The function of tannin in host-parasite relationships with special reference to ribes and Cronartium ribicola

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Title:
The function of tannin in host-parasite relationships with special reference to ribes and Cronartium ribicola
Physical Description:
27 p. : 27 cm.
Language:
English
Creator:
Offord, H. R ( Harold Reginald ), 1903-
United States -- Bureau of Entomology and Plant Quarantine
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United States Department of Agriculture, Bureau of Entomology and Plant Quarantine
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Washington, D.C
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Tannins   ( lcsh )
Ribes   ( lcsh )
Cronartium ribicola   ( lcsh )
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bibliography   ( marcgt )
federal government publication   ( marcgt )
non-fiction   ( marcgt )

Notes

Bibliography:
Includes bibliographical references (p. 23-27).
General Note:
Caption title.
General Note:
"E-518."
General Note:
"November 1940."
Statement of Responsibility:
by H.R. Offord.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 030271190
oclc - 11944926
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lcc - QK898.T2 O34 1940
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AA00023017:00001

Table of Contents
    Table of Contents
        Page 1
    Introduction
        Page 2
    Previous related investigations
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
    Summary of data on tannin content of several western ribes
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Summary
        Page 21
        Page 22
    Literature cited
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
Full Text





E-519 November 1940

United States Department of Agriculture .
Bureau of Entomology and Plant Quarantine


THE FUNCTION OF TANNIN IB HOST-PARASITE RELATIONSHIPS
WITH SPECIAL REFERENCE TO RIBES AND CRONARTIUM RIBICOLA

By H. R. Offord, Division of Plant Disease Control



CONTENTS

Page
Introduction ..................... ............................. 2

Previous related investigations ............................... 3

Chemical properties of tannins............................. 3

The, unction of. tannins in plants .........................

Influence of tannin on hoBt-parasite relations ............ 5

The physiological nature of host-parasite relations in
blister-rust disease ........................... 9
Status of the tannin 'question as it relates to disease
resistance .......................... ...................... 10

Sumnmary of data on tannin content of several *estern ribes.... 11

Qtialitative tests on the tanrnins of R. petiolare and
R. inerme ................. ............................. 11

Histological work.... ............................... 11

Microchemical tests for tannins on greenhouse ribes.. 13

Qiiauntitative determinations of tannin for several western
ribes..... ............................................... 15

Summary, ................................................... 21

Literature cited .............................................. 23







-2-


INTRODUCTION

The purpose of this circular is to summarize unpublished data l_
on the tannin content of Ribes spn., obtained in the course of laboratory
and greenhouse investigations on the chemical eradication of ribes
(36, 37). 2/ 3-1

The original objective of the laboratory and greenhouse work,
undertaken during the period 1927-1930, was to determine whether plant
extractives such as tannins served as protective agents against the toxic
action of herbicides. Evidence was obtained to show that, in general,
the most effective herbicides were those which form soluble compounds or
mixtures with tannins and similar water extractives rather than those which
form immediate precipitates -ith such extractives.

As a by-product of this work, it mas noted that the leaves of the
black currants, which are highly susceptible to blister rust ai~sease
(Cronartium ribicola Fischer), tended to be low in tannin, -hile some of
the resistant species, such Ps Rib7s l'custre, showed a high tannin content,
Laboratory tests on the nture of the tinnins in the leaves of R. petiolare
and R. inerme--t7-o ribs differing in susceptibility to the rust--indicated
that there were differences in the type as well as the quantity of tannin.
It was realized that both quantity and quality of the tannins could be
significant factors in resistance to a fungous disease.



l/ The tannin analyses -rTre made at Berkeley, Calif., chiefly by
G. R. Van Atte, and at ioscow, ideho, by R. P. d'Urbal. Research facilities
-ere made )v-ilabl- at B;eirk-, ey through the cooperation of the Department
of Forestry nd th2 Division of Plant Nut rition of the College of Agriculture,
University )f California, and at iioscow through the Forestry Department' of
the Univrsity of Idaho. iicrochemical tests were made at Berkeley by
I. E. W' bber, with the assistance of F. A. Patty and C. R. Quick, and were
made possible through facilities furnished by the Department of Botany,
University of C!Aifornia. At the tim- that the 7ork on tannin was under-
taken, the blister-rust-control activities vere directed by the Bureau of
Plant Industry. Sinc December 1933 this -ork has b en directed by the Bu-
reau of Entomology and Plant Quarantine.

2/ The generic name Ribes ind the common name ribes are used in this
paper to indicate both curr,1nts end gooseberries.

3j Nubers in parentheses refer to the literature cited at the end
of this circular.





Since the writer was orimarily concernedwith development of
herbicides and not with research on'the physiological relations between
ribes and the blister-rust fungus, the investigations were dropped when
they were no longer of immediate use to practical work on the chemical
eradication of ribes. Recently, however, there have ben a number of re-
quests for a summary of the data on th, tannin content of ribes obtained
during the course of this work. These requests reflect the increasing
interest in the physiological function of tannins and their decomposition
products, esopcially in pathologic relations between host and parasit-
(6, 10, 12, 14, 29,.55), in horticultural and ecological problems dealing
with crop rotation and plant succession (9, 15, 46), and in matters per-
taining to the palatability and nutritive value of forage (4, 7).

Recently published data (14, 25, 29, 34, 35) show that the degra-
dation products of tannin- are the substances most directly concerned in
the protective action against fungi. Since these products have not bean
thoroughly studied and identified, it se-emed preferable to make general
use of the term tannin throughout this report.

In order to indicate the significance of the data on the tannin
content of Ribes spp., th- chemical qnd physiological properties of tannins
and related work on th- role of tannins in host-parasite relationships
will be presented first.

PR OUS RELATED INVESTIGATIONS

Chemica-l properties of t!)nnins

It has been. customary to aply the name tannin $o that portion of
the water-solubLe matter of certain vegetablH materials vhich rill pre-
cipitate gelatin from solution :ind which will form compounds with hide
fiber that are resist,.nt to washing. In this paper, tannin is used as a
collective term for a group of substances having the special properties
just described.

Tannins are amorphous or crystalline solids of astringent taste,
having wide occurrence end1 gierral distribution in the tissue of most
higher plants. They are readily obtained from pulverized or shredded
plant material by extraction with hot wat':r or alcohol. Tannins -xhibit
a marked tendency to absorb oxygen, especially in Plkaline solution.
Fischer (17) synthesized penta-m-digalloyl-beta-glucose, and showed it to
be an isomer of the tannin obtained from Chinese nutgalls, and some recent
w-ork by Russel and coworkers (47, 4S,_ 49, 50) has extended data on the
chemical structure of t.qnnins. Th-* greeter part of the 7ork on tannins,
however, has dealt chiefly with methods for cl:essifying, extracting, and
measuring tannins for use in tho leather industry. Classifications
generally accepted are those of P rkin (38) Pnd Freudenberg (18).









