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AN ABSTRACT OF THE THESIS OF
Sandra Gonzalez-Caldwell for the degree of Master of Science in
Horticulture presented on December 28. 1989.
Title: ROOTING OF PEAR HARDWOOD CUTTINGS USING VESICULAR-ARBUSCULAR
MYCQRRHIZAE. AGROBACTERIUM RHIZOGENES AND ROOTING HORMONES.
Abstract approved: _ . ^ Dr. Porter Lombard
Rooting experiments were conducted with hardwood cuttings of Old
Home x Farmingdale (OHXF) pear rootstock selections 217 and 282 to
determine the effects of rooting hormones, VA mycorrhizal (VAM) fungi
and the bacteria Agrobacterinm rhizopenes applied to the cuttings or
added to the rooting medium.
Mycorrhizal fungus inoculum was generated in pot cultures of pear
roots collected from pear trees at two locations. Pot cultures
contained spores of Glomus fasciculatum. G. microaggregatum. G.
intraradices. G. caledonium. G. mosseae. G. occultum. Acaulospora
trappei. and a Glomus species similar to G^. pallidum: the first three
species were most prevalent.
Inoculation with VA mycorrhizal (VAM) fungus inoculum from pear
roots did not increase, and sometimes reduced rooting of pear
cuttings. This negative influence appeared to be the effects of other
microbial components of the pot culture contents, since few
mycorrhizae formed on cutting roots.
Of three strains of Aprobacterium rhizogenes tested, strain TR105
improved rooting most, compared to untreated cuttings. Rooting
enhancement, however, was only sometimes as good as but never better
than that achieved with rooting hormones.
OHXF selection 282 always rooted better than selection 217, but
both rooted best with the addition of rooting hormones, regardless of
the rate or source. Rooting response was quantified by nine
parameters including root number, root length, percent of root
branching, survival, a rating of rooting based on a scale of 1 to 4,
root dry weight, root area, shoot number, and shoot dry weight.
Hormone treatments alone gave 71-96 % survival of cuttings compared to
8-21 % without hormones.
Some effects were detected between hormone treatments with A.
rhizogenes and/or VAM fungus inoculum, such as increased rooting of
cuttings with A^. rhizogenes when no hormone was applied, and a
decrease when VAM were used in combination with the powder.
These studies demonstrate that OHXF pear cuttings can be
successfully rooted with hormone treatments, but the microbes A.
rhizogenes and VAM fungi need further experimentation to demonstrate
any possible value. Adding inoculum after rooting from hormone
treatments could maybe enhance subsequent growth of roots and shoots.
ROOTING OF PEAR HARDWOOD CUTTINGS USING
VA MYCORRHIZAL FUNGI. AGROBACTERIUM
RHIZOGENES AND ROOTING HORMONES
by
Sandra Gonzalez-Caldwell
A THESIS
submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Master of Science
Completed December 28, 1989
Commencement June 1990
APPROVED:
i.f ■ ' -"tf^ ■—i, - t—
Professor of Horticulture in charge of major
T^T1 a*' Head of Denaxtment of Horticulture
Dean of Gc^ctiate School 'H^^V,
Date thesis is presented December 28.1989
Typed by Sandra Gonzalez-Caldwell
Acknowledgements
I wish to express thanks to my major professor, Dr. Porter
Lombard, for advice and help during this research project.
Deep gratitude goes to Dr. Robert Linderman, my academic co-advisor,
in whose laboratory and greenhouse facilities this research was
accomplished, for moral support, advice and guidance throughout, as
well as technicians, researchers and graduate students from the
Horticultural Crops Research Laboratory who aided me in so many
different ways, enriching my learning experience and making it easier
for me to work in new and unfamiliar areas.
I wish to also thank Dr. James Trappe for help with VAM fungus
identification, and Dr. Larry Moore and his lab for supplying the
Agrobacterium rhizogenes strains and training necessary to work with
these bacteria.
I am indebted to good friends in the Horticulture Department, Dr.
Robert Stebbins and his wife who, thanks to their kindness and help,
have made possible the completion of this degree. In all, my studies
in the United States have been a wonderful opportunity for growth,
both academic and personal aspects.
TABLE OF CONTENTS
I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
Preparation of the VAM inoculum 6
Identification of VA mycorrhizae from pear roots 7
Source and method of application of A. rhizogenes 8
Hormone formulations and concentrations 9
Plant material 9
Rooting media, containers and greenhouse conditions .... 10
Statistical design, treatments 10
Evaluation 11
a. Root production 11
b. VAM colonization 13
c. Retrieval of A. rhizogenes 13
d. Leaf and shoot growth 13
e. Survival 13
III. RESULTS AND DISCUSSION 15
Effect of rootstock cultivar 15
Detection of VAM in rooted cuttings 16
Effect of VAM on rooting 16
Retrieval of A. rhizogenes 17
Effect of A. rhizogenes on rooting 18
Effect of hormones on rooting 18
IV. BIBLIOGRAPHY 37
V. APPENDIX 41
LIST OF FIGURES
Figure No. Page
1. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root number of mist propagated hardwood cuttings of OHXF 217 19
2. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root number of mist propagated hardwood cuttings of OHXF 282 20
3. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root length of mist propagated hardwood cuttings of OHXF 217 21
4. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root length of mist propagated hardwood cuttings of OHXF 282 22
5. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 217 23
6. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 282 24
7. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 217 25
8. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 282 26
9. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 217 27
10. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 282 28
11. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 217 29
Figure No. Page
12. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 282 30
13. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 217 31
14. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 282 32
15. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 217 33
16. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 282 34
17. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 217 35
18. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 282 36
LIST OF APPENDIX TABLES
Table No. Page
1. Effect of VA mycorrhizae, Agrobacterium rhizoeenes. and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 217 41
2. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 282 42
3. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 217 43
4. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 282 44
5. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 217 45
6. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 282 46
ROOTING OF PEAR HARDWOOD CUTTINGS USING VESICULAR-ARBUSCULAR MYCORRHIZAE,
AGROBACTERIUM RHIZOGENES AND ROOTING HORMONES
I. INTRODUCTION
The use of clonal rootstocks, in contrast to seedlings, is of great
value in tree fruit production. Those recommended for each species and
cultivar have been selected for their characteristics such as disease
resistance, performance under unfavorable soil and climatic conditions,
scion compatibility and tree size-regulating capacity. Vegetative
propagation of these clonal materials leads to orchard uniformity, which
greatly contributes to cost-effective production. In cultivars where
layering is not economical, rooting of stem cuttings is the next best
alternative. Use of hardwood cuttings should be an efficient, relatively
simple and inexpensive method of clonal propagation, requiring minimal
investment and substantially less specialized labor compared to other
promising alternatives such as tissue culture, especially when dealing with
limited quantities which are frequently required with perennial tree crops.
Orchards can be economically productive for over 15 years.
Plant materials, even within the Rosaceae family (deciduous fruits),
differ widely in their propensity to root from stem cuttings. Frequently,
rootability is markedly influenced by the mother plant and weather
conditions (Hansen,1987), harvest date, dilution and nature of applied
hormone and rooting bed conditions (Howard, 1968; Hartmann and Kester,
2
1985). Hardwood cuttings of pear are difficult to root because of such
factors.
Commercial rooting of pear rootstock cuttings has involved the use of
lH-indole-3-butanoic acid (IBA) dips on hardwood (Westwood and Brooks,
1963) . Two methods used in their propagation trials of Old Home X
Farmingdale (OHXF) hardwood cuttings were: a) cuttings harvested in the fall
after chilling commenced, prior to full chilling using 100 to 200 ppm IBA in
a 24 h soak; and, b) dormant cuttings pruned in late January, after chilling
requirements were met, treated with 1000 to 2000 ppm IBA in 5 sec dips.
