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This is to certify that the

thesis entitled
THE EFFECT OF HYPOCHLORITE WASH TREATMENTS
ON THE SUBERIZATION AND FIELD PERFORMANCE

OF CUT POTATO SEED.
presented by

Raymond Vander Zaag

has been accepted towards fulfillment
of the requirements for

Masters degree in Sc1ence

 

.2 Mew/f fix/1c...

4 Major professor

May 18, 1984

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THE EFFECT OF HYPOCHLORITE WASH TREATMENTS ON THE
SUBERIZATION AND FIELD PERFORMANCE OF CUT POTATO SEED.

BY

Raymond Vander Zaag

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1984

ABSTRACT

THE EFFECT OF HYPOCHLORITE WASH TREATMENTS ON THE
SUBERIZATION AND FIELD PERFORMANCE OF CUT POTATO SEED.

BY

Raymond Vander Zaag

Hypochlorite wash solutions are used as cut potato seed
treatments on some Michigan farms. The effect of chlorine
wash solutions on the suberization and field performance of
cut seed was investigated.

Histochemical staining showed increasing chlorine rates
caused a thicker suberin layer to form. However, increasing
chlorine rates caused a less effective water vapour and
pathogen barrier to form, as measured by weight lost during
drying and decay of tuber disks inoculated with Fusarium

sambucinum, respectively. A chemical assay of the lignin-

 

like component of the suberin layer did not show any consis-
tent or large effect of increasing chlorine rates. Washes
of 500-1000 ppm Cl did not adverSely affect suberization.

In the field, 2000 ppm Cl washes occasionally reduced
stands, but produced yields equal to the best treatment, a
captan-streptomycin dust. A 500 ppm C1 wash was usually
intermediate. Storing chlorine treated seed in various
typical on-farm environments for 2-5 days before planting

did not adversely affect seed performance.

ACKNOWLEDGEMENTS

I would like to express my sincerest appreciation to
Dr. R. W. Chase for his continuous guidance, support and
friendship during my studies here. I have learned a great
deal from his excellent example, both professionally and
personally, and I am deeply grateful.

I am greatly indebted to Dr.IL Hammerschmidt for his
advice, suggestions, reviews and friendship. I thank him and
his graduate students for sharing their lab space, equipment
and jokes. As well, appreciation is extended to Dr. M. L.
Vitosh for serving on my guidance committee.

I am very grateful to Dick Kitchen for his friendship
and help with the field experiments; he has taught me things
not found in any textbook. Thanks also to Mr. Theron Comden
for his gracious help at the research farm.

Financial support from the Michigan Potato Industry
Commission is gratefully acknowledged.

This page would not be complete without expressing my
deepest thankfulness and love to my family; to my Mom and Dad
for their love and support and the chance to grow up as I did,
to my brothers, especially Peter, for their encouragement and
love, to my cousin Pete for his advice and encouragement, and

finally to Jayne for her unfailing love and friendship.

ii

TABLE OF CONTENTS

LIST OF TABLES O O O O O O O C O O O O O O 0
LIST OF FIGURES O O O O O O O O O O O O O 0
INTRODUCTION 0 O O O O O O O O O O O O O 0 0

CHAPTER 1. LITERATURE REVIEW . . . . . .
Stand problems . . . . . . . . . . .
Causal organisms of seed-piece decay
Effects of chemical seed treatments
Wound healing . . . . . . . . . . .
Use of hypochlorite on potatoes . .

CHAPTER 2. LABORATORY EXPERIMENTS . . . . .
MATERIALS AND METHODS . . . . . . . . . .

Experiment 1. Histochemical measurement of

suberization . . . . . . . . . . .
Experiment 2. Resistance to water loss
Experiment 3. Resistance to decay . .
Experiment 4. Chemical measurement of

components . . . . . . . . . . . .'

RESULTS AND DISCUSSION . . . . . . . . .

suberin

Experiment 1. Histochemical measurement of

suberization . . . . . . . . . . .
Experiment 2. Resistance to water loss
Experiment 3. Resistance to decay . .
Experiment 4. Chemical measurement of

components . . . . . . . . . . . .

SUMMARY . . . . . . . . . . . . . . . . .

CHAPTER 3. FIELD EXPERIMENTS
MATERIALS AND METHODS .
Experiment 1a . . .
Experiment 1b . . .
Experiment 2 . . . .
RESULTS AND DISCUSSION
Experiment 1 . .. . .. . .. . .. .
Experiment 2 . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . .

LITERATURE CITED 0 O O O O O O O O O C O O 0

iii

suberin

Page

iv

15
16
17

18
21

21
24

26
31

32
32
32
36

42
42
SO
56

57

LIST OF TABLES.

Page

The effect of different wash treatments on
the thickness of suberin layer formation . . . . . 22

Seed treatments used in Field Experiment 1 . . . . 33
Timetable for field Operations performed in

Field Experiment 1, and number of days

between Operations . . . . . . . . . . . . . . . . 37

Percent stand and yield produced by seed
given various storage treatments . . . . . . . . . 52

iv

LIST OF FIGURES

Figure Page

2.1. Percent weight lost during drying by
cylinders of tuber tissue treated with
different rates of chlorine . . . . . . . . . . . 23

2.2. Decay ratings of tuber disks treated
different rates of chlorine . . . . . . . . . . . 25

2.3. Decay of tuber disks given various
treatments and suberized 4 days after
treatment . . . . . . . . . . . . . . . . . . . . 27

2.4. Rate of appearance of lignin-like material
on the cut surface of potato tubers of two
cultivars suberized at 18 and 10 degrees
Celcius . . . . . . . . . . . . . . . . . . . . . 28

3.1. Average weekly maximum and minumum temperature
and precipitation at the Montcalm Research
Farm durine the spring and early summer of
1983, and the average of the previous four
years (temperature only.) . . . . . . . . . . . . 35

3.2. Percent stand produced by Atlantic seed
(Experiment 1b) treated with one of 5 seed
treatments and planted on 3 dates . . . . . . . . 43

3.3. Plot decay rating of Atlantic seed
(Experiment 1b) treated with one of 5 seed
treatments and planted on 3 dates . . . . . . . . 44

3.4. Percent stand produced by Atlantic seed
(Experiment 1a) treated with one of 5 seed
treatments, averaged over 3 planting dates . . . . 47

3.5. Plot plant height produced by 3 varieties
treated with one of 5 seed treatments,
averaged over 3 planting dates . . . . . . . . . . 48

3.6. Total and 0.8.41 yield produced by seed
treated with one of 5 seed treatments . . . . . . 49

3.7. Specific gravity of tubers produced by seed
treated with one of 3 seed treatments . . . . . . 51

vi

Figure Page

3.8. Percent stand produced by seed treated with
one of 4 seed treatments . . . . . . . . . . . . . 53

3.9. Total and U.S.#1 yield produced by seed
treated with one of 4 seed treatments . . . . . . S4

INTRODUCT ION

Stand problems in potatoes (Solanum tubersunlIu) due to

 

seed-piece decay occur throughout North America. The occur—
ance of seed-piece decay varies greatly, depending on seed
and soil conditions. Many farmers regularly use chemical
seed-piece treatments to guard against the possibility of
seed-piece decay. Though it is not a substitute for the
prOper handling and storage of quality seed, it is consider-
ed inexpensive insurance that partially protects against
invasion by seed and soil borne microorganisms (62).

Fungicide seed treatments are usually applied as dusts
to the cut seed. The anticipated benefits of such treatment
include the control of seed-piece decay in the soil (usually
caused by Fusarium sppn)‘when adverse weather (usually cool
and wet) follows planting and slows seed-piece suberization,
and the control of seed-piece decay when cut seed must be
stored for a length of time before planting (62).

In Michigan, approximately 40,000 acres of potatoes are
grown annually, having an estimated value of 50 million
dollars. In recent years, several potato Operations have
been using sodium hypochlorite (chlorine bleach) washes to
treat cut seed. Hypochlorite is considerably cheaper than

the fungicides conventionally used as seed treatments, and

is easier and cleaner to use. However, little information
is available on the effectiveness of hypochlorite washes, or
guidelines for its use. This study was undertaken to
provide some of this information.

The objectives of this study were:

1. to determine the effect of different rates of
hypochlorite on tuber wound healing.

2. to determine what rate of chlorine is most
effective in preventing seed piece decay and producing good
stands.

3. to evaluate the effectiveness of chlorine wash
treatments compared to a conventional dust treatment.

4. to determine the effect of storing chlorine-
treated seed for various periods of time under various

environmental conditions.