The arrangement of tannins into class is based on color re-
actions with ferric salts and other reagents, on the presence or absence
of a precipitate with bromine water, on the production of a so-called
"bloom" on leather, and on the products of hydrolysis. For the discussion
presented in this paper, Perkin's 3-group classification will be used,
i.e., alpha group (depsides or gallotarnins): beta group (ellagitannins);
end gamma group (phlobatannins or catecholtannins). The following de-
composition products of tannins are of major importance in their classi-
fication:

(1) Heated alone, they form trihydric phenols (pyrogallol) and
dihydric phenols (catechol).

(2) Heated with dilute acids, they form dextrose, trihydroxyacids
(gallic acid), ellagic acid, and insoluble amorphous anhydrides
called phlobaphenes.

(3) Fused with alkaline hydroxides, they form trihydric phenols
(pyrogallol and phloroglucinol), dihydroxyacids (protocatechuic
acid), and acetic acid.

Tha function of tannins in plants

Although physiological investigations of the role of tannins in
plant life have been hampered by incomplete data on their chemical nature,
it has long been recognized that tannins Pre something more than by-products
in plant metabolism, and a physiological function has been assigned to them
by most -orkers (10, 14, 25, 31, 39)-

Tannins have be-n noted to affect the activity of enzymes. Brown
and 'lorris (5) observed thnt tannin inhibited the action of diastase in
leaves, an I Vinson (56) showed that the solubility of date invertase in-
creased as the fruit ripened, and that these changes were paralleled by
changes in the solubility of the extractable tannins. Tannin has also been
used to absorb ocroxidase (16).

Moore, as noted by Haas and Hill (19), states that tannins may play
an important part in the lignification of cell walls and are tied up with
cork formation, and further that tannin in the epidermis of leaves may in-
fluenc- the opening and closing of stomata. Denny (13) treated seeds of
pumpkin, almond, and peanut Tith solvents for tannins and lipoids, and re-
ported that tannin is one of tye ingredients of the cell wall which affects
its permeability to water. Lloy! (29) and Herszlik (24) present evidence
to sh-vw that in the cell sap an, vacuol-s tannin is linked up or adsorbed
with cell constituents of P cellulose nature and that tannin vacuoles in
cortical tissue are protected by a pectic membrane from contact with
protoplasm.










Quendiac (41), in his studies on oak trees, concluded that early
in spring tannins are localized in areas in which sap flow is restricted,
notably the parenchyma of the sapwood. In May the accumulation zone ex-
tends toward the ends of the branches. Subsequently the sapwood develops
two distinct regions, one normal and one -ith tannins, the accumulation
corresponding with the zone of transition between heartwvood and snp'vood.

The most comprehensive study of the location and function of tannins
In the plant cell is that of Dekker (12), vho used ribes as experimental
material. Using a 5-percent potasslum bichromate solution as a stain, he
studied the distribution of t;onnin in leaves, current-seaion twigs, old
steins, fruits, and roots. -- concluded that tannin accumulated in regions
of strong vegetative growth--for example, in the tips of tw7igs. Tannin in-
variably occurred in those layers of cells which, because of their position,
may be expected to have a protective function. In the young st: Ik this is
the epidermis and its bordering layers; in the older stem and roots it is
the cork tissue. In Ribes sp. the primary vascular bundles in th' earliest
stages of the stqlk are surrounded by a girdle of cells 'rhich are particu-
larly rich in tannin. By covering active ribes leaves 'ith tinfoil and
gelatin paper, and comparing the tannin-bichromote color in covered leaves
with uncovered controls, Dekk-er show ed that light -vas an indispensable fac-
tor in the formation of tennis in the leaf. The results of further stain-
ing tests showed that the tannin bands in the medulla are of significance
in the transport of reducing sugars.

According to hauser (21, 22, 23), tannins act on the cell plasma as
polyvalent phenols, -7ith relatively l mre rolecules tending to prevent the
formation of coarse disper'ions on se'ondqry structures. H,4 ascribes a
regulatory function to tannin corresponding to thet of a protective colloid.
The effectiveness of this protective colloid -ill depend upon its degree of
dispersion ani upon related phenomena, such as p:rmeability, absorptive
power, and cell turgor.

Influence of tannin on host-parnsite r l-Ations

The high concentration of tannin in pathologicglls and in hyper-
trophied tissue resulting from insect injuries cnlld -Prly attention to
the role of tannins as probable protective materials. Ward (57), Cook and
Taubenhaus (10), -nd othemlisted tannins anong th probable substances
having a chemotactic action on the ger-n tubes of fungi. Cook and Taubnhaus
conducted extensive tests on the toxicity rf tannin on a wide variety of
organisms. They point out the necessity of correlating the function of
tannins rith other constituents of the plnnt cell in any consideration of
the reaction of a plant to invading funi.





TT P T










Cook and Taubenhaus also showed that small amounts of tannin may
stimulate production of spores and that specificity of action between
tannin and the various invading fungi was indicated. They show that
Fusariums were more resistant that Gloeosporiums and Collectotrichums; the
Cladosporiums were more resistant than the Fusariums, and Penicillium
oliveceum was the most resistant of all species tested. The majority of
parasites were retarded by 0.1 to percent of tannin.

The fact that Plants which contain unusually high percentages of
tannin nay also be subject to disease indicates that tannin per se cannot
be exclusively charged with the protection of a plant agAinst fungi. The
chemical nature of the tannins, the quantity present, the location of the
tannin-bearing cells, the available food supplies near the point invaded
by the parasite, and the effect of enzymes or hormones on the liberation of
protective substances must all be considered.

In a report of cytological studies on the biochemical aspects of
immunity, Dufrbnoy (14) points out that the susceptibility of a plant to
invading hyphnae depends upon the death rate of the attacked cells, and the
ability of the germ tubes to establish ne7 contacts with adjoining cells.
he states that in v resistant plant the attempt at penetration by a filament
rapidly provokes the formation of phenolic compounds,Tj which resets in
death of the first cells threatened by the infection. Thus the filament is
unable to establish a relation with the plant that it is trying to invade.
In highly susceptible plants the filaments probe between the living cells and
penetrate them without causing a significpnt br-ak-down in the cell plasma.
If the fungus grows rapidly enough to Pstablish itself b-yond the zone of
cells containing toxic concentrations of Phenolic compounds, it can continue
its advance. Conversely, development of the fungus is arrested if it cannot
grow fast enough to keOp established beyond the zone of differentiated cells.