Rooting success in their trials ranged from 10 to .78 %, varying with the
size and condition of the plant material. To be acceptable in commercial
rooting of cuttings, they suggest a minimum of 50 % success. The OHXF
rootstock series has been pointed out as displaying a wide spectre of
propensity to root from stem cuttings, from 0 to 80 % (Westwood, pers.
comm.). There is need to develop treatments which could improve the
adventitious rooting of these difficult-to-root materials, and in the
process, possibly improve the understanding of physiological events
involved. With these purposes in mind, vesicular-arbuscular mycorrhizae
(VAM) and the bacterium Agrobacterium rhizopenes were selected for study.
Mycorrhizal fungi have been found to produce growth-regulating
substances and other organic compounds in the rhizosphere (Cooper,1985;
Ek,1983; Rouillon,1985; Madej,1985) and thus were studied as potential
rooting aids for many types of cuttings (Linderman and Call,1977; Barrows
and Roncadori,1977; Branzanti et al,1985; Cooper,1985; Powell and
Santanakrishnan,1986; Verkade,1986).
3
A. rhizopenes. formerly regarded as a pathogen, displays extraordinary
ability to transfer certain parts of its DNA to the host genome. This
transfer (T) DNA has been shown to confer auxin responsiveness to the
transformed plant tissues enabling root differentiation in the presence of
auxin, an event that did not occurr in untransformed material (Cardarelli et
al., 1987).
Vesicular-arbuscular mycorrhizal (VAM) fungi, so-called because of the
structures formed within root cortical tissues (arbuscules and vesicles) are
fungal symbionts in the roots of many plants. Vesicles are storage organs
formed on internal hyphae while arbuscules are complex haustoria-like
structures involved in nutrient exchange within the plant cell. VA
mycorrhizae differ from ectomycorrhizae in that the fungal symbionts are
obligate, penetrate plant cells, and do not significantly alter root
morphology. They achieve long term survival in soil by producing thick-
walled chlamydospores.
Aprobacterium rhizogenes. first studied as the causal agent of 'hairy
root' disease (Suit, 1933) in dicots (DeCleene,1981), is a bacterium which
causes great proliferation of roots in wound sites, by means of a plasmid-
mediated genetic transformation (Moore, 1979). Aside from the impressive
potential that the Ri plasmid and its peculiarities represent for molecular
biology (Birot, 1987; Klee, 1987; Zambryski, 1989), the capability of
eliciting prolific rooting in host plants could prove to be a useful tool in
plant propagation. This has been attempted with success (Diana,1986;
Strobel,1985,1987; Bassil and Proebsting, pers. communic), thus harnessing
the very quality (Ark, 1961) that caused it to be considered pathogenic.
Tepfer's (1984) finding that A^ rhizogenes- transformed plants displayed
4
shortened internodes (dwarfing) merits verification for fruit trees;
rootstocks displaying such traits are rare and desirable.
A number of works confirm the dynamic interactions occurring between
rhizosphere microorganisms and mycorrhizae (Linderman, 1988; and Linderman
and Paulitz, a review in press). In many cases, rhizobacteria and
mycorrhizae were shown to exert a mutually positive influence on each other,
either directly by increasing availability of certain nutrients or growth
substances (Barea,1975; Raj,1981; Leyval,1988) or indirectly by suppressing
potentially deleterious organisms (Cook and Baker,1983). Whenever such
situations arise, it is valid to assume that the host plant would also
benefit, whether from the absence or decreased populations of certain
pathogens and/or the added effect/s that mycorrhizae and associated bacteria
may exert, either directly or through mutual complementation.
In the case of a stem cutting, the potential for adding beneficial
microorganisms is even more significant, since in the absence of roots it is
severely stressed. Cutting survival depends on rapid morphological changes
in response to small variations in many factors, the most important being
phytohormones (Hartmann and Kester, 1985). Rhizospheric microorganisms
which could consistently stabilize host response (and interact
mutualistically) leading to enhancement of adventitious rooting processes
could be extremely valuable, not only for improving the percentage and
quality of rooting but also for safeguarding against potential stress
problems, many of which cannot be detected until too late (e.g. pathogenic
infection of plant tissue, drought, nutrient stress, etc.). Caesar and Burr
(1987) showed that various bacterial strains promoted growth of clonal
rootstocks of apple. Many species of higher plants have been shown to form
metabolites called phytoalexins when exposed to fungi or mechanical wounding
(Keen,1981). Certain phytoalexins present in concentrations below their
antibiotic activity have been shown to act synergistically with lH-indole-3-
acetic acid (IAA) to promote adventitious rooting (rooting cofactor
activity) in mung bean bioassays, (Yoshikawa et al.,1986). This fact might
partially account for the positive effects of certain bacteria and fungi on
plant rooting processes.
The purpose of this study was to answer the following questions:
a. Is rooting of Old Home x Farmingdale pear hardwood cuttings influenced by
VAM?
b. Does Aprobacterium rhizopenes affect rooting?
c. How do different concentrations and formulations of plant growth
regulators compare to the above treatments?
d. Do any of the above factors interact in any way in adventitious rooting?
II. MATERIALS AND METHODS
An experiment to study the rooting of hardwood cuttings of the pear
rootstock series Old Home x Farmingdale (Westwood, 1982), developed in
Oregon and unique for its resistance to the diseases pear decline and fire
blight, was begun in the greenhouse in November 1988. The treatments
included VAM fungi collected from pear orchards, three strains of
Aprobacterium rhizopenes and three phytohormone treatments.
Preparation of the VAM inoculum:
Pear roots were removed from trees in several orchards during fall and
winter 1987/1988, washed in water, cleared and stained for detection of VAM
colonization (Phillips and Hayman, 1970). Colonized roots were surface-
sterilized using 0.5 % sodium hypochlorite and cut into 1 cm pieces to be
used as inoculum to multiply the symbionts in plant pot cultures. The
latter were started in the following way: half-gallon pots filled with
pasteurized (air-steamed 1 h at 60 C) sand in which three cavities, 2.5 cm
wide x 10 cm deep, were made. These cavities were 75 % filled with VAM root
inoculum, topped with sand. One seed of subterranean clover (Trifolium
repens L.), previously surface-sterilized 10 min. with .5 % sodium
hypochlorite solution, was placed on each inoculum core. Cultures were
maintained under greenhouse conditions for 3.5 months and watered and
fertilized weekly with Long Ashton Plant Nutrient Solution (P content
modified to 11 ppm). The pots were allowed to dry and were then harvested.
Aerial plant parts were discarded, the roots were cleared and stained to
determine the degree of VAM colonization. The entire volume of sand and
roots divided into 1 cm pieces was thoroughly mixed and stored under dry,
7
cold conditions (4 C) for later use. VAM - free medium for controls was
made by washing sand from pot cultures with water, sieving the resulting
slurry through a 38 micron metal sieve and blending the sieved liquid with
pasteurized sand.
Identification of VA mvcorrhizae from pear roots:
The pot cultures of VAM that served as inoculum for the experiment were
made from surface-sterilized pear roots gathered from orchards in Medford
and in different locations at the National Germplasm Repository [USDA-ARS],
Corvallis, OR. Dr. James Trappe, from the Department of Forest Science,
Oregon State University, kindly indentified the VAM in those cultures based
on examination of spores contained therein. The findings were as follows:
Sample(Location) VAM species
1. (Medford) Glomus fasciculatum (Thaxter) Gerd. &
Trappe emend. Walker & Koske.
2. ( " ) a.G. microapprepatum Koske, Gemma & Alexis.
b.G. ca'ledonium (Nicol. & Gerd.) Trappe &
Gerd.
3. ( " ) G. fasciculatum.
4. ( " ) G. fasiculatum ? (if so, spores very
young).
G. fasciculatum.
None.
G. intraradices Smith & Schenck.
G. fasciculatum.
5. ( tl )
6. ( 11 )
7. ( 11 )
8. ( II )
Sample(Location)
9.