CHAPTER 1. LITERATURE REVIEW

Stand problems. The reported severity of stand

 

problems varies greatly from area to area and year to year
in North America. In 1967, Cross and Ohms (14) reported
that stand reduction in eastern Idaho was due primarily to
seed-piece decay. In a four year survey of over 600 farmers
they found 18% of the fields had stands of less than 70%,
28% had stands of 70-79%, and 40% had stands of 80-89%. The
average yield for these classes was 160, 181, and 197 cwt/a
respectively. Vruggink et a1 (70) reported that the occur-
ance of even 3.5% plants infected with Erwinia reduced
yields. This was due not only to plant death, but because
some symptomless plants produce smaller tubers. In a study
of yield compensation for misses in potato crops, Hirst et
al (30) found yield decreased 0.33% (+-0.15%) for every 1.0%
of plants removed at emergence.

Other studies have minimized the importance of seed-
piece decay in causing stand reductions. James et a1 (33)
found only 2.5% of all misses were caused by rotted or
diseased seed-pieces in a 1973 survey of New Brunswick
potato fields. There was an average of 32% misses in the
fields, the majority due to missing or misplaced seed-

pieces. In Washington, Thornton (69) reported that 90% of

misses were due to lack of proper seed-piece placement.
Even though there is considerable variability in the
occurance of seed-piece decay between areas and years, the
large number of studies investigating the benefits of
chemical seed-piece treatments attests to the need to
improve stands. Miska and Nelson (48) have compiled a
bibliography of 640 papers published between 1930 and 1975
investigating various aspects of seed-piece decay.

Causal organisms 9f seed—piece decay. Seed-piece decay

 

 

is typically caused by the dry rot fungus Fusarium solani

 

'Coeruleunfl and/or Fusarium sambucinum, often in association

 

with the soft rot bacteria Erwinia carotovora var.

atroseptica (E; 9: var. atroseptica) and/or var. carotovora

 

 

(18, 29, 49, 52). Recently, Davis et al (18) have
suggested a synergistic effect between E; g;'var.

atroseptica and F; sambucinum in causing seed-piece decay

 

 

in Russet Burbank potatoes in Idaho. When these pathogens
were inoculated together, the severity of tuber rot was
significantly greater than when either was inoculated alone.
Similarily, they also interacted to reduce yield. Blackleg
symptoms, however, were observed in less than 1% of the

plants. Inoculation with E; 9: var. carotovora and E;

 

sambucinum, together or individually, did not reduce yield.

 

Several other studies have reported the control of blackleg
with fungicides (17, 50, 67) or the induction of blackleg
following inoculation with E; solani (67). Thus,

synergistic effects between Fusarium spp. and Erwinia appear

to occur. Corsini and Pavek (11) found that temperature
influenced the development of disease caused by two Fusarium

species. Fusarium sambucinum caused more disease at 10° C, F;

 

‘gz'Coeruleum'lcaused more disease at 21° C, while both
species caused a similar amount of disease 16° C. They also
reported that the cultivar Russet Burbank was very

susceptible to E; sambucinum.

 

The environmental conditions that favor development of
seed-piece decay are cool (4-6o C), wet soils, which have
low oxygen and elevated carbon dioxide concentrations, or
very dry soils. Under these conditions suberization of the
cut seed-piece is slowed, sprout emergence is delayed and
infection by the pathogens is promoted (24, 29, 45, 51).

Seed-borne inoculum appears to initiate most disease
deveIOpment.(29, 41, 52, 58) and almost all seed stocks
carry some level of Erwinia spp. (57, 58, 59, 67) and
Fusarium spp. (52, 53, 64). Thus the benefit of chemical
seed treatment depends on the degree of seed contamination.
(69), and it is important for seed producers to keep these
diseases at low levels (14, 70). However, both organisms
can overwinter in the soil (19, 29, 52, 64), and soil
insects feeding on seed tubers can cause seed infection (23).

Effects of chemical seed treatments. There are many

 

studies which have investigated the benefits of various
chemical seed-piece treatments. This review will only
summarize some of their findings, since the purpose of the

present study was not to evaluate the many chemicals

currently used as seed treatments, but to investigate the
use of hypochlorite as a seed treatment. Due to the
variable occurance of conditions favoring disease deveIOp-
ment, chemical seed treatments have had mixed results.
Stands and yields are often improved, especially when cool,
wet weather follows planting (9, 10, 15, 71), or if cut and
treated seed is held several days before planting (8, 20).
If seed is cut, treated and planted immediately into soil
with conditions favoring rapid emergence, often no benefit
is observed (15, 20, 44, 63, 71).

An overall benefit from seed treatment was found in a
1967 study of 936 eastern Idaho fields (14). Fields that
received no seed treatment (61%) yielded an average of 183
cwt/a, while treated seed produced an average of 195 cwt/a.
Several studies have found no benefit under any of the
conditions tested (21, 32).

Seed treatments are generally applied as a dust.
However, applying seed treatments as a liquid with a
mechanical fog generator has been reported to be as
effective as chemicals applied as dusts (40).

Wound healing. The formation of a suberin and wound
periderm layer over the cut surface of the seed-piece is of
prime importance. Suberin and periderm formation prevents
excess water loss from the seed-piece and prevents pathogen
entry. Both of these factors are necessary for producing
vigorous stands. Numerous studies have shown that

resistance to pathogen entry and subsequent decay increased

as suberin and wound periderm formation progressed after
wounding (5,' 35, 54, 65). O'Brien and Leach (55)
reported that the mode of active resistance of potato tubers

to E; sambucinum was the formation of a continuous suberin

 

and wound periderm layer around the infection site. 'They
found the cultivar Russet Burbank, which is susceptible to
dry rot, tended to form a suberin layer more slowly and less
uniformly than a numbered resistant line.

Numerous studies have also been conducted to determine
the Optimum conditions for wound healing; The optimum
temperature is between 20 and 30° C; the rate of healing
decreases as the temperature decreases below 20° C (5, 19,
54, 72). High relative humidity (between 70 and 100%) is
also required for rapid suberization (3, 5, 54, 72). Oxygen
levels lower than, and carbon dioxide levels higher than
atmospheric levels also tend to inhibit suberin synthesis
and wound periderm develOpment (72).

There are numerous reported methods that have been used
to measure the extent of wound healing. Often, thin
sections perpendicular to the cut surface are made and the
thickness of the stained suberin layer is measured with a
microsc0pe (5, 16, 26, 65, 72). The weight of water lost by
tuber pieces under a specific drying regime can be measured
(34, 36), as the resistance to water loss in potato tissues
is directly proportional to the amount of suberin deposited
(39). A later study reported evidence that waxes associated

with the suberin polymer and not the suberin polymer itself,

formed the major water diffusion barrier (66). Wound heal-
ing can also be measured by inoculating healed tissue with
Fusarium spores or Erwinia cells, and measuring the extent
of decay (35).

Chemical techniques have also been used to identify and
measure the compounds deposited during suberization. These
studies have shown that suberin molecules are polymers
composed of phenolic components, similar to lignin, and
aliphatic (lipid) components. Waxes are also deposited in
association with the suberin polymer (37, 38, 66L. Suberin
deposition can be measured by following the amount of
certain lipids released after depolymerization with LiA1H4
or BF3, or by the phenolic aldehydes released by alkaline
nitrobenzene oxidation (38). Kolattukudy has successfully
applied traditional lignin chemistry techniques, such as
alkaline nitrobenzene oxidation, to the study of suberin,
and found lignin-like materials were deposited in parallel
to suberin aliphatics and increases in diffusive resistance
(12, 13, 39). Thus lignin chemistry techniques can be used
to measure the lignin-like components of suberin.

One of the factors that suggested the possible useful-
ness of hypochlorite as a seed treatment was a study by
Grigg and Chase (26). They found that increasing rates of
hypochlorite to 5000 parts per million (ppm) in the wash
water promoted the development of a thicker and more uniform
protective layer on the cut surface of potatoes. Tubers

washed in water produced a protective layer with an average

thickness of 76 um after six days, while those washed in
100, 500, 1000, and 5000 ppm chlorine had layers 81, 95,
127, and 196 um thick, respectively.

The use 9: hypochlorite 93 potatoes. Sodium

 

hypochlorite is one of the most widely used compounds for
surface sterilization Of seeds and other plant tissues for
use in biochemical studies and pathogen isolation (1, 2).
Hypochlorite is also widely used as a disinfectant in the
wash water in potato packing plants (22, 26). The germicidal
efficiency and stability of hypochlorite varies with tempera-
ture, pH, concentration and catalyts, though researchers
using hypochorite often do not consider these variables (2).