Ros, ( 4, 45) reported that apple bark carrying blister canker caused
considerably wore oxiation with oyrocotechin, guaiacol, and benzidine than
healthy bark, and attributed the phenomenon to the influence of tannin on
oxilaso activity.






Q In a report before thg American Association for the Advancement
of Science, G. A. Gr-athouse and N'. E. Rigler "Rhowed that natural and
derived ph-nolic co'ipounds are toxic to the root-rot fungus. Isomerism
was found to play an important port in toxicity." Science, vol. 91,
p. 16, Feb. 2, 1940. Published later in Amr. Jour. Bot. 27: 99-108 (1940).


-6-





The specificity of phenolic combinations (tannins) in reactions
with invading fungi, the high toxicity of certain types of these phenolic
combinations, and their propensities for oxidation-reduction action in the
'plant 6ell have been confirmed and further developed by the work of
Kargapblova(25). He investigated wheat species and varieties extremely
resistant or susceptible to Puccinia triticin. for their phenolic content
and derivative substances. Susceptible varieties contained mere trrc'es, or
only about one-third to one-quarter as much of the phenolic compounds as
the resistant varieties. Qualitative reactions showed that the tannin
fraction contributing most strongly to immunity was of the pyrocAtechin
rather than the pyrogallol type. It is known that the pyrogallol series of
tannins are readily hydrolized by enzymes (such as those carried by yeasts
and fungi) into nontoxic materials, whereas the pyroc..techin series are
more stable, and are thus more persistent in their protective and precipi-
tating reactions on proteins. Kargapolova's tests of the effective action
of pure phenols on the spores of P. triticina also showed that the separate
phenols vary in toxicity. Of the phenols tested, the most toxic for the
rust were hydrochinon and pyrocntechin. The acetic-ethyl fractions, when
tested directly on spores of P. triticina, were more toxic when prepared
from immune than from susceptible varieties of wheat plants.

Thornberry (55) reported that tnnnic acid, if applied prior to inocu-
lation, inhibited the tobacco mosaic in direct proportion to its concen-
tration. Stapp and Bortels (54) noted. that not all fungi were able to split
tannins; in their tests the ability to decompose tannins was restricted to
several types of Aspergillus and Peniciilium. Rippel and Xeseling (43) re-
port that Penicilliuim, Citromycs, and Ap _erillus spp. were the only molds
examined that were able to use tannin as a sole source of carbon. Bavendamm
(4) and Davidson(ll) describe methods fdr the differentiation of wood-
decaying fungi, based on their growth reactions on media containing gallic
or tpnnic acid.

Naumann (32) described experiments to show that: uingi ca nnot pro-
duce tannin, but may take it up and utilize it as food when decomposed.
Certain fungi do not absorb tannin and are injured in presence of excessive
amounts. Polyioreae contained 0.034 to 0.400 percent tannin and Agaricaceae
0.041 to 0.060 percent tannin. Parasites usually contain more tannin
(0.10 to 0.40). Tannin is chemically decomposed in fungi."

Specific physiological responses following, fungous attack are also
indicated by transpiration phenomena. Reed and Cooley (42) reported that
apple leaves infected -ith Gymnosporangium juniperi-virginianae showed
lower transpiration loss than healthy leaves. Lowered transpiration for
cocklebur infected with Puccinia xpnthii was reported by Weaver (59).
There appear to be many instances, ho,7evar, heree fungous attack is followed
by a marked increase in transpiration rate. Weaver (5s) ha- suggested that
increased transpiration on the part of infected plants might be attributed
to changes in the permeability of cell walls brought about by substances
secreted by the fungus.


-7-










The physiological nature of host-parasite
relations in blister-rust disease.

The resistance of the host plant to an attacking parasitic fungus
is generally physiological rather than anatomic-l in nature. Early work
by Ward (57) )nd more recent work by Dufrdnoy (14), Kargapolova (25),
Sta1cman (53), Pnl Allen (1) have established this observation for a wide
variety of plants and fungi.

The same conclusions were reached by the writer, following a histo-
logic study 5j of the internal and external anatomy of several ribes of
widely differing susceptibility, such as the highly susceptible R. petiolare
and the immune Viking red currant (syn. Rd Hollpindsk Druerips.). The
number of stomata, the thickness of protective tissue, and other histologic
charecters frequently varied -is much between ecologic forms of one species
as between resist-ant and susceptible varieties.

The recent -ork of Anderson (2) pro, ides confirmatory evidence that
the resistance of Ribes sp. to Cronartirmn ribicola is physiological in
nature.

In a cytological study of the immune Viking curr,-nt Anderson found
(2) that the entrance phenomenon of the hyphan was essentially the same for
the immune currant and for th susceptible Ribes nigrum, R. sativum, and
R. hirtellum, Stnkman (53), i1'wton (33), and Allen (1) also report no
essential difference in entrance? phenomnna of the rust hyphae in resistant
and susceptible vo)rieties of wheat attacked by Puccinia grnminis Pers. and
P. gLrinis tritici Erikss. Anderson (2) confirmed the previous conclusions
of H.?hn (20) thnt the blister-rust fungus was abl., to enter the leaves of a
Viking current hut filed to propagate itself. About 49 to 72 hours after
inoculati,:i of the Viking currant, the contents -f the invading hyphae
shoved v:, >oles znd become granular, pro-13ing evidence of marked degener-
ation. WiVithin 96 hours the myceliuim had broken down and disappeared.

Inv-?-sgtigtors -orking 7-ith resistant wheat plants (1, 33, 53)
report tn: d-=ath -f host clls rec d s that of the parasitic hyphae,
'ith ti t .tt -d;:th from starvation of the invading organisms. In the
Vikin 1_ indeir-ri observed that the death of hyphae preceded that of
the2 ht ciis by several days, and for this reason he postulates a physio-
logical incompatibility.





5/ See unpublishe-d reports on the morphology of Ribes sp. by I. E.
Webt r, C. R. Quick, and F. A. Patty on file with the Bureau of Entomology
and Plant Quarantine, Division of Plant Disease Control at Berkeley, Calif.
and 'Xn)shinpto)n, D. C.