10.
11.
12.
13.
18.
19.
20.
21.
22.
23.
24.
" )
" )
Corvallis)
14. ( ' It )
15. ( It )
16. It )
17. 11 )
VAM species
G. microagpregtatum.
Too few spores to identify.
Spores found mainly in roots, probably G.
intraradices.
Acaulospora trappei Ames & Linderman.
a.A. trappei.
b.Glomus sp. (too few spores to identify)
G. cf pallidum Hall or poss. undescribed.
G. mosseae. plus possibly one or two other
immature or otherwise unidentifiable spp.
a.G. fasciculatum.
b.G. mosseae.
G. occultum Walker.
G. fasciculatum.
G. intraradices.
G. fasciculatum.
Source and method of application of A. rhizopenes:
Strains A 4, A 4783 and TR 105 were provided by Dr. Larry Moore,
Department of Botany and Plant Pathology, Oregon State University. Strain A
4 was originally isolated by Dr. P. Ark (Univ. of California, Berkeley) from
9
roses, A 4783 was discovered by M. Canfield (Oregon State Univ.) from
carrot, and TR 105 was of unknown origin. Petri dishes with mannitol
glutamate medium (Keane et al., 1970) were inoculated with the bacteria and
incubated for 72 h at 25 C. A cell suspension of each strain was made by
pouring sterile water on these dishes and rubbing with a glass rod. The
resulting cell suspension was poured into tubes and vortexed until
homogenized. Sterile water was added to obtain the desired reading of 70 on
a Klett colorimeter (approx. 3 x 10 colony forming units/mL).
Approximately 30 plates were used to prepare 2.25 L of bacterial suspension
for each strain. The suspensions for inoculation were prepared on the
daythey were used; 5 mL of each was pipetted into the center of each pot
shortly before cuttings were 'stuck'.
Hormone formulations and concentrations:
Indole butyric acid (IBA), dissolved in 50% ethanol, was used at 5000
and 2500 ppm (Westwood and Brooks, 1963) as was a dry hormone preparation,
referred to as Hormodust, containing 1000 ppm each of IBA and NAA in talc.
Cuttings were dipped to a depth of 0.5 cm for 5 sec in the solutions or
powder, (Howard and Nahlawi, 1970), excess liquid or powder was flicked off
immediately, and the cutting was then stuck.
Plant material:
Cuttings of the pear rootstock series Old Home x Farmingdale (OHXF),
genotype selections 217 and 282, provided by Patchwork Nursery, Forest
Grove, Oregon, were harvested on October 22, 1988 and hand defoliated as
necessary (Westwood and Brooks, 1963). The topmost and lowest 10 cms from
each cutting were removed. Stems with calipers less than 0.7 cm and those
greater than 1.0 cm were discarded (Marini, 1983). The bottom cut was made
10
perpendicular to the stem, 0.5 cm below the first leaf node, resulting in a
final length of 15 cm. All cuts were made just before sticking.
Rooting media, containers and greenhouse conditions:
The rooting medium consisted of 4 parts perlite, 3 parts Douglas fir
sawdust and 1 part steam sterilized Hypnum peat (Biermann and
Linderman,1983) blended in a twin-shell blender and adjusted to pH 7 with
CaC03 (Davis et al.,1983). Black plastic 'band' pots measuring 6 x 6 x 13
cm were placed in plastic flats on a greenhouse bench equipped with bottom
heat cables (24 C) and intermittent mist. The bench was covered entirely
with a plastic tent rising 120 cm above the cuttings. Air temperature
during the first three months was maintained at 10 C, but thereafter the
temperature was allowed to rise during daylight hours.
Bands were filled with rooting media, leaving a central cavity of 7 cm
deep x 2 cm wide, where 18 mL of VAM inoculum from the pear root sources or
non-mycorrhizal medium were added. Cuttings were stuck so that their lower
ends were in the inoculum.
Statistical design, treatments:
The design was a factorial in a randomized block for each pear genotype
with 32 treatments of 24 cuttings per treatment, totalling 1536 units. All
cuttings were placed on the same greenhouse bench, randomly arranged in 4
replicates of six cuttings each. The treatments were applied to each
rootstock cultivar, as follows:
11
Treatment VAM HORMONE A. rhizopenes
1. 2. - - A 4 3. - - A 4783 4. - - TR 105 5. - IBA 2500 - 6. - IBA 2500 A 4 7. - IBA 2500 A 4783 8.. - IBA 2500 TR 105 9. - IBA 5000 - 10 - IBA 5000 A 4 11. - IBA 5000 A 4783 12. - IBA 5000 TR 105 13. - 'HORMDST' - 14. - 'HORMDST' A 4 15. - 'HORMDST' A 4783 16. - •HORMDST' TR 105 17. + - - 18. + - A 4 19. + - A 4783 20. + - TR 105 21. + IBA 2500 - 22. + IBA 2500 A 4 23. + IBA 2500 A 4783 24. + IBA 2500 TR 105 25. + IBA 5000 - 26. + IBA 5000 A 4 27. + IBA 5000 A 4783 28. + IBA 5000 TR 105 29. + 'HORMDST' - 30. + 'HORMDST' A4 31. + 'HORMDST' A 4783 32. + 'HORMDST' TR 105
Evaluation:
The following observations were made:
a. Root production - Root number, average root length, root area, rooting
rating and presence of lateral roots were quantified. Each cutting was
extracted individually from the rooting medium and carefully washed in cold
water. The roots were severed from the cutting and placed on a sheet of
clear celulloid under a white background and photocopied (Collins et al.,
1987). All separate roots were placed in the same direction. This provided
12
a permanent record of the configuration, number, average length and lateral
branching of roots. The root images, as photocopies, were later analyzed
with a Delta-T brand area meter (Harris and Campbell, 1989) and the
measurement obtained is hence referred to as root area. The area meter was
calibrated to read with precision to 0.1 cm on a cm2 scale.
An artificial scale to rate rooting response, ranging from 1 to 4 was
used as follows:
1= base of cutting unchanged, no rooting.
2= presence of callus only, no rooting.
3= rooted, root lengths less than 5 cm
4= rooted, root lengths over 5 cm
Lateral branching of roots was estimated as a means of detecting'hairy
root'-like proliferation, a feasible occurrence in roots treated with A.
rhizoeenes. It involved a subjective evaluation of the profusion,
ramification and size of lateral roots emerging from roots originating at
the cutting base, expressed in percentage (100 % corresponding to the
maximum and 0% to the minimum). In order to standarize readings the root
photocopies were used instead of the actual roots, thereby confining the
observations to one physical plane.
Total fresh weight of roots from each cutting was then measured.
However, representative samples for clearing and staining for VAM
colonization were removed after that step, and the remainder oven dried at
70 C for 72 hours. Sample fresh/dry weight ratios were used to calculate
total dry weight. This conversion was used to add back the weight of the
roots removed for VAM colonization analyses.
13
b. VAM colonization - VAM colonization was evaluated microscopically after
clearing and staining with Trypan blue in lactoglycerin (Phillips and
Hayman,1970). The percent root length with VAM was determined by the method
of Biermann and Linderman (1981).
c. Retrieval of A. rhizogenes - Various techniques were used to retrieve A,
rhizopenes from roots and/or rooting medium rhizosphere samplings. Another
approach used was with water-washed root macerates, surface-sterilized
(using 0.5 % sodium hypochlorite solution) or not, prepared either with
mortar and pestle or by applying the hand-held 'tissue disrupter' (Agdia
Corp.; Elkhart, Indiana 46154) over roots placed for that purpose in plastic
self-locking bags into which 10 cc of sterile water was initially placed.