The strength of hypochlorite solutions is measured as
parts per million available chlorine (Ppm available C1) or
percent available chlorine (% available Cl.) Available
chlorine is a measure of the oxidizing power of the chlorine
solution, not the actual hypochlorite concentration (2).

In addition to the study Of the use of hypochlorite in
packing plant wash water already mentioned, several other
studies involving hypochlorite and potatoes have been
reported. In New Zealand, hypochlorite has been used to
decontaminate potato tubers bearing nematode cysts (73).
10,000 ppm chlorine solutions destroyed cysts in 30-45
minutes. The rate of destruction was independent of pH
above 7.0, while the presence of soil increased the time

required for destruction. The amount of Rhizoctonia solani

 

sclerotia adhering to the tubers was also reduced, though

10

some viable sclerotia remained.

The effect of such chlorine treatments on seed potato
dormancy, emergence and yield was also studied (74). Whole,
dormant seed of the variety Ilam Hardy was treated by immer-
sion in 10,000 ppm Cl for two hours , rinsing in water and
drying. Treated seed emerged faster, produced more stems
and tubers and yielded an average 20% more that untreated
dormant seed. The length of storage before or after treat-
ment did not affect the treatment benefits. Emergence was
reduced when sprouted seed was used because the chlorine
burned the sprouts. This reduction was eliminated by treat-
ing sprouted seed 3-4 weeks before planting. When seed was
treated for longer than two hours or with higher concentra-
tions of NaOCl, emergence and yield were usually reduced.
Chlorine treatment (two hours in 10,000 ppm chlorine two
weeks before planting) did not influence emergence or yield
of three common varieties. However, the emergence of the
cultivar Rua and the yield of the cultivar Sebago were
reduced.

A second study in New Zealand by Bedi and Genet (7) did
not produce such successful results. Dormant tubers of
three main crOp varieties, Rua, Whitu and Katahdin, har-
vested 1-2 months previously, were submersed in 10,000 ppm
Cl for 1 to 3 hours and planted four months later. Sprout-
ing of Rua was not increased, emergence was decreased, and
yield was reduced by 10% compared to water dip or no treat-

ment. Treatment of the cultivar Whitu reduced yield. With

11

both cultivars, yield was further reduced by longer treat-
ments. The cultivar Katahdin was not affected by any treat-
ment. Thus there appears to be varietal difference in
response to hypochlorite treatment.

In Michigan, Grigg and Chase (25) found both pre- or
post-storage washing of whole seed tubers with chlorine
solutions improved stand counts. The varieties Russet
Burbank and Arenac showed a 10-20% increase in stand when
washed with 100-1000 ppm chlorine solutions.

Potter has reported a number of seed treatment trials
evaluating the use of hypochlorite (unpublished, reported in
the 1977-1981 Montcalm Farm Research Reports, Michigan State
University). In 1977 with the variety Monona, and in 1978
and 1979 with the variety Sebago, the hypochlorite treat-
ments (2 min. dips) resulted in stands and yields as good
(1978) or better (1977 and 1979) than no treatment, and as
good or better than other chemical treatments. All treat-
ments were applied the day of planting. In 1980 with the
variety Sebago, the hypochlorite treatment (2 min. dip in
500 ppm C1 solution) produced stands and yields better than
no treatment and equal to those produced by dust treatments
when applied the day of planting. When applied 2 or 14 days
before planting, the hypochlorite treatment reduced stand
and yield compared to other treatments. For all treatments,
stand and yield were reduced as the time between treatment
and planting was increased.

In 1981, Potter cut and treated Monona seed 14, 2 and 0

12

days before planting. For two weeks before planting, all
cut and treated or whole and untreated seed was stored at
either 4-6° C or 19-210 C at a relative humidity of 90%. As
in 1980, stand generally decreased as the time period
between cutting/treatment and planting increased. Seed
stored at the colder storage temperature produced a poorer
stand than seed stored at the warmer temperature, the
largest detrimental effect occuring at the longest storage
period. The hypochorite dip produced a stand as good as no
treatment or the captan-streptomycin treatment at the same
temperature-storage period combination. Unlike 1980, it did
not reduce stand or yield when applied 2 or 14 days before
planting.

Yields did not always correlate with stands in 1981.
There was no significant difference between any of the
hypochlorite temperature-storage period combinations. For
any particular temperature-storage period combination,
except seed stored at 19-21° C and cut/treated 0 days before
planting, the yield produced from the hypochlorite treatment
was as good as that from the captan-streptomycin treatment.
In all cases, the hypochlorite treatment was as good or
better than no treatment, and better than the water only
treatment. I

Potter‘s studies suggest that the 500 ppm Cl'
hypochorite treatment is as good as a conventional dust-
treatment in improving stands and yields over no treatment,

at least when applied immediately before planting. There

13

appears to be a possible detrimental effect of hypochorite

when applied 2 to 14 days before planting, but this may be

due to the method of application, as the hypochorite treat-
ment did not reduce stand or yield compared to a water only
dip treatment.

The usefulness of the results of these studies to the
present study are limited, however, since the hypochlorite
treatments were applied as a 2 minute dip. This is not a
practical application method that can readily be adopted in
a farm-scale production system. In addition, the 2 min.
submersion in a liquid appears to have some detrimental
effect on the seed-piece.

Several studies have reported the effectiveness of
hypochorite against bacterial pathogens, including potato
pathogens. Odlang (56) found a 90% reduction in cell popu-
lations for most organisms after a 10 second exposure to
hypochlorite solutions of 5 to 15 ppm Cl. Bacterial spores
required longer exposures to hypochlorite. Letal et al (43)
found a 5 minute exposure to a 5000 ppm hypochlorite solu-
tion completely killed all ring rot bacteria
(Corynebacterium sepedonicum) on metal, wood and burlap
surfaces. This same treatment was also 100% effective
against Erwinia carotovora on metal and burlap surfaces, but
not completely effective on wood surfaces. Lund and Wyatt

(46) inoculated tubers with E; carotovora var. atroseptica

 

 

and subsequently dipped them in hypochlorite solutions for

five min. Hypochlorite concentrations of 600 ppm Cl (pH 9.4)

14

and 2000 ppm Cl (pH 9.7) considerably reduced the amount of
bacterial rot. However, even a 30 min. dip in 1000 ppm (pH
9.4) did not completely prevent decay.

Research in India has shown that Stable Bleaching

Powder (SBP) can also effectively control E; carotovora and

 

blackleg (61). A 1000 ppm formulation of SBP inhibited the
bacteria both in gitrg and on tuber cylinders. Treatment of
inoculated seed tubers prior to planting with 1000 ppm SBP
gave good germination, disease control and yield as compared
to the comparable control. Treated, uninoculated tubers
planted in inoculated soil produced poorer results. Soil
drenching at 12.5 kg/ha gave better germination, disease
control and yield than seed treatment. Lower concentrations
of SBP were less effective. The authors report that SBP
contains about 35% chorine, but it was not clear if the
given concentrations of SBP are in parts per million (ppm)
available chlorine or ppm total product.

A comprehensive study into the potential of hypo-
chlorite solutions as a potato seed-piece treatment has
never been reported. This appears to be an area where

investigation is needed.

CHAPTER 2. LABORATORY EXPERIMENTS

The effect of chlorine on suberization was investigated
using several methods reported in the literature.
MATERIALS AND METHODS
All potato tubers used in the laboratory experiments
were of the cultivar Russet Burbank, unless otherwise noted.
Tubers were stored at 3° C since harvest and were warmed at

room temperature 1 to 7 days before use.

 

Experiment I; Histochemical measurement 2:

 

suberization The effect of chlorine on suberin deposition
was investigated using histochemical staining techniques.
Six to eight ounce tubers were cut in half along their long,
axis with a sharp knife. Each of the following treatments
was applied to 12 half-tubers:

a. 30 sec. dip in water

b. 30 sec. dip in 100 ppm available chlorine.

c. 30 sec. dip in 500 ppm av. Cl.

d. 30 sec. dip in 1000 ppm av. Cl.

e. 30 sec. dip in 5000 ppm av. Cl.
Six half-tubers were left untreated as controls.

Immediately after treatment, the excess liquid was

shaken off and the half-tubers were placed on elevated racks

in a tub containing an inch of water to provide high

15

16

humidity. The tub was covered with a sheet of plastic, and
the half-tubers were allowed to suberize at room temperature
(20-220 C). After 5 days, 10-20 um sections were made per-
pendicular to the cut surface of each half-tuber, one-third
of its length from the apical end inside the vascular ring,
using a Hooker rotary microtome. The sections were extract-
ed and dehydrated in methanol for several days, then stained
with 0.01% toluidine blue (28). The thickness of the
stained suberin layer was measured using an ocullar micro-
meter at 3 randomly chosen points on the section, and the
values averaged to give the mean suberin layer thickness of
that half-tuber.