The differences noted by Anderson in the response of young, fully
expanded leaves and fully matured, hardened leaves slso point to tie
physiological nature of resistance. Germ tubes per etrated the leaves
through stomata in both types of leaves, but only the young loaves per-
mitted the temporary development of hyphae. The chemical and physiological
properties of tannins, as previously pointed out, are such that quantita-
tive 9nd qualitative differences may be assuided in various plant parts
where differences occur in age and metabolic activity. That the suscppti-
bility of ribes leaves and pine needles to blister rust varies with the ag'
of leaves and needles has previously been demonstrrtei by the work of
Spaulding (52), Snell (51), Lachmund (27), and 1Mielke ( 0). For ribes,
Spaulding (52), summarized many of thc,se data as follows:

"The ago end relative maturity of th3 leaf has much to do
with its susceptibility. It has been th- general ?xperience
that Ribps le ves may be ovrm;ture :nd also may bi too young
to take the disease.. Infection does not occur on l~sves of
a given species of Ribes until they hpwy reached P c ?rtpin
degree of mturity. L,?- veq pro,9ucei by buds ew reloping in
late summer or fall even if very smll, readily become in-
fected. The different species of Ribes vary ruch in this
regard. Ribes ni&rup show a gr rit r:n-.e in its Pj: of
susceptibility, whil r-sistant species b-com inlfectel only
on leaves of a certain maturity. The most fraorable stage
of growth sems to be about when the leaf attAins full size
but has not become hardened and leathery as it does later."

Under field conditions it is difficult to establish with 9ny degree
of accuracy an order of susceptibility of ribes to blister rust disease,
especially if the list is intended to col,< r ribs of Jifferknt geogr,,phic
and ecologic regions. Kimmey (26) showed that susceptibility to direct
inoculation varied among species end -ven in the sn;me speci .s for open,
part-shade and shade forms. Tn- -bility to produc telia pres-nted a
different order of susceptibility than tht pro-iDcd by the concept of
ability to take infection, and. the ecolo,;ic influences wI-re not always
followed by a consistent increase or decrease in reaction to the disease.
Generally the more susceptible plants produced the rmost telin, and vice
versa. Within a species, the pnrt-shads form was the most susceptible
and produced the most telia; the open form 4s pst susceptible arnd pro-
duced the least telia. This genernl reaction to th: rust is consistent
with the physiology of tannin production in the pl> nt, in-snuch ss sun-
light and high plant metabolism tend to optimum production of t.annin.

Hahn (20) has recently made an interesting contribution to stuIies
on the susceptibility of ribs to blist-r rust liseas-, by o,'onstrnting
the immunity of a steminz te clone of z/ib alpinum. This obs r-ration
would indicate theft susceptibility is sex-linked, and thpt a horrlone or
enzyme may be responsible for initiating subsequent protective responses
of the plant.


-9-







-10-


Status of the tannin question as it relates to disease resistence

In presenting the foregoing discussion, the purpose has been to
assemble data from published records which relate to the chemical and
physiological properties of tannins and to the effect of tannins and
their degradation products on the C11rowth of fungi. No attempt has been
made to discuss generally the biological aspects of disease resistance in
plants. A recent review of this subject by Brown (0) shows all too clearly
the size of such a task and the mass of data--most of it analogous in nature--
which might be taken as support for one of several theories.

The case for the tannins must certainly be considered in the light
of their widespread distribution and occurrence, frequently in large amounts,
in plants *hich are readily attacked by fungi. On the other haxfd, the tox-
icity of many tannin extracts, as expressed in their precipitating action
on albuminoids and in direct tests on the growth of parasitic fungi, has
been well enough Pstablished to be generally accepted. The Dresence of
tannin in solution in the vacuoles of the normal plant cell, however, would
presumably r-quire (a) that the urotoplast be protected against immediate
contact with the r active tannin by a limiting layer of a secondary adsorbed
compound, or (b) that the concentration of tannin in solution be too low to
precipitate albuminoids or (c) that the toxicity of tanain be indirect and
caused by degradation products released through enzymic action upon contact
of host and parasite.

In th, writer's opinion, the mechanism of the tannin-parasite
relationship, in agreement with most of the direct evid-nc-, is the follow-
ing: An en2zyir-i. or hormone secreted by the fungus initiates the penetrative
action into living cells. The first tbrri-r to succesful contact of hnus-
toria and call contents is the adsorbsI tannin of the cell -all. At this
point the complex nature -f thw t:ennins offers plenty of scope for specif-
icity lasod on the lock-and-key simile of enzyme and substrate.

If fhe cliemicnl composition of tne hectic ;.nd tanninllk substances
of the c311 wall cannot be converted into ;oluble or us oble decomposition
products by the enzymrn or hormone secreted by the fungus then the haustoria
fail to develop and tnese sp-cialized1 feeding branches die Thile the plant
cells are still turgid. This type of r,-.istance is probably that noted by
Anderson (2) in the caqe of the immune Viking current. Sho uld the haustoria
be able to penetrnte into th- plant cell, then th- soluble nnd oxidizable
tannins )f the cell sp mpy offer usalle nutrients to the fungus, vith the
kesultnt stimulation of its growth (10, 11, 32, 43). Or, thf- tannins may
form irngredie ntq upon Tcopoition --ich :3re toxic to fungi (10, 14, 25).
Failure of thei haustoriq to develop, or disorganization andl death of the
plant cells prior to thnt of tile u'austoria as not l by Stakman (53) and
Allen (1), "ould sn t9 depend on whether the antag-onistic action is
exerted at the surfac, layer )f the plnnt cell or within the cytoplast.
FollDn'ing contact "If thp haustoria *vith cell contents, subsequent growth
end developm-nt of the invatling organism may be retarded or encouraged by
the nature )f the toxic or oxidiz ble tannins occurring in solution in the
sap vcuoles.





U


ll -

The salient facts just sumrize .may n& be used to interpret
the results of the tannin studies Made by the writer.