The supernatants of each of the resulting samples, both rooting media or
root macerations, were made into acqueous supensions diluted to 10-1, 10-2
and 10-3, and plated on Petri dishes containing two different agar selective
media (Brisbane and Kerr, 1983), one containing malachite green (selective
for Biovar II), the other gentian violet (Biovar I specific).
d. Leaf and shoot growth:
Numbers of shoots per cutting were recorded, and the total lengths
recorded for each cutting. Freshly severed leaves and shoots laid flat
under a clear glass sheet were measured with the Delta-T area meter,
calibrated to read with precision of 0.1 cm on a cm2 scale. Shoot and leaf
fresh weight was recorded, then dried for 72 h at 70 C for dry weight
determination.
e. Survival:
Survival was determined by counting, within each replicate, those
cuttings that have achieved sufficient root development to ensure survival.
14
This rating was subjective in that it did not consider cuttings which had
only callused or had very sparse rooting; it aimed to evaluate survival
capacity if transplanted to a larger container or into a field situation
15
III. RESULTS AND DISCUSSION
All the above parameters were evaluated, but shoot length was
disregarded because variation was too great for it to be considered a
reliable indicator of treatment effects. The values for shoot area
indicated a positive trend in treatments which included rooting compounds,
but the variability within treatments precluded them being statistically
significant.
A parameter resulting from the artifact of multiplying average root
length x root number for each cutting was tested as an additional estimation
of root development, but was later disregarded because it showed the same
patterns as root length measurement.
Effect of rootstock cultivar
Cuttings of OHXF 282 had higher survival and rooting ratings than OHXF
217, after 3.5 months in the rooting chamber (Figures 7, 8; and 9, 10
respectively, and Appendix Tables 3 and 4). The parameter "survival" refers
to the 'take' of each cutting, while rooting rating, though related, is more
descriptive of morphological changes which occurred (or not) at the base,
(e.g. callusing; formation of short, stubby roots; or the long, healthy ones
essential for good quality nursery stock). The survival index means for
OHXF 217 and 282 were .62 and .71, respectively, giving a clear indication
that the 'take' of OHXF 282 is inherently the better of the two. The rooting
rating means also reflect that tendency, at 2.99 and 3.27 for 217 and 282,
respectively.
Root number averages for each rootstock were 3.86 and 5.82; root length,
5.66 and 6.27; root branching, 13.06 and 18.79, the higher of each pair of
16
values invariably corresponding to OHXF 282. These data confirm what the
nurseryman who provided the cutting material communicated, regarding the
better rooting qualities of OHXF 282 over 217. Westwood and Brookes (pers.
communic.) also indicated a wide range of adventitious rooting ability
within this line of rootstocks.
Detection of VAM in rooted cuttings:
Roots of OHXF 217 cuttings yielded no detectable VAM colonizations,
while those of OHXF 282 showed root length colonizations ranging between 5
and 10 per cent in four root samples, corresponding to treatments 19, 20, 21
and 28. It is assumed that the levels of colonization would have been
higher should the cuttings have been evaluated at a later date, but such
practices are not economically feasible for commercial nurseries. The low
rates of colonization found made it impossible to identify the species of
VAM involved.
In this study, inoculation with VAM fungi had no effect in most cases.
Since the inoculum was generated in pot culture from colonized pear roots,
we assume the fungi would be compatible with roots of pear cuttings.
However, since practically no mycorrhizae were detected in cutting roots,
other factors could have precluded their formation. Possible explantions
for lack of colonization could be factors affecting inoculum potential
including medium pH, spore dormancy, physical placement in relation to
roots, delayed formation of roots, etc.
Effect of VAM on rootinp:
Controls without VAM fungus inoculation actually rooted better than
those containing VAM inoculum for the parameters survival and rooting rating
(Figures 7, 8, and 9, 10, respectively; Appendix Tables 3 and 4). Values
17
for root number followed the same trend, with overall means for VAM
treatments being 4.45 compared to 5.26 for controls.
VAM fungus treatments which included the powdered hormone formulation
showed lower responses for rooting rating, shoot dry weight, root length,
root number and root branching at the P-0.001 level of significance. For
instance, means for root number were 6.33 for no VAM plus powder formulation
versus 3.75 for VAM with powder (Figures 1 and 2). In contrast, results for
IBA at 5000 ppm were 7.26 and 6.66, respectively, without and with VAM
inoculum.
Rooting rating results reflect the same tendency, as shown by the means:
3.41 (no VAM inoculum plus powder) versus 2.88 (VAM inoculum plus powder).
There was little difference between the rooting rating for the other
hormones with or without VAM fungus inoculum. An explanation could be that
the VAM inoculum exerted some negative effect on the hormone contained in
the powder formulation, either through a degrading or immobilizing action.
The fact that this is the only formulation studied which contains NAA
invites speculation as to the possible effect on the rhizosphere flora or
the microbes contained in the VAM pot culture inoculum.
A second relationship was found between VAM inoculum and Agrobacterium
which indicated that rooting rating was decreased with VAM added to the
rooting medium in the absence of bacteria or with strain TR 105 (Figures 9
and 10; Appendix Tables 3 and 4). It could be speculated that, since no
other root parameters are affected, the factor being influenced is the
formation of callus, which is only evaluated by rooting rating.
Retrieval of A. rhizogenes:
18
All attempts to recover and identify Aprobacterium strains failed due to
the extensive proliferation of great numbers of bacterial colonies after 72
h incubation at 26 C, most of which did not have Agrobacterium
characteristics. Controls responded in the same way as did treatments. A
strain marked for antibiotic resistance might be considered for future
studies.
Effect of A. rhizopenes on rooting:
The inclusion of any strain of A^. rhizopenes in the rooting medium
increased survival, rooting rating and root dry weight, (Figures 7, 8, 9,
10, 11 and 12; Appendix Tables 3 and 4). The most effective strains were A
4783 and TR 105. The survival rate of cuttings with any bacterial inoculum
ranged from 65 to 77 per cent; average survival on cuttings without
bacterial inoculum was 58 %. The positive effect of the bacterial
suspension could be due to enhanced initiation of adventitious roots due to
transformation of cells within the cutting. Confirmation of transformation
by opine analysis was not done, however.
An relationship between Agrobacterium and rooting compounds was found
for the parameters root number, root length, root branching, rooting rating,
shoot number and shoot dry weight. When Agrobacterium was applied to the
rooting medium without hormones, there was a slight increase in root
initiation and development.
Effect of hormones on rooting:
Use of IBA at 2500 or 5000 ppm or the dry hormone formulation increased
the rooting of both cultivars of pear cuttings regardless of the parameters
measured (P=0.001). In contrast, none of the biological agents were as
effective.
OLD HOME X FARMINGDALE 217: ROOT NUMBER 19
A)
10.00
9.00
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10.00
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7.00
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OLD HOME X FARMINGDALE 217: ROOT LENGTH 21
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VAM
Fiq. 3. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on the root length of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: ROOT LENGTH ??