A logrithmic transformation was used during analysis by
Duncanfs multiple range test. Means presented are not
transformed.

Experiment 3; Resistance to water loss. The effect of

 

 

chlorine on suberizaton, as measured by resistance to water
loss, was investigated using a weight-loss method (34, 36).
Six cylinders of tuber tissue, 8.5 mm in diameter and
extending 25 mm into the center of the tuber, were cut from
the midsection of each tuber, avoiding eyes, using a cork
borer. Sufficiently large tubers were used to avoid
including the vascular ring of the opposite side of the
tuber. Each cylinder was given one of six treatments:

a. no treatment

b. 10 sec. dip in distilled water

c. 10 sec. dip in 200 ppm available chlorine.

17

d. 10 sec. dip in 600 ppm av. Cl.
e. 10 sec. dip in 1800 ppm av. Cl.
f. 10 sec. dip in 5400 ppm av. Cl.

Immediately after treatment, excess liquid was shaken
off and the cylinders were placed on elevated racks in a tub
containing an inch of water to provide high humidity. The
tub was covered with plastic, and the cylinders were suber-
ized at room temperature (20-22o CL. They were then weighed
before and after drying for 3 hours at 36-38° C in a con-
vection oven, and the percent weight lost during drying was
calculated.

The experiment was repeated 3 times, twice with 5 days
Of suberization and once with 11 days. Each experiment was
arranged as a randomized complete block, with tubers as
blocks and 8 replications. .An arcsin transformation was
used during analysis by Duncanfls multiple range test.i Means

presented are not transformed.

Experiment 3; Resistance tquecayg The effect of

 

 

chlorine on suberization, as measured by the ability of
chlorine-treated tuber tissue to resist invasion by the dry

rot fungus, Fusarium sambucinum, was investigated. Six

 

disks of tuber tissue (15 mm in diameter and 4 mm thick)
were cut from the center portionlof each tuber using a cork
borer, avoiding the vascular ring. Each disk was given one
of six treatments used previously (see Experiment 2).
Immediately after treatment, excess liquid was shaken

off and the 6 disks from the same tuber were placed together

18

in an inverted Petri plate containing c. 20 ml of 1% water
agar (34, 47). The water agar provided high humidity.
After two hours, 1 day, 2 days, 4 days or 8 days of suber-
ization at room temperature (20-22° C), 0.1 ml of distilled
water containing 2.5*104 spores per m1 of Fusarium

sambucinum was placed on each disk in 8 plates.

 

Spore suspensions were prepared from 6-10 day cultures
by flooding the culture surfaces with distilled water,
dislodging the spores with a glass rod, and filtering the
suspension through 4 layers of sterile cheesecloth. Final
spore concentration was adjusted using a hymacytometer.

After 6 days at room temperature, the disks were rated
for decay, using a 1-5 scale (1=0-5% of the disk volume
decayed, 2=5-25% of the disk decayed, 3=25-50% of the disk
decayed, 4=50-75% of the disk decayed, and 5=75-100% of the
disk decayed).

Each suberization period was arranged as a randomized
complete block with tubers and petri plates as blocks and 8
replications. The experiment was repeated twice, with a
seventh treatment (1 sec. dip in 5400 ppm available ClJ
added the second time, to determine if the time of exposure
to chlorine affected suberization.

NO statistical analysis was performed because of the
non-homogeneous variance in the data, and the clear trends
in the results.

Experiment _4_._ Chemical measurement 9_f_ suberin

 

 

components. The effect of chlorine on suberization was

19

measured using a chemical assay of suberin components.
Tubers of the cultivars Atlantic and Russet Burbank were cut
in half along their longitudinal axis with a sharp knife.
One half of each tuber was given one of the following
treatments:

a. no treatment

b. 1 sec. dip in distilled water

c. 1 sec. dip in 150 ppm available chlorine.

d. 1 sec. dip in 300 ppm av. Cl.

e. 1 sec. dip in 600 ppm av. Cl.

f. 1 sec. dip in 1200 ppm av. Cl.

A 1 second dip was used since this more accurately reflects
exposure times occuring on farms that use spray nozzles to
apply chlorine wash treatments.

Immediately after dipping, a plug of tissue (13 mm in
diameter) was cut from a random location inside the vascular
ring using a cork borer. The top 0.5 mm of the plug was
removed with a razor blade and the slice was placed in
methanol. The half-tubers were placed in a pan lined with
wet paper towel, covered with aluminum foil, and allowed to
suberize in a growth chamber at 18° C or 10° C. Water was
added as needed to keep the paper towel wet. Relative
humidity in the pan, measured using a fan operated pyschro-
meter, was approx. 70-80% at the higher temperature and 80-
85% at the lower temperature.

With the half-tubers suberized at 18° C, plugs of

tissue were again cut from inside the vascular ring 1 day, 2

20

days, 4 days, 8 days and 16 days (cv. Atlantic only) after
treatment. The top 0.5 mm of the plug was removed with a
razor blade and the slice placed in methanol. The same
procedure was carried out 1 day, 2 days, 4 days, 7 days and
11 days after treatment with the half-tubers suberized at
10° c.

The slices were extracted for at least 72 hours in 3
changes of methanol. After extraction, the slices were air
dryed and then assayed for lignin-like compounds using a
thioglycolic acid procedure (27). Each slice was placed in
a test tube and 0.25 ml of thioglycolic acid (Sigma Chemical
Co., St. Louis, MO.) and 2.5 ml of 2N HCl added. The tubes
were capped with glass marbles or a glass plate and heated
at 95° C for 4 hours. After cooling, the liquid was dis-
carded, the slices were washed in 5 ml of water, and the
water wash was discarded. The slices were incubated in 2.5
ml of 0.5M NaOH for 16-18 hours in order to solubilize the
lignin thioglycolate. The liquid extract was collected into
15 ml conical centrifuge tubes, the slice washed with 2.5 ml
of water, and the water wash was combined with the liquid
extract. After adding 0.5 ml of concentrated HCl, the
acidified solution was held at 4 C for 3.5-4 hours to allow
ligninthioglycolic acid precipitation. The precipitated
ligninthioglycolic acid was collected by centrifugation
(1000xg, 5 mind. The pellet was washed twice by resuspen-
sion and centrifugation in 0.1N HCl (2.0 ml per wash). The

final pellet was dissolved in 2.5 ml (or multiples thereof,

21

depending on the amount of ligninthioglycolate present) of
0.5M NaOH. The final solution was centrifuged (1000xg, 3
flux») to remove any insoluble material, and the absorbance
of the solution at 280 nm was measured.

Each suberization period was analysed as a separate
experiment using Duncanfs multiple range test, and the
response to increasing chlorine rate was analysed using
orthogonal polynomials.

The thioglycolic acid technique was also applied to two
treatments from Experiment 3. The tOp 0.5 mm of tuber
disks treated with 200 ppm C1 or 1800 ppm Cl and suberized
for 14 days (8 days before inoculation, and 6 days after
inoculation-no decay was observed) was removed using a
potato peeler, and assayed for lignin-like compound using
the above technique. IEight disks of each treatment were

used. Results were analysed using a t-test.

RESULTS AND DISCUSSION

Experiment I; Histochemical measurement 2f

 

 

suberization. Increasing rates of chlorine produced a

 

thicker suberin layer (Table 2.1). 'This is in agreement
with the results of a previous study by Grigg and Chase (26).

Experiment 2;’ Resistance 29 water loss. Tissue

 

 

 

cylinders treated with increasing chlorine rates lost more
weight (water) during drying than those treated with water
or low rates of chlorine (Figure 24”. This indicates that

high chlorine rates cause a less effective water vapor

22

Table 2.1. The effect of different wash treatments on the
thickness of suberin layer formation.

 

 

Treatmenta Suberin layer thicknessb
(um)

no treatment 82 aC

water 81 a

100 ppm Cl 78 a

500 ppm c1 94 b

1000 ppm Cl 96 b

5000 ppm c1 167 c

 

aThirty second dip.

bAfter 5 days of suberization at 20-22° C. Means are the
average of 12 tubers (except no treatment, which is the
average of 6 tubersJ The value for each tuber was the
average of the thickness of the stained suberin layer at 3
random points on a section taken from inside the vascular
ring, one-third of its length from the apical end.

