vesicles attributable to vesicular-arbuscular mycorrhizal fungi. Seven species were sometimes colonized by fungi with septate hyphae, which often formed intracellular sclerotia, here called septate endophytes. The associations observed are compared with previous reports and discussed in relation to legume systematics.