A) 10.00
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9.00
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Fig. 4. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on the root length of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 217: ROOT BRANCHING
A)
^ 30.00
2? 25.00
E 20.00 TO
♦J 15.00 o o SH
x 10.00
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25.00
20.00
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Fig. 5. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: ROOT BRANCHING 24
A)
30.00 -
00 25.00
c CTJ t-l
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30.00
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15.00
10.00
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30.00
25.00
20.00
15.00
10.00
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Fig. 6. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 217: SURVIVAL
A)
x
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Fig. 7. Effect of VA mycorrhizae, Aarobacterium rhizogenes and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
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27 OLD HOME X FARMINGDALE 217; ROOTING RATING
A) 4.00
oo c
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. 1.00 > <
0.00
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NO HORM IBA 2500 1BA 5000 POWDER
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3.00
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I NO BACT [IZ1A4 ^A4783
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I / / / /
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4.00
- 3.00
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NO HORM IBA 2500 IBA 5000 POWDER
VAM
0.00
Fig. 9. Effect of VA mycorrhizae, Aqrobacterium rhizogenes and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: ROOTING RATING 28
A)
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TR105 *9
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be "I /
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4.00
3.00
2.00
1.00
0.00
4.00-
3.00
2.00
1.00
0.00 NO HORM IBA 2500 IBA 5000 POWDER
VAM
Fig. 10. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
29
A)
B)
> <
OLD HOME X FARMINGDALE 217: ROOT DRY WEIGHT
0.25
0.20
00
« 0.15
o o
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0.10
0.05
0.00
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M 0.20
M -" 0.15 a) 3
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ITR105 v
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(7)
D
f-i f-i [71 f-;
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NO H0RM IBA 2500 IBA 5000 POWDER
NO VAM
ESS NO BACT HDAA EZZA4-7S5
ITR105
;9j
/
hJ 9J
/ / / /
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NO HORM IBA 2500 IBA 5000 POWDER
VAM
0.05
0.00
0.25
0.20
0.15
0.10
0.05
0.00
Fig. 11. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: ROOT DRY WEIGHT
A) 0.25
S 0.20
« 0.15
•a
o o SH
0) > <
B)
0.10
0.05 -
0.00
I NO BACT □ A4 ^A4783
ITR105 su ^ J
/
/ / / / /
NO HORM IBA 2500 IBA 5000 POWDER
NO VAM
NO HORM IBA 2500 IBA 5000 POWDER
VAM
0.25
0.20
0.15
0.10
0.05
0.00
0.10
- 0.05
0.00
Fig. 12. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
31 OLD HOME X FARMINGDALE 217: ROOT AREA
A)
B)
NO HORM IBA 2500 IBA 5000 POWDER
NO VAM
£
> <
NO HORM IBA 2500 IBA 5000 POWDER
VAM
Fig. 13. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: ROOT AREA
A)
E o
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NO VAM
ESaNO BACT .CZIA4 EaA4783
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VAM
25.00
20.00
15.00
10.00
0.00
25.00
20.00
15.00
10.00
5.00
0.00
Fig. 14. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 217: SHOOT NUMBER
A) 1.80
1.60.
■i->
z. 1.20 o
^ 1-00
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B)
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0.00
ESS NO BACT IZDA4 ^A4783
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NO HORM IBA 2500 IBA 5000 POWDER
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NO HORM IBA 2500 IBA 5000 POWDER
VAM
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Fig. 15. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
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NO NORM IBA 2500 IBA 5000 POWDER
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B)
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0.80
0.60
0.40
0.20
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Fig. 17. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
OLD HOME X FARMINGDALE 282: SHOOT DRY WEIGHT
A) 1.20
1.00
DO
w 0.80
£ 0.60
| 0.40 in
% 0.20
0.00
1.20
B) 1.00 t—*
4J 0.80 3
■o 0.60
o i 0.40
0) >0.20
0.00
E^ NO BACT
^A4783 BETR105
I
hi hi
NO H0RM IBA 2500 IBA 5000 POWDER
NO VAM •
ES NO BACT IZHA4 ^A4783 OMTRIOS
ad
i
1 /d9
NO HORM IBA 2500 IBA 5000 POWDER
VAM
1.20
1.00
0.80
0.60
0.40
0.20
0.00
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Fig. 18. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.
37
IV. BIBLIOGRAPHY
Ark, P.A. and Thompson,J.P. (1961). Detection of hairy root pathogen, Agrobacterium rhizogenes. by the use of fleshy roots. Phytopathology, 51:69-71.
Barea.J.M.; Azcon.R. and Hayman.D. (1975). Possible synergistic interactions between Endogone and phosphate-solubilizing bacteria in low-phosphate soil. In Sanders,F.E.; Mosse.B. and Tinker,P.B.(eds), Endomycorrhizas. Academic Press, London, pp.409- 417.
Barrows,J.B. and Roncadori.R.W. (1977). Endomycorrhizal synthesis by Gipaspora margarita in poinsettia. Hycologia 69 (6): 1173- 1184.
Biermann.B. and Linderman,R.G. (1983). Effect of container plant growth medium and fertilizer phosphorus on establishment and host growth response to vesicular-arbuscular mycorrhizae. J. Am. Soc. Hortic. Sci. 108 (6): 962-971.
Biermmann.B. and Linderman, R.G. (1981). Quantifying vesicular- arbuscular mycorrhizae: a proposed method towards standarization. New Phytol. 87, 63-67.
Birot.A.M.; Bouchez.D.; Casse-Delbart.F.; Durand-Tardif,M.; Jouanin.L.; Patout.V.; Robaglia.C.; Tepfer, D.; Tepfer.M.; Toumeur.J. and Vilaine.F. (1987). Studies and uses of the Ri plasmids of Agrobacterium rhizogenes. Plant Physiol. Biochem. 25: 323-335.
Branzanti.B.; Cristoferi.G.; Zocca.A. and Zambonelli.A. (1985). Ectomycorrhizal fungi and IBA effects on fruit rootstock rooting. In Proc. VI N. Am. Conf. on Mycorrhizae, p. 324. June 25-29, 1984, Bend, OR. 471pp. Brisbane,P.G. and Kerr, A. (1983). Selective media for three biovars of Agrobacterium. J. Appl. Bacteriol. 54:425-431.
Caesar,A.J. and Burr.T.J. (1987). Growth promotion of apple seedlings and rootstocks by specific strains of bacteria. Phytopathology 77: 1583-1588.
Collins,R.P.; Gregory,P.J.; Rowse.H.R.; Morgan,A. and Lancashire,B. (1987). Improved methods of estimating root length using a photocopier, a light box and a bar code reader. Plant Soil 103, 227-280.
Cook.R.J. and Baker, K.F. (1983). The nature and practice of biological control of plant pathogens. The American Phytopathological Soc. Press, St. Paul, MN. 539 pp.
38
Cook.R.J. and Baker, K.F. (1983). The nature and practice of biological control of plant pathogens. The American Phytopathological Soc. Press, St. Paul, MN. 539 pp.
Cooper,K.M. (1985). Physiology of VAM associations. IV. Hormonal effects. In "VA Mycorrhizae', Eds. Powell,C.L. and Bagyaraj,D.J. CRC Press, pp. 173-186.
Davis,E.A.; Young, J.L. and Linderman.R.G. (1983). Soil lime level (pH) and VA-mycorrhiza effects on growth responses of sweetgum seedlings. Soil Sci. Soc. Am. J. 47:251-256.
De Cleene.M. and De Ley.J. (1981). The host range of infectious hairy-root. The Botanical Review 47: 147-194.
Diana,G. (1986). [The transmission of Aprobacterium rhizogenes plasmids in propagation of olive cuttings.] Trasmissione di plasmidi di Aprobacterium rhizogenes nella propagazione di talee di olivo. Annali dell'Istituto Sperimentale per 1'Olivicultura (1981/1983) 7, 1-11.
Ek,M.; Ljunquist.P.O. and Stenstrom.E. (1983). Indole-3-acetic production by mycorrhizal fungi determined by gas chromatography- mass spectrometry. New Phytol. 94: 401-404.
Hansen.J. (1987). Stock plant lighting and adventitious root formation. HortScience 22 (5): 746-748.
Harris,G.A. and Campbell,G.S.(1989). Automated quantification of roots using a simple image analyzer. Agronomy Journal (Nov.1989, in print).
Hartmann.H.T. and Kester.D.E. (1985). Plant propagation: Principles and practices. Prentice-Hall Inc., Englewood Cliffs, New Jersey. 622 pp.
Howard,B.H. (1968). The influence of IBA and basal temperature on rooting of apple rootstock hardwood cuttings. J. hort. Sci. 43, 23-31.
Howard,B.H. and Nahlawi.N. (1970). Dipping depth as a factor in the treatment of hardwood cuttings with IBA. E. Mailing Res. Stn. R. 1969.