SUMM, ARY OP DATA WiC TA M,"IIlN C TIT OF I, : '. T ,-< ft' "

Qualitative tests on the tannins of itibes petiolare and i{. iner-,e

Laboratory investigations conducted at "'erltey in t e fall f
1927 sl owed differences in the chemical properties of ccnstit"e nts (If
the tannin aggregate in R. petiolare ard R. iner.o. The tarnnins of
R. petiolare belong to the group that gives blue-black arscioittcs
with ferric salts, while R. inorme tarnins give a : ixturo of b(,th blw3-
black and green precipitates. On careful hcating7, R. potiolaro tannLn
showed the characteristic flakeliko crystals of i;,yrogallol, while
R. inerme tannin change over into a black amorphous .ass characteristic
of the phlobaphenes. Acid hydrolysis cf R. Teticlare tainin yilded
only a trace of dextrose, considerable rsllic acid, and so Chlohamheno.
Aci-I hydrolysis of R. in rme train howed1 a fair quanrtitv of dextrose,
no gallic acid, anl a i3rzc an:ount cf phlobaphene. fusion v.ith -otassium
hydroxide indicated -0,ro:allcl in the case of the former aid >lorc-
glucinol for the latter. During t'e oc,'src of a -roxi' tcnalysis
of the leaves and ctens. of R. Veticlcre, R. inerme, i.. viscosissimun., and
R. lacustre, it was noted that ess than 1 rcent ot .nin of
R. petiolure was obtaine in an alcohol fraction, while about CO Tocrcent
of the tannin in R. lacustre, R. inerme, and R. viscol-isl-imumr wes roovod
by alcohol. Thus it is seen that tlI taninj con Olex of ribeS coprises
ingredients which differ widely in chemical comoosition a-nJ vhvsical
character.

On the basis of these tests it a3 'cars tint A.)es
belong chiefly to the alpha group containi.- thl, depsids or gi!lotannins
(Perkin's classification) which yield phonolics of the pyrog-Ilol tlpc,
while the R. inenne tannin cornplex colitains representatives of buth the
alpha and garmia typos. As Kargarolovt pointed out, the ->yrop- llol phenols
and their tannins (alpha group) ,re rt;adilv hydrolyze l by msuny or zy's of
fungi into alcohols and'nonactivu phenol acids and arc tier-for lcss
reliable protective substances than the cat,3chol tannirs (ga: na group),
which are loss easily hydrolyzed by enzy n-es and WqLCh thus their
pover of precipitating proteins.

Histolopical work

The histologic methods used by Dekkor (12) wore atiployod at
Berkeley in the winter of 1928 to study the distr'buticn of tznin in
Ribespetiolaro Dougl.. R. lacustre (Thers.) 7,ir, R. viscosissimmn Iursh.
and R. inerme Rydb. Leaves and y ouni, stens cf 7roenhcuse -Froir- plants were
sectioned, stained with 5-percent nctassium bichromatc ..nd hcto"icrofrraphic
records were made. In noting the distribution of tannin and allied
substances, it was observed that--










(a) In leaves such substances nay occur in epiderraal cells,
epidernal hairs, scattered coll s of palisade and spongy parenchyma,
border r..r. chyma, ar. vascular tissues.

(b) In ycur4g stets such substances mrny occur in t1 epidermis and
its appendages, scattered cells of the cortex, the poricycle, phloen,
scattered cells cf the xylem, aid scattered cells of the pith.

(c) T1ho followinG data pertain to leaves: Ribes petielare showed
traces of tannin in the epiderzal layers andA only small Laounts in the
vascular tissue cf t-c midrib; R. lacustre ar. R. viscosissimum had tannin
rather evenly dis tributed in eq]-dermal layers arT spongy parenachypra;
R. in rre showed less tannin in eDiderLal layers than R. lacustre and
77 viscosissi-um and about equal amounts in the spongy parenchyrm.

The a::ount of such substances -resent was not constant for any of
the s>,cius. Free-hand sections of leaves and stems taken from greenhouse
ribs and stained 'or tannins with potassiun dichromate showed considerable
variation in anount of t: nnin b:tweon individuals as vell as between species.
Variation was aparontly due to several factors, Df u:hich differences in
illuminaticn seemed to be onc.

Dckker's ex7)erinents with tinfoil-and gelatin-covered leaves were
rcoeatod, using several sets cf leaves frola different positions on a
number cf pla.nts. After 48 hours under artificial illmiam'tion, tho loaves
wore stained in biclronmato solution, and in every case the gelatin-coerod
and th. uncovered leaves were darker showingg t1h presence of more taxnins)
than thc tinfoil-covered leaves. Fresh ribes loaves taken front the plants
follcvirc several cloudy days ;wcro much lo;er in tannin content than
t field sa ples first tested. After two suniy days, R. lacustre showed
an increase in t1c anourt of tannin as indicated by the dichro-mte staining
reacti on.

An attempt was to study the quantitative distribution of
different ty'es cf tannin by i..uersi:g freshly picked ribes leaves
in various s .l jents, nrior to staining with potassium bichromate, Ob-
ser~ati'ns from these tests are sum.. rized below.








-13-


icrocho.,ical tests for tannins on grecnhcusc ribs

Leaves gathe-red at end of dy of full sunshine frm rrtu~ro
plants grown on sand cultures:

R. peticlare--
(1) Directly into the bichroente.
Tannin re, cticn in the vascular tissue r, ther -:".rkd, quite
well marked through the spongy parenchyiaa, ind ncticeable
in so2e cells of both upper and lower epidermis. Practically
none in the palisade parenchyma.

(2) Directly into absolute alcohol for 24 hcurs, then into biclcXon:te.
Tannin reaction slight in the phloom region. Not noticetable
elsewhere.

(3) Directly into water, kept at 540 F. for 24 hours, then into
bichromnate.
Tannin reaction as in (1) above, but less _rrkcd, particularly
in the spongy parenchyma.

R. lacustre--
(1) Directly into bichronate.
Tannin reaction exceedingly w,,ell -.rId in the region of the
vascular tissue and in a fow cells throughout spongy parenchyna
al palisade ce1ls. PCrochyma ". ol is give fairly well rmrked
reaction in lover and upoor o-,idcr.Iis.

(2) Absolute alcohol aid then bichromate as above.
Tann.in reaction well Iharked in vascular tissue; a fewv colls in
spongy paronchyria, nxd som. palisado cells give fairly well mrked
reacti on.

(3) Watcr and bichrcmate as above.
Slight ta'nin reaction in the vascular tissue, practically none
elsewhere.

R. inerme--
(i) Directly into bichronrte.
Fairly well .rrked tannin reaction in the vascular tissue,
'ractically none elsewhere.

(2) Absolute alcohol and bichrornte as above.
Slight to= _nin reaction in the vascular tissue.

(3) VTatcr and bichromate as above.
Slight tann-in reaction in vascular tissue.