CMeans followed by the same letter are not significantly
different at the 5% level using Duncan's multiple range test.

23

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBERIZED 5 DAYS c
30 '-—-iq
20> b _b_ .
a
E l . T a “—1
-1 IO —‘ .
.—
1: .
2
u:
3 0
,\° SUBERIZED II DAYS
30 " d .4
' C
20 . ‘--—1
b
._____ b
‘F—q
a a
'0 T—-—w ‘F— .
O NO HATER 200 600 I800 5400
T MT PP") PPm PP'“ PPm
Cl CI Cl Cl
TREATMENT

Figure 2.1. Percent weight lost during drying
by cylinders tuber tissue treated with different
rates of chlorine. Cylinders were suberized 5
or 11 days before drying. Treatments with the
same letter are not significantly different at
the 5% level using Duncan's multiple range test.

24

barrier (suberin layer) to form, possibly due to lack of wax
synthesis. Waxes are the vapor barrier in the suberin layer
(66). The retardation was evident even after 11 days of
suberization (Figure 2.1). However, Grigg and Chase (26)
found post-harvest washing of whole tubers with 100 to 1000
ppm Cl wash solutions did not cause any significant differ-
ence in weight lost during 4 months of storage, compared to
untreated tubers.

Untreated tissue lost more weight than water or 200 ppm
Cl treated tissue. There was no significant difference
between the water and 200 ppm Cl treatment (and the 600 ppm
Cl treatment after 5 days of suberization). This suggests
any beneficial effect of treatment compared to no treatment
was due to the washing effect of the water, and not the
chlorine present in the washes.

Experiment 3;_ Decay resistance. With disks suberized
2 hours after treatment, increasing rates of chlorine
allowed more Fusarium decay than 200 ppm Cl, water or no
treatment, which allowed only slight decay (Figure 2.2).
As the suberization period increased, the amount of decay
decreased and the difference between the high and low
chlorine rates became less. After 8 days of suberization,
essentially no decay was observed amoung the treatments,
except for disks treated with the highest chlorine rate
(5400 ppm ClJ Unlike the inhibition of water-vapor barrier
formation, the inhibition of pathogen barrier formation by

chlorine was not long lasting.

25

 

SUBERIZED 2 HOURS I——‘ T

I‘M—WITH l

’ SUBERIZED l DAY

I I
fimlfiflfl

. SUBERIZED 2 DAYS

-h30hb(fl

 

 

 

 

 

—NU-§U’

 

 

 

 

—N0‘-bUi

SUBERIZED 4 DAYS

 

DECAY RATING

—~u¢m

' SUBERIZED 8 DAYS *

 

 

 

. 4
NO WATER zoo soo l800 5400

 

TMT PP!“ PP'“ PPm PPm
cu CI cu cu
TREATMENT

Figure 2.2. Decay ratings of tuber disks
treated with different rates of chlorine.
After suberization, 0.1 ml of water contain-
ing 2.5x10 macroconidia per m1 of Fusarium
sambucinum was placed on each disk. 1=0-5%
decay, 2=5-25% decay, 3=25-50% decay, 4:50
-75% decay, and 5=75-100% decay.

26

There was no significant difference at any suberization
period in the decay rating of disks dipped for 10 seconds or
1 second in 5400 ppm Cl (data not shown). Grigg and Chase
(26) found no difference in the effect of 15 second to 2
minute dips in chlorine (rates of 100 to 1000 ppm Cl) on
tuber blackening.

Observation of the disks after several days of suberi-
zation showed browning of the disks treated with higher
chlorine rates, and some browning of the untreated disks.
Disks dipped in water or low chlorine rates remained light
yellow. With disks suberized for 4 or 8 days before inocu-
lation, 6 days after inoculation the drop of water contain-
ing Fusarium spores remained beaded on the surface of disks
treated with water or low chlorine rates (Figure 2d”.

Disks treated with high chlorine rates or not treated did
not have the water drOp remain beaded on their surface.
These observations suggest high chlorine rates adversely
affect some hydrophobic property or compounds that are part
of the suberin layer.

Experiment & Chemical measurement 93 suberin

 

 

components. The thioglycolic acid assay was originally

 

develOped to measure lignin deposition in disease resistance
studies (27). Since some of the components of suberin are
similar to lignin (38) , an attempt was made to use this
assay to measure suberization.

The rate of appearance of lignin-like compounds after

cutting (averaged over wash treatments) (Figure 2.4) was

27

 

Figure 2.3. Decay of tuber disks given various
treatments and suberized 4 days after treatment.
Disks were inoculated with 0.1 ml of water

containing 2.5x10 macroconidia per ml of
Fusariunlsambucinum after suberization and
Incubated at room temperature for 6 days.

28

 

 

 

 

 

/
I.8 F — RUSSET BURBANK /
--- ATLANTIC . [I
/
/
LG - [I
p
/
1.4 » [I
E I l8°C
‘ I
o /
8 I.2- I
I
{3’ /
z I
35 Lo- /
a: /
0 I .
I3 - I
I
l
I
.6 _ I
I! 2°
. /
’ /
4 / //
I
I ‘ x/
/ ,’
2 I ,5
. I /
I "/'/
,3 /'
0 ~11..- 1 . J . a n L n 1
I 2 4 7 8 ll

SUBERIZATION PERIOD (DAYS)

Figure 2.4. Rate of appearance of lignin-like
material on the cut surface of potato tubers of
two cultivars suberized at 10 and 18° C. Results
are the average of all wash treatments.

29

consistent with published reports (12, 66). Also con-

sistent with reported results were the Observations that
lignin-like material appeared faster at 18° C than at 100 C (5,
19, 54, 72), and the cultivar Russet Burbank suberized faster
than the cultivar Atlantic (42, Hammerschmidt, personnal
communicationh. The consistency of these findings with other
researcher‘s results confirm that this procedure is likely
valid for measuring suberin components.

The effect of the wash treatments on the amount of
lignin-like material that was deposited was highly variable,
with an average coefficient of variability of 43%. There
were no significant overall differences (data not shown).
Occasional significant differences between individual treat-
ments and occasional significant trends were observed:
however, they were not consistent between suberization
periods, suberization temperatures and varieties. The large
variability was probably due to tuber to tuber variations.
Several studies have shown considerable differences in the
healing ability of different tubers of the same variety from
the same field (4, 72). The procedure of taking disks of
suberized tissue randomly from the inside of the vascular
ring should not have caused the large variability, since no
differences in the rate of healing have been found between
areas within the vascular ring (4, 60).

There was no significant difference in the amount of
lignin—like material in disks (from Experiment 3) treated

with 200 ppm C1 or 1800 ppm Cl. Disks treated with the

30

higher chlorine rate had 95% as much lignin-like material as
those treated with the lower chlorine rate. There was much
less variability in the amount of lignin-like material in
disks from Experiment 3: the average coefficient of
variability was 8.4%. In Experiment 3, the tissue disks
were all taken from the center of the tuber, and the experi-
ment was randomized with tubers as blocks. This is further
evidence that the large variability in Experiment 4 was due
to tuber to tuber differences.

These results suggest that the occasional significant
differences observed in Experiment 4 are due to the large
variability in its results, and suggests no significant
differences in suberin deposition due to chlorine treatment.
Further experimentation with greater control of between
tuber variability is needed to provide a definite answer to
this question.

The two physiological measures of wound healing are
undoubtably the most meaningful in terms of the real life
effect of chlorine on cut seed potatoes. Prevention of
water loss and pathogen entry are the two vital functions of
the suberin layer. Contrary to the results of the anatomical
and chemical techniques used, which indicated chlorine
increased or had no effect on suberin layer formation, the
two physiological methods used showed that treatment with
increasing chlorine rates cause a less effective water vapor
and pathogen barrier to form. This apparent contradiction

may be explained by the results of Kolatakudy and coworkers

31

(66), who found that waxes associated with the suberin
polymer, and not suberin itself, form the major water diffu-
sion barrier. These studies support their findings, suggest
chlorine inhibits wax deposition, and suggest waxes may also
play a role in preventing pathogen entry. However, the
effect of chlorine on these two functions is not identical,
as the detrimental effect of chlorine on resistance to water
loss appeared to be long lasting, while its effect on resis-
tance to decay was not. The chlorine treated disks in
Experiment 3 did become resistant to pathogen entry in
parallel to the appearance of lignin-like compounds

observed in Experiment #4. Possibly waxes are important in

preventing pathogen entry until a suberin layer forms.

SUMMARY.