Keane.P.J.; Kerr.A. and New, P.B. (1970). Crown gall of stone fruit II. Identification and nomenclature of Agrobacterium isolates. Aust. J. biol. Sci. 23:585-95.
Keen, N.T. (1981). Evaluation of the role of phytoalexins. In Plant Disease Control, Staples, R.C. ed., J. Wiley & Sons, New York, pp. 155-177.
39
Klee.H.; Horsch.R. and Rogers,S. (1987). Aprobacterium-mediated plant transformation and its further implications to plant biology. Ann. Rev. Plant Physiol. 38: 467-486.
Leyval.C. and Berthelin.J. (1988). Interactions between ectomycorrhizal fungi and phosphate-solubilizing bacteria: Phosphorus mobilization from different inorganic phosphates. In: Proc. of the 7th Intntl. Symp. on Environmental Biogeochemistry. Viterbo, Italy, Sept. 1985 (in press).
Linderman.R.G. (1986). Mycorrhizal interactions with the rhizosphere microflora: the mycorrhizosphere effect. Phytopathology 78 (3), 366-371.
Linderman.R.G. and Call.C. (1977). Enhanced rooting of woody cuttings by mycorrhizal fungi. J. Am. Soc. Hortic. Sci.102 (5): 629-632.
Linderman.R.G. and Paulitz.T.C. (1989). Mycorrhizal- rhizobacterial interactions. In: Biological control of Soil-borne Plant Pathogens. Hornby,D.; Cook.R.J.; Henis.Y.; Ko.W.H.; Rovira.A.D.; Schippers.B. and Scott,P.R. (eds.). CAB International (in press).
Madej.A. and Haggblom.P. (1985). Radioinmunoasssay for determination of indole-3-acetic acid in fungi and plants. Physiol. Plantarum 64: 389-392.
Marini.R.P. (1983). Rooting of hardwood cuttings as affected by shoot position and thickness. HortScience 18 (5): 718-719.
Moore.L.; Aichele.M.D.; Millikan.D.F. and Johnson,H.G. (1980). Reevaluating hairy root disease. Amer. Nurseryman, Jan.: 10-11 and 116-117.
Moore,L.; Warren,G. and Strobel.G. (1979). Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2, 617-626.
Nelson,S.D. and Clough.K.S. (1989). Rooting, growth and infection of Cotoneaster and Cornus cuttings by three Glomus species. In Mycorrhizae in the next decade; eds. Sylvia,D.M., Hung.L.L. and Graham,J.H. U. of Florida. P. 297.
Phillips,J.M. and Hayman.D.S. (1970). Improved procedure for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans, of the Brit. Mycol. Soc. 55, 158-161.
Powell,C.L. and Santhanakrishnan.P. (1985). Effect of mycorrhizal inoculation and phosphorus fertilizer on the growth of hardwood
40
cuttings of kiwifruit (Actinidia deliciosa cv. Hayward) in containers. New Zealand J. of Agric. Research, 29:263-268.
Raj, J.; Bagyaraj,D.J. and Manjunath.A. (1981). Influence of soil inoculation withn vesicular-arbuscular mycorrhizae and a phosphate-dissolving bacterium on plant growth and P32-uptake. Soil Biol. and Biochem., 13, 105-108.
Rouillon.R.; Gay.G.; Bernillon,J.; Favre-Bonin,J. and Bruchet.G. (1985). Analysis by HPLC - mass spectrometry of the indole compounds released by the ectomycorrhizal fungus Hebeloma hiemale in pure culture. Can. J. Bot. 64: 1893-1897.
Strobel,G.A. and Nachmias.A. (1985). Aprobacterium rhizopenes promotes the initial growth of bare rootstock almond. J. of Gen. Microbiol. 131, 1245-1249.
Strobel.G.A.; Nachmias.A. and Hess.W.M. (1987). Improvements in the growth and yield of olive trees by transformation with the Ri plasmid of Aprobacterium rhizopenes. Can. J. Bot. 66: 2581-2585.
Suit, F.R. (1933). Pseudomonas rhizopenes (R.B.W.K. and S.); its host relations and characteristics. Iowa State Coll. J. Sci. 8: 131-173.
Tepfer.D. (1984). Transformation of several species of higher plants by Aprobacterium rhizopenes: sexual transmission of the transformed genotype and phenotype. Cell 37, 959-967.
Verkade,S.D. (1987). Effect of Glomus fasciculatum on rooting of Viburnum dentatum during propagation. In Mycorrhizae in the next decade; eds. Sylvia,D.M., Hung.L.L. and Graham,J.. U. of Florida, p. 298.
Westwood.M.N. (1982). Rootstocks for pear. Proc. of Oregon Hort. Soc. 73, 64-79.
Westwood.M.N. and Brooks, R.M. (1963). Propagation of hardwood pear cuttings. Proc. Intnl. PI. Prop. Soc. 13, 261-268.
Yoshikawa.M.; Gemma,H.; Sobajima.Y. andMasago.H. (1986). Rooting cofactor activity of plant phytoalexins. Plant Physiol. 82, 864-866.
Zambryski,P.; Tempe.J. and Schell.J. (1989). Transfer and function of T-DNA genes from Aprobacterium Ti and Ri plasmids in plants. Cell 56: 193-201.
A)
41
TREATMENTS PARAMETER
ROOTING AGROB. COMPOUND PHIZ.
CONTROL CONTROL ■■ A4 " A47B3 " TRIOS
IEA 2500 CONTROL .. A4 " A47S3
TR105 ISA 5000 CONTROL
" A4 " A47e3
TR 1 05 POWDER CONTROL
.. A-;
.. A47S3 " TR105
ROOT NUMBER
(.) 25 0 S5 0 46 i 29 0 58 1 38 rt 78 4 94 0 25 0 85 0 4£, 1 29 0 58 1 38 2 71 4 94 7 21 7 25 ^ 21 6 89 3 17 4 48 7 04 6 06 4 50 3 71 4 17 5 08 i, 13 4 42 3 21 o 20
ROOT LENGTH
MEAN ST D
8 00 0 00 S 00 1 00
11 00 4 36 6 18 1 60 o 68 2 45 8 38 2 09 o 50 3 26
10 77 2 84 10 06 2 69 9 47 1 69 o 92 1 S3
10 68 3 42 10 21 2 32 12 79 4 95 11 79 2 2B 16 67 17 IS
ROOT BRANCH.
20. 00 6. 67
10. 00
1 4 . 38 19. 44 14.71 22.78 24. 00 19. 17 28.42 17.37 40.71 32. 11 16.67
12 19-.
15
0/ 47
IS
B)
TREATMENTS PARAMETER
ROOTING AGROB. COMPOUND RHIZ.
CONTPCL
IBA 2500
POWDER
CONTROL A4
A47S3 TR 105
CONTROL A4
A47S3 TR105
CONTROL A 4
A47S3 TR 1 05
CONTROL A4
A4783 . TR105
ROOT NUMBER
ME :AN ST D
0 00 0 00 1
— — o ■?-?
0 88 1 4S 13
4 25 4 48 4 03 4 95 5 17 4 67 5 96 4 S5 5 83 6 79 3 67 4 25 6 75 6 6° ■7 46 5 80 2 00 2 59 1 79 2 30 2 83 3 98 I 42 2 17
ROOT LENGTH
MEAN ST D
0 00 0 00 7 50 1 90 7 50 2 00 8 14 0 69 10 "70 3 89 9 87 3 02
10 50 3 50 10 19 2 66 10 67 1 99 o 43 2 98 10 12 1 41 11 53 2 e>5 14 27 3 ,"*'.'? 11. 08 3 55 o 60 2 46 o^ •?2 1 92
ROOT BRANCH.
JT D
6 00 5 I £.
1 2 50 17 53 7 14 4 SS
36 43 "^S 72 30 00 21 7 i
35 00 25 -73
r«3 33 20 OS 26 67 23 81 13 57 23 4 1 24 71 15 46 39 47 29 34 41 o^ 37 37
15 S3 15 64 23 00 24 52 17 78 10 93
Table 1. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.