Leaves gathered late in the afternoon from young, vigorous plants
exposed to sunlight plus artificial illumination. ?lants grown on water
culture:


|








-14-


R. inerme--
(1) Directly into the bichromate.
Tannin indicated by strong staining reaction in upper and lower
epidermal cells, -landular hairs, border parenchyma, a few
sc-.ttered cells in phloem, radial rows of cells in the xylem,
scattered cells in palisade and spongy parenchyma.

(2) Directly into water, kept for 24 hours, then into bichromate.
Tannin indicated by medium orange color in glandular hairs,
pale orange in a few epidermal cells and occasional palisade
cells.

(3) Directly into ethyl acetate for 24 hours, then into bichromato.
Tann in indicated by medium orange-brown coloration in scattered
phloem cells, radial rows of cells in xylem, and rather pale
orange-brovn coloration into border parenchyma.

R. petiolaro--
(1) Directly into bichromate.
Tanin indicated by deep orange-brown coloration of upper and
lower epidcIr:ial cells, in scattered subepidcrmal -,nd parenchyma
cells in region of the midrib, border parenchyma, scattered
cells in phloe: ard radial rcws* of culls in xylem, few scattered
cells in palisade parenchynra and numerous cells in spongy
parenchyma.

(2) '.ter and bichronate as above.
Light orange-brov'a color indicates tannin in scattered phloem
cells a-rd radial cells in xylem.

(3) Ethyl acetate aind bichrorkate as above.
Tan-in indicated by orange coloration in glandular hairs.
OCnarge-brown scattored cells in spongy a palisade parenchyma,
r' there deep orare-brovrn coloration in border parenchyna, in
phloem cells, and radial rows of cells in xylem.

R. il custre--
(1) Directly into the bichronato.
T--in indicated by orange-brown coloration in epidermal cells,
glaadular hairs, border parenchy a, phloemi cells, and radial
rows of cells in the xylem.

(2) Lthyl acetate ad bichrote as above.
Tamin indicated by brownish coloration in the vascular tissue.

Thse tests sho;cd that young, fully developed leaves contained
more taLiin thaa oldcr, hardeed leiaves, aIn further confirmed the im-
portance of light for optimm production of tannin. The intensity and
actu1 distribution of tL stains indicated that the solvents water,
-solute alcohol, ard ethyl acetate had reacted differently. These tests
merely confirm the diff2rucs previously poted in the chemical properties
of the tanin aggr g to previously noted for R. potiolare and R. iner-oie.








-15-


Quantitative determinations of tannin for several western ribs

In September 1927, leaves of Ribes petiolare, R. ineriie, R.
viscosissimum, and R. lacustre were collced from several reas within
the white-pinc belt of northern Idaho. These leaves were air-dricd on
wire trays and subsequently analyzed for total tannin. The following year
fresh leaf and stem material of the same four ribes sipecics was collected
and preserved in alcohol for proximate analysis.

'In 1930 a more comprehensive collection of ribs rf-terial was
made in northern Idaho, western Oregon, arld central California. Leaves,
current-season stkuis, old stems, and roots wore separately collected,
air-dried in the shade on wire screens, aid then packed in paper bags
for subsequent tannin analysis.
The official hide powder method (3, pp. 119-129) ws used for
all of the tainin analyses reported in tables 1 anjd 2. Fixable tan-Lin
was deterined by the revised Viilson-Korn r cthed (59, pp. 290-295) for
several samples from the 1928 and 1930 collections, to compare the
quantities of so-called fixable and total talnin. Tha total tannin
(official -ethod) was from 30 to 50 percent higher than the fixable
tannin (Wilson-Kcrni method) for all the sca.,plos tested. Since the lat-
ter data are incomplete they will not be Lcludod in this report.
For the collections rrdo in 1930, ...n atto2pt ws -:d to obtain
a representative sample containia, a.oo L ocual arnounts cf healthy plants
of both shade and sun forms. bhile it as realized, from' preliminary
tests'made in 1928 aA 1929, that there would be colisiderable variation
within a single species for shade a-..d sun fcrrois of that plant, the
purpose of the 1930 survey was to obtain qualititative data which could
be taken as indicative of the spOcies as a whole.

For the colloctiorn xride in 1930, detailed reports on file at
Berkeley sh weather data, and remarks on insulation, age, and vigor
of bushes, as well as notes on site and associated vegotation. These
field notes add nothing to the possible interpretations from the quanti-
tative data shown in table 1 and are therefore omitted from this report.

The results of the tannin deter:minations for the 1930 collections
and a su-mary of data for the taniin content of the leaves of the 1927,
1928 and 1930 collections of the four principal ribs of northern Idaho
are given in tables 1 and 2. In all cases the figures shown are averages
for at least two deter.irtions.









-16 -


Table l,--Seasonal variation in tannin content of leaves, stems, and
roots of several Ribes species


Date of :,
collection


Portion
of plant
analyzed -/


Tannin content /
(bone-dry basis)


Percent


ibe s petiolarei/




Do.




Do.




R. ine rme3/


Do.




Do.




00.


1i30
June 2,3,4



July 25




Aug. 29




1930
June 3, 4


June 23,24,
25,26



July 21




?ept. 1


!/L = leaves, CSS = current-season stem, OS = old stem, and R = roots.

2/F'ach figure shown is the average of two determinations.


3/Collecte. at Renf'ro Creek, Santa, Idaho.


Spe cie s


L
CSS
OS
R

L
CSS
OS
R

L
CSS
OS
R

L
CSS
OS
R

L
0SS
OS
R

L
CSS
tS
R

L
C


5.87
3.93
3.07
4.07

5.94
3.74
2.19
4.00

4.06
1.29
0.92
3.64

5.52
0.43
2.14
3.55

5.85
0.65
2.26
1.53

3.12
0.75
0.61
1.33

3.49
0.89
1.04
2.92






-17-


Table 1 (contd.)


Date of
collection


Species


Portion
o0f Q1ant


Ta-,nin content/
(bone-dry basis)


R. viscosissimiUm3/




Do.




Do.


R. lacustreo/




Do.




Do.




R. nevadense4/


Do.


Do.


1.930
July 2


July 25




Sept. 1


1930
June 26,27,
28


July 23




Sept. 1




1930
June 10


July 22,23


Sept. 9


_ Collected at South For]z of Stanislus River below w Strawberry, Stanislaus
N. F., Calif.