Laboratory experiments indicated increasing chlorine
rates caused a less effective water vapor and pathogen
barrier to form. Anatomical and chemical measurements
showed increasing chlorine rates increased or had no effect
on the amount of suberin deposited. This indirectly
supports previous findings that waxes are the primary water
barrier in the suberin layer and suggests they are also
important in preventing pathogen entry and are inhibited by
chlorine. Washes of 500 ppm Cl, as might be used by

farmers, did not adversely affect suberization.

CHAPTER 3. FIELD EXPERIMENTS

During the 1983 growing season, two field experiments
were conducted to evaluate the effect of hypochlorite

(chlorine) wash solutions on cut potato seed performance.

MATERIALS AND METHODS.
The experiments were performed at the Montcalm Research
Farm, Entrican, MI, on a McBride sandy loam soil.

Experiment 1a. This experiment evaluated the effect of

 

several chlorine wash treatments on 3 varieties of potatoes
over a range of spring soil and weather conditions.

On three dates, cut seed of three varieties was given
one of 8 seed treatments 2-4 hours before planting.
Certified Atlantic seed from New Brunswick, Canada,
Foundation Russet Burbank seed from Prince Edward Island,
Canada, and Certified Monona seed from Michigan were used.
Seed was obtained in mid-April and stored at 3° C; Thirteen
days before planting, the seed was moved to 18° C. Seed had
13-25 mm sprouts at the time of treatment. Seed was hand
cut to 42-56 gram size. The 8 seed treatments are listed in
Table 3.1. ~

All treatments (except the captan-streptomycin treat-

ment) were applied to the cut seed as a liquid spray

32

33

Table 3.1 Seed treatments used in Field Experiment 1.

 

1. no treatment inoculated with Fusarium macroconidiaa
2. water ” ' ' '

3. 500 ppm chlorineb ' ' ' '

4. 2000 ppm chlorine ' " ” ”

5 captan-streptomycinC " " " "

6. water not inoculated

7. 500 ppm chlorine " '

8. 2000 ppm chlorine ' '

 

aWhole tubers were inoculated approx. 18 hrs. before treat-
ment by submersion for 2-3 min. in water containing 1500
spores/ml of Fusarium sambucinum. Spore suspensions were
obtained by washing 5-7 day old culture plates with distilled
water followed by filtering through 4 layers of cheese-cloth.
Spore concentrations was determined using a hemacytometer.

 

bThe NaOCl source was 5.25% household bleach. Available
chlorine was determined by the titration method of Hoffman et al
(31). However, by comparing this method with those of
commercial test kits obtained later (Taylor Chemicals, 31
Loveton Circle, Sparks, MD. 21152), an error was found. Each ml
of arsenite solution required to neutralize 5 ml of chlorine
solution equals 2 g/l available chlorine, and not 1 g/l.

CCaptan-streptomycin dust (1.0 kg/quintal seed) containing
7.5% captan and .01% streptomycin by weight. Hopkins Ag.
Chemical, Box 584, Madison, WI. 53701.

34

solution using a centrifigal pump and flat-fan nozzles as
the cut seed passed over a steep decline on a chain
conveyor. (This operation caused the seed-piece to roll,
providing complete surface coverage») The spray solution
was applied at a rate that wetted the seed-pieces just past
dripping (approximately 1.5 l/quintal.) The captan-
streptomycin was applied by shaking the dust and seed-pieces
together in a plastic bag.

The experimental design was a 3 factor factorial
randomized complete block, with one split and 5 replica-
tions. Planting date was the whole plot factor, and variety
and seed treatment the subplot factors. Each subplot
consisted of one 1.83 m row; rows were 0.86 m apart; and
each whole plot consisted of 4 15.3 m rows, each with 6
subplots in it.

Eight seed-pieces were hand planted 23 cm apart in each
subplot. Plots were separated within each row by 1 or 2
hills of a red-skinned variety, and one red-skinned hill was
planted at the end of each row. Planting dates (May 4, May
16 and June 1) were chosen in an attempt to have the treat-
ments undergo typical early, midseason and late spring
weather and soil conditions (See Figure 3.1 for weather
conditions during this periodJ Seed was covered to a depth
of 10-13 cm within a half hour of planting. Fertilization
was as follows: 280 kg/ha of 0-0-60 at spring plowdown, 560
kg/ha of 20-10-10 banded with the planter (which Opened the

rows), and 84 kg/ha of 46-0-0 sidedressed twice during the

3S

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

30 "“4 YEAR AVERAGE
E3 ‘-'I983
”25»
DJ
(I
.320
.4
a:
3315*
2
Lu
F- (C) .
5 .
o- 1
18.5.
-5 t . 65
I P-
4E-
, e
8
12c:
‘l ‘ a
.— o

3 I0 I724I a I52229 s {2 I826 3 IO
APRIL MAY JUNE JULY

Figure 3.1. Average weekly maximum and minimum temper-
atures and precipitation at the Montcalm Research Farm
during the spring and early summmer of 1983, and the average
of the previous four years (temperature onlyJ

36

growing season. Aldicarb was also applied at planting at
3.36 kg a.i. per hectare. The previous crop was alfalfa.
The plots were sprayed with herbicides, insecticides and
fungicides, and irrigated, in accordance with normal potato
production practices.

Five to six weeks after planting (see Table:3.2), plots
were evaluated for stand and vigor. Plot plant vigor was
determined by measuring the above-ground plant height of
each hill in a plot and summing. The number of missing
hills was also determined. All missing hills were subse-
quently dug up to determine if the seed-piece was decayed.
If the seed-piece was not decayed and appeared blind, or had
produced little tubers, it was not counted. Percent stand
was calculated from the number of missing hills.

Plots were harvested 125-126 days after planting. A
vine desicant was not applied. At harvest, all the culti-
vars were close to or at maturity, except for the Russet
Burbank cultivar at the first two harvest dates. Yield Of
under 51 mm, 51-83 mm, over 83 mm and No. 2 tube‘rs was
determined for each plot. From these, total yield, UJL#1
yield, and the percent of total yield in each category was
calculated. For three seed treatments (dry, 500 ppm and
2000 ppm inoculated), the specific gravity was also

determined.

Experiment lb. .At each planting date, a separate

 

planting of only the Atlantic treatments was made. These

plots were not allowed to grow to maturity: instead the

37

Table 3.2 Timetable for field Operations performed in Field
Experiment 1, and number of days between Operations.

 

Planting Date

Rating Date

Harvest Date

 

Exp. 1a May 4
May 16
June 1

Exp. 1b May 4
May 16
June 1

June
June
July

June
June
July

16 (43la
27 (42)
6 (35)

16 (43)
27,28 (42,43)
6 (35)

Sept 7 (126)b
Sept 19 (126)
Oct 4 (125)

June 20,22 (47,49)
June 29,30 (44,45)
July 8 (37)

 

aNumber of days between planting and rating.

bNumber of days between planting and harvest.

38

plots were ”harvested” after approx. 6 weeks and the seed-
pieces rated for decay. The experimental design was a 2
factor factorial arranged as a randomized complete block,
with one split and four replications. Planting date was the
whole plot factor, and seed treatment the subplot factor.
Each subplot consisted of one 3.7 m row; rows were 0.86 m
apart: and each whole plot consisted of 2 15.3 m rows, each
containing 4 subplots.

Fourteen seed-pieces were hand planted 25 cm apart in
each subplot, and covered with soil to a depth of 10-13 cm
within one half hour. Five to six and one half weeks after
planting (see Table 3.2), cumulative plot height was deter-
mined. Several days later, the plots were “harvested”.
Total plot vine fresh weight was determined by cutting off
all vines at the soil surface and immediately weighing.
Each hill was dug up, and the seed-piece was rated for
decay. The rating scale used was: 1=0-5% of seed-piece
decayed, 2=5—20% decay, 3=20-60% decay, and 4=60-100% decay.
Individual ratings were summed to Obtain a plot decay
rating. The total number of stems per hill was also
recorded, and from this the average number of stems per
emerged hill was calculated.

Data was analysed using Duncan's multiple range test
and a planned comparison between the 3 inoculated and 3
corresponding non-inoculated treatments. When data con-
sisted of small whole numbers, such as for decay ratings or

number of missing hills, a square root transformation was

39

used to increase variance homogeneity. However, all data
presented are not transformed.