A)
42
TREATMENTS PARAMETER
ROOT INC- AGRCB. ROOT NUMBER ROOT LENGTH ROOT BRANCH. COMPOUND RHI 2 .
MEAN ST P MEAN ST D MEAN ST D
CONTROL CONTROL 0.54 1.4! 10.75 2. £3 7. 50 0. 57 A4 25 2 1 1 7 90 1 91 10.00 21 60
" A47S3 1 i7 2 37 E 00 2 45 15.00 i 7 SO " TR105 3 Oi 3 SI 7 £3 -? 91 9.47 13 53
IBA 2500 CONTROL . 7 21 5. 82 o 91 4 43 1 7. 39 18 15 „ M £ 33 6 55 7 94 2 33 IS.24 9 51 .. A47e- i. 00 5 75 e 44 2 £0 22.78 IS .'tO
.. TR105 Z I 3 3 58 9 63 1 99 23.64 28 17 IBA SOOO CONTROL 10 13 7 87 o 77 2 83 25. 00 1 £ 2£
•i A 4 o 04 10 74 S 55 1 99 IS. 50 IS 43 " A4783 "7 25 4. 87 o 27 3 71 17. 39 IS 15
TR 1 05 g 04 £ 08 10 47 2 34 4 1 . OS 24 24 POWDER CONTROL E 33 c o.t o 58 1 77 38. 95 28 65
AJ S 54 o 27 S 61 2 6'= 31.67 20 65 ., A47S3. o 96 e 03 o 35 1 76 24.00 1 6 36 „ TR 105 5 33 n 76 o 61 -? 68 27. S3 25 04
B)
TREATMENTS PARAMETER
ROOTING AGR03. ROOT NUMB ER- ROOT LENGTH ROOT BRANCH. COMPOUND RHI2.
MEAN ST D MEAN ST D MEAN ST D
CONTROL CONTROL 0.46 1 . 38 8. 33 1.53 13.33 5.77 " A4 I or- -2 95 o 00 2 87 £.67 7 07 " A47S3 3 75 *=: 94 9 oc. 4 24 20.00 24 83 " TR 105 2 63 3 35 o ^c- 3 05 10.71 12 07
IBA 2500 CONTROL 5 I "T ~ 48 10 75 1 97 31 . 50 27 00 „ A4 8 — w1 y 33 10 59 3 89 37. 90 24 £3 „ ■ ,. A4783 7 21 7 33 1 x ■< o 5 47 31.43 22 £5
TRIOS 6 S3 6 41 1 1 22 2 36 36. £7 22 49 IBA 5000 CONTROL 4 13 5 07 11 36 2 4" 40.00 23 21
„ A4 10 i 3 9 r?9 10 50 3 14 72. 27 -?.;-, 4 6 ,. A47S3 6 54 6 42 9 90 2 75 22. 11 15 i ">
„ TR105 3 3^ o 35 10 21 I 25 '"?9. ^^ 1 O 5° POWDER CONTROL *"-" 75 5 £1 1 '■"' 9 3 2 52 33.75 25 00
„ A4 5 50 i 23 1 *■> 00 2 9° 4t=. 29 "9 21 " A4783 5 -70 3 82 1 1 47 2 17 29.47 '-'O 94
TR 1 05 A 92 A -7£ 11 00 2 i^. 35.29 27 39
Table 2. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cutting of OHXF 282, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.
43
A)
TREATMENTS
ROOT I t-JG COMPOUND
A5R0B. RHIZ.
JURVU'AL
MEAN ST D
PARAMETER
ROOTING RATE.
MEAN
ROOT DRY WT.
MEAN 5T D
CONTROL CONTROL 0.03 0 17 1 96 0 -7^. 0.01 0 02 " A4 0. 2? 0 4S 2 21 1 06 0. 02 0 03
A47S~ 0.46 0 09 2 46 I 10 0. 04 0 03 '• TRIOS 0.75 0 rt i ~ i y. 0 99 0.05 0 03
I BA CS'l"'.' CONTROL 0.71 0 37 ~. 63 0 82 0. 16 0 18 .. A4 0.67 0 27 3 04 1 40 0. 12 0 1 1
A47S3 0. 7? 0 25 3 46 1 02 0. 13 0 16 TRIOS- O. 79 0 OS 3 38 1 10 0.12 0 13
IBA 5000 CONTROL 0.75 0 09 3 oo 1 27 0. 13 0 1 2 " A4 0. 62 0 ■TC-. 2 92 1 44 0. 10 0 1 1
A47S3 0.54 0 37 2 58 ; 50 O.OS 0 10 TR 1 OS 0.67 0 36 3 50 i 06 0. 19 0 19
F'OWDER CONTROL 0.7"? 0 16 3 33 1 "^5 0.12 0 IS " H4 0. 75 0 21 3 OS 1 25 0. IS 0 20
A47B.7. 0.79 0 OS 3 46 1 10 0.21 0 IS TR! 05 0.63 0 25 3 21 1 14 0. 11 0 15
B) TREATMENTS
ROOT INS AGROB. COMPOUND RHIZ.
SURVIVAL 7.
PARAMETER
ROOTING RATE. ROOT DRY WT.
CONTROL 0 00 0 00 A 4 Q 63 0 21
AH 783 0 46 0 16 TRIOS- 0 46 0 1 £.
CONTROL ,-) 54 0 28 A*1 0 i T 0 44
A4 7S3 0 S3. 0 34 TRIOS 0 87 0 09
CONTROL 0 £.•3 0 37 A 4 0 63 0 •70
A^783. 0 79 0 16 TRIOS 0 79 0 16
CONTROL 0 4 6 0 23 A4 r> SO 0 20
A4 7S3 0 46 0 25 TP105 0 54 0 25
1 91
79 58
0.
1
1.
^ 04 or- 1.
3 50 0. 3 71 0. 3 13 1.
3 29 1.
->
2
46 58 ■7^.
67
1
1. 1.
2 63 1.
10 r> 04 0 06 14 0 04 05 23 0 1 -3 0 1 9 44 0 14 0 1 6 98 0 17 0 1 7
S6 0 16 0 14 1 9 0 12 0 14 29 0 08 0 10 23 0 14 0 1 1 10 0 23 0 20 38 0 12 0 20 3-3 0 OS 0 15 24 0 03 0 1.3 28 0 It 0 49
Table 3. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.
A)
44
B)
TREATMENTS PARAMETER
ROOT INS ASRCB. COMPOUND RHII.
CONTROL CONTROL " A4 •■ A47e3
TR 1 05 IBA 2500 CONTROL
,. A4 A47S~
.. TR105 IBA 5000 CONTROL
Afl „ A47S; „ TRIGS
POWDER .CONTROL At
A47S3 " TR105
MEA!. ST D
0. 21 0. 17 0.34 0. 14 O. 09 0. 1 4
0. 17 O. 16 O. 17 0. 24 O. 32 0. 21 0. 1 4
ROOTING RATE.
ST D
04 1 04 54 1 32 83 1 09 54 0 °S SS 0 61 29 1 20 76 1 i -?
75 0 85 S3 0 57 53 1 02 75 0 85 50 1 06 4fc 1 14 33 1 20 50 1 14 88 0 61
ROOT DRY WT.
MEAN
0. 01 0 02 0. 03 0 06 0. 04 0 05 0. 07 rjp OS 0 09 0 05 0. 07 0 07 0 09 0 '07 0. 12 0 i: 0 1 1 0 08 0. 1 1 0 1"?
0 12 0 14 0. 16 0 11 0. 14 0 12 0. 1 1 0 1 i
0 14 0 14
TREATMENTS
ROOT ING COMPOUND
AGROB. RHII.
ci ipi; T \ ;<:
MEAI.'
iL :-:
ST D
PARAMETER-
ROOTING RATG.