L
OF,
0SS

R

L
CSS
0S
1)
R

T

IS



L
CS6




L
CSS
OS
R

L


R

T,
(-
OS
B


134











0S


L
CSS
OS
R


Po rcert

11.30
8.23
2.12
7.0

9.75
5.70
3.12
5.61

8.79
3.51
2.01
4.16

16.70
5.74
1.61
4.85

17.95
3.74
1.54
4.28

18.00
1.,..-3
1.21
4.04

12.10
5.22
4.67
9.20

12.72
8.78
3.96
7.66


11.29
6.93
5.22
4.43





- 1 11 -


Table 41 (contd.)


Date of
collection


Fcrtion
of plant
analyzed/


Tannin content!/
(bone-dry basis)


Percent


R. roezli2!




D 0.




Do.


1930
June ii



July 16, 17




.;ept. i


racteusu -/ 19.0
AVV. 18




. erythrocarpumT7/ 1i30
,ur. 20



R. lacustre 5
July 22



Do. Sept. 6





Iui um1928
Jun~e i1[{


L
CS



L
CS2

R

L
Czi
OS


T


R


12.71
4.04
2.47
4.48

12.80
6.85
4.47
9.22

13.58
6.15
4.32
7.71

5.11
2.64
2.29
8.27

16.95

4.76
7.50

14.93
4.13
0.72
4.58

15.21
1.27
1.02
1.92


4.70


8.80


ry :'0


Collected u I t Itill r,:ek .xj,
;olIectel t AcIi Jreek, toe 1( ivor -. I F.
The amount of cArr t-seas readily collected,


L
OSS
R


oecies


iZ. a p[,:~)







- 19 -


Table 2.--Tannin content of te leaves of four principal Ribes soecies
of northern liaho


Date of collection


NLn)o r
of
collec-
tions


I method of
prese rvir :
field
specimens


Tannin
in leave s /
(hbrne-dr-f
basis)


1)t rent


R. petiolare


Do.

Do.


R. iPe rmo

Do.


Do.


Sept. 1927


i av 1928

June 4, July 25,
Aug. 29, 1.930


ScrIt. l~2~

~~av 1928


June 3,23; July 21;
Sept. 1, 1930


R. viscosissimun Sept. 1927


Do.

Do.


R. lacustre


Do.

Do.


I'a y 1928

Jul. *:, 25 ;
Sept. 1, 1930


Sept. 1927


lay 1928

June 26, July 23,
Sept. 1, 1930


Air dried

In alcohol


Air dried

Air dried

In alcohol


Air dried

Air dried

In alcohol


Air dried

Air dried

In alcohol


Air dried


L/ Tannin figures shown for the 1927 and 1928 collections are averages
of duplicate tests; data for the 1930 ruterials are seasonal averages taken
front table 1.


Species


1.6


4.9


5.29


3.4


4.50

11.9

9.2


9.95

13.8

11.2


17.55








-20-


Data in table 1 shcw that, with a few exceptions, the tannin con-
tent cf ribes leaf tissue, current-season stem, rect, and old stem de-
creases for the various plant parts in the order named. Over the period
of collection (June to September) no substantial variation is shown in the
tanunin ccnternt of leaves. The last collections for the Idaho and California
Ribes species were made just prior to the beginning of leaf fall when leaves
wore nature and well hardened. In no case were leaves collected early
enough to provide a correlation with the observations of Lachnund (28) re-
garding the high susceptibility of young leaves to blister-rust disease.
Gener-lly speaking, there is a slight reduction in the tannin content of
ribes leaves after midseason.

Insofar as host-parasite relationships for ribes are concerned,
the t.-min content cf tLhe leaf would be most directly involved in any
physiological reactions. Attention is therefore directed to loaf-tannin
data. If all the ribs listed in table 1 are takcn as a group, there is
no direct correlation between the quantitative tannin data and known sus-
ceptibility to blister-rust disease. Although the highly susceptible
black currants Ribcs petiolare, R. bracteosur, n*dR. nigrun arc generally
low in tannin and --o highly rosist.iit R. 7Vcustre (Oregon and Idaho) is
the highest in ta .i l, so~c of the other species are out of line. Most
noticeable is R. rr ozli (one of the most susceptible of the ribes listed
in table 1), which ontained 12 percent of tannin, R. alpinu is general-
ly cc nceded to be a resistant ribes on the order of 7. laoustre, and in
table 1 it occupies fan intermediate position in regard to ta;nmin content.
There is, hoadever, a recent report by Hahn (20) in which he shows that the
susceptibility of the dioccious R. alpine varies significantly between the
st a nate and pistillate foris. -he loaf sample of P. alPinum sent to the
writer presunbly contained m trial from both staminate and pistillate
plants. It would be interesting to compare the quantity and nature cf the
tannins in leaves taken from staminate and pistillate specimens of R.
alpinium.

Data for the four principal species of northern Idaho, as s=mmarized
in table 2, are nore c .11Iplete than those for the remaining species, and
comparative susceptibility to blister-rust disease has been most clearly
eetablishcd for those species. Mielke and coworkers (30) show that Ribes
potil(lare and R. inerrie -re much more susceptible than R. viscosissinum
a d R. lcustro. Under natural conditions, R. viscosissinun has been
more ative in spreading and intensifying tT- rust in northern Idaho than
R. inorric, but factors uther than direct susceptibility have combined to
bring this ab ut. For these four species, quantitative data for leaf
tannin shoa a go.Lral correlation with susceptibility to blister-rust
disc:so. For the ccnplete group (f ribes studied, however, the quantity
of tann:-in in loaf tissue ca mot be taken as a direct neasuroment of sus-
ceptibility to blister-rust disease.









-21-


Variations in the quantity and chemical properties of tannins in
the Ribes species described in this report indicate that the tannins
would be a profitable subject for further investigative viork on the sus-
ceptibility of ribes to Cronartium ribicola. Data from such a study
would bear on the highly important matter of the mechanism of disease re-
sistance in plants. In this field of work Ribes species and Cron'artium
ribicola would be convenient experimental material for the following
reasons: A pure stock of experimental plants can be maintained, because
ribes are readily propagated from cuttings and may be conveniently cared
for under greenhouse conditions. Detached ribes leaves may be preserved
on nutrient solutions in petri dishes for use in inoculation tests accord-
ing to the technique described by Clinton and McCormick (8). For reaction
tests the complete range of susceptibility to the rust is provided by the
highly susceptible black currant on the one hand and the immune Viking
currant on the other. The racial purity of the aeciospores used to in-
fect ribes could be maintained. Finally, a vast amount of data is al-
ready available on the susceptibility of ribes and pine under field con-
ditions, and on the histology and ecology of ribes.