Experiment 2;. This experiment was conducted in

 

COOperation with a Montcalm County potato grower, and was
designed to evaluate the effects of a commercial chlorine
wash treatment used by that grower, as well as several post-
seed-treatment storage conditions. Up to and including the
seed cutting operation, Certified Monona seed was handled in
bulk in accordance with the grower‘s regular seed handling
practice. :Before cutting, the seed had been warmed to 10-1
13° C for approximately 1 week. At the back of the cutter,
one of four seed treatments were applied to the cut seed as
it passed over the sliver-removing rollers. The four treat-
ments were: (1) no treatment (dry), (2) water ((7
l/quintal), (3) 2400 ppm chlorine (.7 l/quintal), and (4)
captan-streptomycin dust (7.5% plus .01%, .75 kg/quintal of
seed). The two liquid treatments were applied with a single
cone nozzle over the rollers, and the dust was applied by
hand-sprinkling the dust on the seed-pieces as they passed
over the rollers. A 1.2 meter auger-conveyor behind the
rollers ensured good mixing of the treatments. Four 45 kg
burlap bags were filled with each treatment.

Nine post-seed-treatment storage treatments that
reflected a series Of possible on-farm seed—handling condi-
tions were evaluated. These treatments were:

. plant treated seed within 4 hours.
. hold seed for 2 days at 11°C with good ventilation.

. " poor ventilation.
. " ” ” ” " " ambient temp. with good vent.

ubUNH

4O

5. hold seed for 2 days at ambient temp. with good vent.

6. hold seed for 5 days at 11° C with good ventilation.

7. ' ' ' ' ' poor ventilation.
I!
II

' " ambient temp. with good vent.
I II I

8. n n u
9. " " " poor vent.

Forty 42-56 g. seed-pieces of each seed treatment were
randomly selected immediately after treatment and planted
the same day. The four bags of each seed treatment were
then randomly distributed , one to each of the four
temperature-ventilation conditions. The 11° C temperature
treatment was performed in one of the insulated storage bins
of the COOperating grower. The ambient air temmperature
treatment was performed in an uninsulated drive-in shed (the
doors were closed at nightJ The temperature in the drive-
in shed fluctuated with the diurnal temperature changes:
consecutive daily highs were 20, 20, 22, 14 and 15° C, and
daily lows were 8, 10, 12, 6 and 3° C. Temperatures at both
locations were recorded using a recording thermograph. The
poor ventilation treatment was achieved by sliding a plastic
garbage bag, which had the bottom cut open, over the outside
of the burlap bag, while the good ventilation treatment was
achieved by leaving the burlap bag uncovered.

After 2 days, 40 42-56 g. seed-pieces were taken from
the center of each bag (2/5 of the bag height down from the
top of the bag) and planted. Three days later, 40 more
seed-pieces were taken from the center of each bag (3/5 of
the bag height down from the tOp of the bag) and planted.

If decayed seed-pieces were selected, they were graded out,

and the percent rotted seed-pieces in that treatment was

41

calculated.

The experimental design was a 2 factor randomized
complete block, with one split and four replications. The
whole plot factor was storage treatment, and the subplot
factor was seed treatment. Each subplot consisted of one
3.0 m row; rows were 0.86 m apart. Each whole plot con-
sisted of two 6.0 m rows, with 2 subplots in each row.

Ten seed-pieces were hand-planted 30 cm apart in each
subplot. All plots were separated by 2 hills of a red-
skinned variety, and one red-skinned hill was planted at the
end of each row. Planting dates were May 11, 13 and 16.
Seed was covered within'a half hour of planting to a depth
of 10-13 cm. Fertilizer and aldicarb application was the
same as in Exp. 1. The previous crop was alfalfa, and
herbicide, insecticide and fungicide, and irrigation appli-
cations were made as required.

On June 28 and 29, six to seven weeks after planting,
plots were evaluated for stand and vigour, using the method
described in Experiment 1. Plots were harvested on Sept.
15, 122-127 days after planting, and yield of under 51 mm,
51-83 mm, over 83 mm and No. 2 tubers was determined for
each plot. From these, total yield and U&L#1 yield was
calculated, as well as percent of total yield in each
category.

Data was analyzed using Duncan's multiple range test.
Storage treatment means were analysed using planned compari-

sons, to allow determination of the main effects of storage

42

period, ventilation and length. When data consisted of small
whole numbers, a square root transformation was used to
increase variance homogeniety. All data presented are not

transformed.

RESULTS AND DISCUSSION

Experiment I; There were no significant differences

 

between the inoculated and non-inoculated treatments in
almost all cases in both Experiment 1a and 1b. Any signifi-
cant differences observed were not consistent. Therefore
new means, averaged over inoculation treatments, were calcu-
lated for the 3 seed treatments that were both inoculated
and non-inoculated. Duncanfls multiple range test, adjusted
for the unequally replicated means (68), was used to compare
means.

In the Atlantic planting (Exp. 1b), different responses
to seed treatment occured at each planting date. In the May
4 planting, the captan-streptomycin treatment produced a
better stand than the water, 500 ppm Cl and 2000 ppm Cl
treatments, and untreated seed produced a better stand than
the water treated seed, which had only 80% stand (Figure
2L2). The captan-streptomycin and 500 ppm C1 treatments had
a lower plot decay rating than the water and 2000 ppm Cl
treatments (Figure 3;”. Under the wet, cool soil
conditions following the May 4 planting, the captan-
streptomycin treatment was clearly the superior treatment,

and the water and 2000 ppm Cl treatments were the poorest.

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IOO ' MAY 4 a ‘
I____1
{ ab ‘ bc
90~ fl +fl+ I,__.bc I
80 I- C d
‘V I [ l ‘P 1?
IOO . MAY l6
0
Z ‘ a ‘ .
I: 90 ‘ a I---I __2_1 .__.a._1
U) r a
:3 80+ «
I- ..
4 1 II, ‘4 4 4 ‘r
Ioo~ JUNE I ‘
a a q
. a II__2__ ‘
90 L .
80 * '
b .I
I: I I I I ;——;. I.
NO WATER 500 2000 CAP-
TMT ppm ppm STREP.
CI Cl

SEED TREATMENT

Figure 3.2. Percent stand produced by Atlantic
seed (Exp. 1b) treated with one of 5 seed
treatments and planted on 3 dates. Treatments
with the same letter are not significantly
different at the 5% level using Duncan's
multiple range test.

44

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IHAY 4
40-
30 I a ‘ ‘ a ‘
I ab 6
20 » r—l"
d J J Jr ‘ J,
,9 MAY I6
.2. 40 ~
h
< . a
m ab b I ab I
3‘. 30~ ,—---p I. _ I'—
U r-
S
20~
" l
3 I, J 4,. . , J I . l, I,
n.
JUNE I a
40 " ab
T———'
C ‘ bc _b.9_.1
30-

20 r

4 J, Li in J, J, J A A,

 

 

 

 

 

 

 

 

 

 

 

 

 

NO WATER 500 2000 CAP-
TMT ppm Cl pmeI STREP.

SEED TREATMENT

Figure 3.3. Plot decay rating of Atlantic
seed (Exp. 1b) treated with one of 5 seed
treatments and planted on 3 dates. Treat-
ments with the same letter are not signif-
icantly different at the 5% level using
Duncan's multiple range test.

45

In the May 16 planting, there were no significant
differences in stand. Untreated seed and seed treated with
500 ppm Cl had a lower plot decay rating than seed treated
with captan-streptomycin.

The 2000 ppm Cl treatment produced a poorer stand than
the other treatments in the June 1 planting. It produced a
higher plot decay rating than the water, 500 ppm Cl and
captan-streptomycin treatments. Untreated seed produced a
higher plot decay rating than the water treated seed. It
appears the high chlorine treatment significantly damaged
the seed under the warm dry conditions that occured after
June 1 (see Figure 3.1).

The effect of seed treatment on plant height and vine
fresh weight was very similar to its effect on percent stand
and plot decay rating; therefore data for plot height and
vine fresh weight are not presented.

Seed treatment had no effect on the number of stems per
hill on the first two planting dates. On the third planting
date, seed treated with captan-streptomycin produced fewer
stems per hill than untreated seed. The rates of chlorine
used do not appear to effect the number of stems treated
seed-pieces produce.

In the main planting (Exp. 1a), the Russet Burbank and
Monona varieties produced excellent stands (averaging over
99%), and there was no significant seed treatment effect on
stand. Therefore, stand data for the Atlantic plots was

analyzed separately. There were no planting date x seed

46

treatment interactions. Averaged over the 3 planting dates,
the captan-streptomycin treatment produced a better stand
than the water and 2000 ppm Cl treatments (Figure 3d“.
These results are similar to those found in the Atlantic
planting.