MEAN ST D
ROOT DRY WT.
MEAN
::ONTPC'L
IBA 2500
IBA 5000
POWDER
CONTROL A 4
A47Sr. TR 1 05
CONTROL H4
A47E3 TR105
CONTROL H4
A47S3 TR 105
CONTROL A4
A4783 TR105
0. 35 0.34
0.14 0. 1 4
O. 40 0. 1 o
0. 40 0. 1 6 0. 29 0. 1 6 0. 03
13
SS
. SO
54 i 06 58 0 88 67
1 21 0 99 79 0 72
oc
88 1 42 00 1 35 88 1 39 38 1 14 — * 1 29
0 01 0. 02 0 03 0. 05 0 09 0. 13 0 07 0. 11 0 12 0. 10 o. 16 0. 1 ~ 0 15 0. 13
0 16 0. 13 0 12 0. 14 0 22 0. 1 6 0 10 0 10 0 16 0. 17 (•-. 12 0 14 0 16 0. 1 9 0 15 0 13 0 14 0. 13
Table 4. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on survival index, rooting rating, and root dry weight of nist propagated hardwood cuttings of OKXF 282, A) no VAM, B) VAN.. Each mean represents the mean of 24 cuttings.
45
A)
B)
TF.EATMEWTS PARAMETER
F.OOTINE A6F.0B. ROOT AREA SKOOT NUMBER SHOOT DP- t' WT. COMPOUND RHIZ.
MEAN ST D MEAN ST D MEAN ST r
CONTROL CONTROL 2. 17 5. 13 0. 13 0.4 5 0.08 0.27 H4 1 48 7 O "* 0.4 2 0. 53 0. 24 ','.■ 72
A47S~ 3. 91 c 07 0.63 0. 77 0. 25 0 ~i
" TR1C'5 1 1 73 12.30 0.53 0.83 0. 39 0 7■'~,
IBA 2500 CONTROL 4? ~~ 24. 50 0. 96 0.69 6. 17 i. ^.'w
A4 32 i y 21.74 1. 04 1. 12 5. 33 -7 ■09 " A4 7S7 30 34 23. 25 0.96 0.69 0.65 0 51 .. TR105 28 f:2 23.48 0.96 I . 00 0.63 0 4 4
IBA SOOO CONTROL 27 67 22. 40 1.54 1.22 9.67 7 64 H4 24 25 24. 43 0.83 0.76 0.66 0 ~C
.. A47P.3 10 00 15.36 0.S3 0.92 0.59 0 55 TRI05 41 50 28. 27 1. 33 1 . 09 0.99 0 5"
POWDER CONTROL IS 58 16. 27 1.13 0. 95 0. 66 o ■a.i
A4 4 0 —— 24. 13 0.33 0. S7 0. 63 0 5° ,. A47S-7 33 ?e 26.41 0. 3S 0. 74 0.6° 0 52
TF:105 12. 57 15.20 1.13 0.95 0. 53 0 4 ^-
TREATMENT PARAMETER-
ROOT IM5 AC-ROB. ROOT AREA SHOOT NUMBER SHOOT DRY WT. COMPOUND RHIZ.
MEAN ST D MEAN ST D MEAN ST D
CONTROL CONTROL 0. 00 0.00 0 08 0.41 0.84 0 a i
ft 4 o 20 9.02 0 59 O.SS 0 36 0 79 -.■3 7?- 8 73 10.37 0 50 0. 59 0 4 7 0 49
•■ TPIO'5 5 23 7.67 0 50 0.S3 0 34 0 J0 IBA 2500 CONTROL •31 25 27.71 0 9"* 0.93 0 59 0 54
A4 29 67 24. 69 0 83 0. 74 Oi 53 0 52 " A47S3 29 10 28.24 1 08 0.86 0 80 0 53
TR105 27 58 16. 18 1 46 1. 06 0 96 0 57 IBA 5000 CONTROL 18 42 28.04 1 04 1.08 0 77 0 65
„ A4 1 3 02 18.57 0 92 0.93 o 72 0 7.2 " A47E-. 27 17 24. 00 1 25 1 . 07 0 77 0 4 9
TRIOC 4 3 59 29. 72 1 25 0. 99 0 91 0 66 c'OWDER' CONTF;^" 30 7.7 33. 18 0 58 0. 77 0 47 0 55
„ A 4 16 42 23.49 0 63 0.83 0 33 0 42 .. A4783 7 75 17.67 0 75 0. 99 0 43 0 63 " TR105 12 62 19.95 0 75 1 . 03 0 36 0 J7
Table 5 Effect of VA mycorrhizas, Agrobacterium rhizogenes, and rooting'compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.
46
A)
TREATMENTS PARAMETER-
ROOT ING AGROB. ROOT AREA SHOOT NUMBER SHOOT DF Y WT. CONFOUND RHI Z .
MEAN ST D MEAN ST D MEAN ST D
CONTROL CONTROL 0.58 2. 02 0.33 0. 70 0.21 0.36 " A 4 4 98 7. 61 67 0. 92 0. 19 0.28
64787- 8 38 9. 86 0 67 0.96 0. 1 8 0.27 " TR1C5 S S3 S.96 79 0.78 0. 32 0.2S
IBA 2500 CONTROL 17 45 9.42 67 0.87 0.67 0.29 .. A4 < e- 58 12.87 29 1 . 04 0.56 0. 50
A47S3 IS 0£ 12.54 25 0.85 0. 69 0.50 TR105 15 57 10. 40 33 0.82 0. 65 0.4 1
IBA 5000 CONTROL 24 58 14.13 29 0. 86 0. 67 0.33 A4 24 li. 21. 18 54 1. 02 0. 62 0.44
.. A47E7 21 18 18.77 42 0.83 0.61 0.3S TRIOS- 32 58 12. 18 08 0.93 0. 59 0.47
c-OWDER CONTROL 27 OT 2~. 1 6 08 0.88 0.65 0.46 „ A4 20 90 15. 83 96 0.86 0. 57 0. 44 " A4787- ~*3 42 24.72 -7S 1.11 0. 72 0.41
TR10S 1 ? 16 15.24 21 0.83 0. 58 0. 32
B)
TREATMENTS- PARAMETER
ROOT !N6 AGROB. ROOT AREA SHOOT NUMBER SHOOT DRY WT. r-nMC-Q' !M[} F;KI Z.
MEAN ST D MEAN ST D MEAN ST D
ni--' IT=0L CONTROL 1.42 4.91 0 08 0.28 0. 09 0.31 ■' H4 4 66 6 87 0 50 0.72 0. 20 0. 2"'
A4 7S7 17 92 i p. 03 0 42 0. 72 0. 23 0. 27 7R105 o 68 14 70 0 92 1. 14 0.38 0. 40
IBA 2500 CONTROL 24 46 14 05 1 38 0.97 0.74 0.49 „ A4 ~o 67 21 93 1 38 0.97 0. 72 0. 46 ., A4783 25 S3 15 45 1 33 1.01 0.59 0.39
TRIOS- 33 75 19 86 1 29 1.12 0.S8 0.58 ;BA 5000 CONTROL 27 76 "^3 5-4 0 92 0.97 0.51 0.49
.. A4 4 0 92 19 32 1 46 1. 06 0.72 0.44 A47S7 18 72 18 23 0 88 0.74 0.50 0. 45
„ TRIOS 38 73 28 24 1 04 1. 04 0.60 0. 52 FOWDER CONTROL 21 08 22 93 0 9'*1 1. 02 0. 56 0.62
.. A4 33 23 23 76 0 96 1. 12 0. 60 0.61 " A4787. 26 58 21 53 1 04 0.91 0.64 0.4! „ TR105 22 00 18 4 5 0 02 0.93 0.41 0.34
Table 6. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.