Although blister rust is generally conceded to be heterothallic
(40), the question of whether biotypes in the blister-rust fungus do exist
has never been answered satisfactorily. This point has not bccn of great
importance in large-acale blister rust control work because so far it h's
not been practical to propagate and plant resistant varieties of white
pine. Nevertheless a careful technical study of the susceptibility of
ribes could not be undertaken without the assurance that leaves were being
infected by a pure strain of aeciospores. because of the difficulties that
have been experienced experimentally in preventing the urediospores from
going into telia, it would probably be necessary to maintain the purity of
the rust strains on ribes by making single spore inoculations. Such a study
also would be advisedly carried out in regions whore thu disease is already
established, and where there would be no hazard from the control standpoint
in having the work in progress.

S TJ 11' 4ARY

Evidence shows that the chemical properties of tannins and their
degradation products, their probable function in the plant, and their
action on parasitic fungi lend themselves to an explanation of the speci-
ficity exhibited in host-parasite relationships. A theory is offered which
postulates a toxic action of tannin initiated and conditioned by enzymes or
hormones secreted by the fungus. The ultimate toxicity of th< tanliiis would
appear to depend partly on the typo of phenolics and other potentially toxic
constituents formed by the reaction of host and p'-rasite, and partly on the
quantity and manner of di stribution of the tannin mass.

Previously published data shay that the susceptibility of Ribes
species to blister-rust disease must be attributed to physiological
rather than to anatomical characteristics.








- 22 -


Quantitative data for the seasonal tannin content of leaves,
current-season stems, roots, and old stems of six far western Ribes
species showed decreasin. amounts of tannin for these rlant arts
in t-e order naned. The six species included in these seasonal
analyses were: R. petiolare, R. ineine, R. 'isoosissi-u, K. lacustre,
R. nevadense, andi R. roezli. In general, there was an increase in the
snniin content of th-ese Ribes species up to the nid-nortion cf the
roin : season. Additional data are c:iven for single collections of
leaves of 1. netiolare. R. inertia, R. viscosissimrwn and R. laoustr6
(T1~ho': for leaves, steris and roots of R. bractecsmi. R. errthro-
cereuzr, an,. R. l.acustre (,.re'j.: ani for leaves of R. ni-'rum and Z.
alpine'. AltLou~> the leaves of t 'e ighly susceptible black currants
tenled to be low in tanrin, te present investic-ation showed that the
qn eatity of tannin Per se cannot be used alone to detr-,ine the
suscetibilit, e' ribes to blister-rust disease. The chorioal nature
of the tannins, narticularly the tyrpe ,-f j-rotective substances which
FiJ_'ft be fCrme1. through interaction of enz1,.es an: tannins, the
location c f t nni"-b ring cells, and the available fool supplies
near t1c noint inv, ded byv t e parasite Trust all be co-sidered.

A study of the decomposition oroduots of t'Co tnlin rass of
R. poticlare highlyh susceptible) and inormeo (moderately susceptible)
shoed the latter to contain more of the catochol tannin s, which
probably contribute to a higher specific toxicity to the blister-rust
fungus, than thoso of R. petiolare, which were pr'doninantly those-
of the gallotrnin type. Differonces wore also noted in the ratio
of a lcohcl to water-solublo tannins between R. petiol!rre on the one
hand and a group of loss susceptible ribos (R. in ,rme, 2. lacustre,
and R. viscosissimLL-) on the ot:Aor.

I icrochcical tests for tannin in eioves of Ribes potiolaro,
R. inerirc, aKd R. lacustre sho-.d that the t-=Jns wero concentrated
in opideror.al layers and around -rascul-r bundles so as to falcilitatc
a prototive action.

%oausa of thE clearly established wide viriatic' in
susce*tibilit,, different Ribes srocieo the ease7 with which a
nure stock cf ribcs plants mav be *iairta'ned un-7r reeonhouse con-
ir th., nractiobility of ....in.. sinI-le snore inocul' tions,
it is co cluled th t ribs :nd the blister-rust fungus have social
advent cs for research or tl e mechanism of disease resistance in
o 1;icnt S.










- 23 -


L~Pif I ~ T "7 )'.7T,


1i) Ionlen, Ruth 7.
i2 Cytclovical st:&iie L2 i:foztir Irr oni
Thiur LLivts b, r z"--:ci rrari2! trlt1ci r: I TI
and 7I'. Jo-r. gr. rcs. 26: 5i- P., jilus.


(2) Andcrson, 0. C.
1039. Y cvtolo i ifoctin Ij v e r
20- illus


ctz r. 2r


(3) Association of (fficial i ,,ric;itural ,: i's.
1935'. Official 'nd tntati .......o s .f ais.
illus. ashi'ntn, D. C.


F a 710 pp
... ,IP


10-28. Ul-er ia-< vorkommrl-n -,nd don ianchwiv;-- nov- -
holzzcrA6t r-
hr: ,ic n ilzen. 7uschr. Pui" uz ...... .
Jf.AnZn hchmbz 3.9: 257-L73, il37ji.

(5) 3ron ,_Tj T.,anl rris, G. >.
1 93S' A ctribA ion to tho chor is171 nd s -ihy:i(lof c e]_,l.'c


(3) Rravrn, o
19'34. ic anist of. f2t Tr-. s r it
Syvoo1. ')'c. 19: 11-3.

(7) 19-rk 1. 7., a uuii. t'
103c. a s l v':.ri,-ti;1-n "-. c-c "to spn: t: scric-,a.
Jo. -r. -.es. 5 1 1- 1 i 1. .1


Clinto,-, and .: .,c 1 C. 11.
!0I1. -Lust infce-tio. oil leavc,_ in '. tr,12his.
Bull. 260: 4,,-(j1 i us.


*.,~ ~ ~ r.


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(10)


Cook, 1. T., .d Thubc. haus, I. I.T
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(a)








- 24 -


(1i) Davidson, Ross Vi., ompboell, J. A., and Blaisdell, Dorothy J.
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- 25 -


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- 26 -


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(35) 5r ,
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(41.5)


jpnnsparan giua.


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( ;r ) -~~~ :-, (.It -4 *
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(37)








- 27 -


(47) Russel,
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(43)----
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(49) Russel,
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and Todd, John
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Alfred, and Todd, John
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(50) - -Todd, John, and !'ilson, Cecil L.
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(51) Snell, V. H.
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(53) Staknan, E. C.
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(54) Stapp, C., and Bortels, hi.
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(56) Vinson, A. 1.
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(59) Wilson, J. A., and Merrill, -. B.
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512 pp., illus. 'New York mid London.




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