There was no significant effect of the seed treatments
on plant height with the cultivar Russet Burbank. The
captan-streptomycin treatment produced a larger plant height
than the 500 ppm Cl and 2000 ppm Cl treatments with the
cultivar Monona, and a larger plant height than the water,
500 ppm Cl and 2000 ppm Cl treatments with the Atlantic
cultivar (Figure 3.5).

The effect of seed treatment on yield did not interact
significantly with the effect of variety or planting date.
The overall yields produced by the seed treatments, averaged
over varieties and planting date, are shown in Figure 3.6.
The captan-streptomycin dust treatment produced the highest
total and UALtl yield. This was better than all treatments
except the 2000 ppm Cl treatment. The 2000 ppm Cl treatment
frequently had the most detrimental effect on seed perfor-
mance (see above), but this effect is not reflected in the
yields (Figure BJH. The yield from the water-treated seed
was significantly lower than the captan-streptomycin and
2000 ppm C1 treated seed. From this experiment, it appears
that any benefit of the chlorine wash treatments is not due
to the washing effect, but rather to the chlorine in the

wash solution, since the water treatments often produced the

47

 

 

 

 

 

 

 

 

 

 

 

 

 

H30 -
a I
f
c: 96 r
z:
'< .
.u—
a) ,
23 92,. , ab ‘ rial.
’ , b _ 7.9..
88 r
A, A, p ' A, , J, 4
NO HATER 500 2000 CAP-
TMT ppm CI ppm CI STREP.

SEED TREATMENT

Figure 3.4. Percent stand produced by
Atlantic seed (Exp. 1a) treated with one
of 5 seed treatments , averaged over 3
planting dates. Treatments with the same
letter are not significantly different at

the 5% level using Duncan's multiple range

test.

 

48

 

200 P ATLANTIC .
IBOI
I60 .
I40 . p a p 1

 

 

 

'20_ ab b b b 4
I00- I II II II I J

 

200. RUSSET BURBANK

I80 ' f__‘3 a r—afi —_Ia I F—fl
I60 '
I40 *
I20 ’ *

IOO’ '
l,l W, .l .

PLOT PLANT HEIGHT (cm)

 

 

 

 

 

 

 

 

 

 

 

 

200, MONONA I

IBO'
I60> .
I40 .

Eflflflfl.i

 

 

 

 

 

wATER 500Cl 2000 CAP.-
ppmc lppm CI STREP.

SEED TREATMENT

Figure 3.5. Plot plant height produced by 3
varieties treated with one of 5 seed treat-
ments, averaged over 3 planting dates.
Treatments with the same letter are not
significantly different at the 5% level
using Duncan's multiple range test.

49

 

 

 

 

 

 

 

350 . [:1 TOTAL YIELD I
El U.S."| YIELD a
340» ' «
ab
bc ‘rfl
a _ b
< 320 c , c
E r—
.‘3 300~
<
I'-
.2.
g zeor
v a
o I I
-I 260~ «
E: ‘ ab ‘
b
240» Y b b \ 'I
220 " \ -I
J 4\ 4\ J J\J JR J J.\ , J

 

 

 

 

 

 

 

 

 

 

N0 WATER 500 2000 CAP-
TMT ppm Cl ppm Cl STREP.

SEED TREATMENT

Figure 3.6. Total and 0.8. #1 yield produced
by seed treated with one of 5 seed treatments.
Means are the average of 3 varieties and 3
planting dates. Treatments with the same letter
are not significantly different at the 5% level
using Duncan's multiple range test.

50

poorest stand and resulted in the poorest yield.

The average specific gravity increased slightly as the
chlorine concentration increased (Figure 3.7). This
increase is not likely due to any direct effect of the
chlorine treatments, but possibly to more vigorous stands
produced by the chlorine-treated seed.

Experiment gL' After storage, untreated seed stored 5

 

days at ambient air temperature with poor ventilation had
13% rotted seed-pieces by weight. Under the same storage_
conditions, seed washed with water or 2400 ppm C1, or dusted
with captan—streptomycin did not decay, suggesting all 3 of
these treatments were beneficial. The decayed seed-pieces
were graded out before planting; this should be considered
when evaluating subsequent stand and yield data. This was
the only treatment in which storage decay was observed.

Results for stand and yield are presented in Table 3.3
and Figures 3.8 and 3.9. There were no seed treatment x
storage treatment interactions. Therefore only seed treat-
ment means, averaged over storage treatment, and storage
treatment means, averaged over seed treatment, are
presented.

Effect of seed treatment. The captan-streptomycin and
chlorine wash seed treatments produced equivalent stands,»
and total and U.S.#l yields (Figure 3.8 and 3.9). Both
increased stands and yields compared to untreated seed.
There was no significant difference in stand or yield

between the water and chlorine wash treatments, suggesting

51

 

 

 

 

 

 

 

 

 

 

 

 

 

L076
>, 4
':.
‘>t l.074 ~ 3
s I . I

b a
O .
E; L072
0
m
(L
‘3 L070“-
I- d
nose-
NO TMT 500 2000

ppm Cl ppm CI
SEED TREATMENT

Figure 3.7. Specific gravity of tubers
produced by seed treated with one of 3
seed treatments. Means are the average
of 3 varieties and 3 planting dates.
Treatments with the same letter are not
significantly different at the 5% level
using Duncan's multiple range test.

52

Table 3.3. Percent stand and yield produced by seed given
various storage treatments.

 

Treatment Stand Total Yield U.S.#1 Yield
(%) (quintals/ha) (quintals/ha)

 

Storage Period

0 days 98.7 aa 318 ab 293 ab

2 days 97.6 a 309 b 284 b

5 days 98.7 a 335 a 309 a
Storage Temperature

11° c 97.0 a 324 a 300 a

ambient air temp. 98.9 a 319 a 293 a
Storage Ventilation

good 98.1 a 321 a 295 a

poor 97.8 a 321 a 299 a

 

aMeans are averaged over seed treatment and other storage
treatments. Means, within the same column and storage
treatment, followed by the same letter are not significantly
different at the 5% level.

STAND

“A

53

 

 

 

 

 

 

 

 

 

 

I00 I
99 . a ‘ a ‘ ~
ab
Ir_____
98I- -
97'- .
b
96'- .
‘ J J J, A J J,

 

N0 WATER 2400 CAP-
TMT ppm CI STREP.

SEED TREATMENT

Figure 3.8. Percent stand produced by
seed treated with one of 4 seed treatments.
Treatments with the same letter are not
significantly different at the 5% level
using Duncan's multiple range test.

 

[:1 TOTAL YIELD
360 ~ 53 US"! YIELD ‘

 

 

 

 

 

 

 

 

 

 

 

HATER 2400 CAP-
ppm Cl STREP.

SEED TREATMENT

g 340 - .
\ ' a
3 ab 1,—2—
‘< 320 - J
I-
z b
S 'r——1 a a
0 300 - ab < < -
‘3. I.
I_a_J 280 - “T .
>.
260 _ \ \ \ \ ‘
1'0
114T

Figure 3.9. Total and 0.8. #1 yield pro-
duced by seed treated with one of 4 seed
treatments. Treatments with the same
letter are not significantly different at
the 5% level using Duncan's multiple range
test.

55

that in this experiment the washing action of the water was
responsible, in part, for the beneficial effect on seed
performance of the chlorine wash treatment. There was no
significant effect of seed treatment on plant height.

The captan-streptomycin treatment caused a shift in the
tuber size distribution, producing a smaller percentage of
51 to 83 mm tubers than the other treatments and a larger
percentage of over 83 mm tubers than the untreated and
water-treated seed.

Effect of storage treatment. Percent stand, plant
height and yield produced by the nine storage treatments
were not significantly different. When the experiment was
analyzed using planned comparisons to determine main effects
of the 3 storage variables, the only significant effect
found was due to storage period (Table 3.3). Seed stored 5
days between treatment and planting produced larger total
and Uéhtl yields than seed stored 2 days. The reason for
this is not clear; the longer storage period may have
allowed the cut surfaces to heal more completely before
planting. The observation of no significant effect of
storage ventilation suggests that: 1) the plastic bags did
not severely restrict ventilation; 2) the seed carried low
levels of disease inoculum; 3) the holding period was too
short. However, the long storage period and poor ventila-
tion treatment did result in 13% seed-piece decay during
storage of untreated seed held at ambient air temperature.

A second reason caution must be used in interpreting the

56

effects of storage treatment is that the storage treatments
were not replicated, due to the large amount of seed that

would have been required.

SUMMARY.

Under some field conditions, the water and 2000 ppm C1
washes produced poorer stands than the best treatment
(usually the captan-streptomycin treatmentJ However, the
2000 ppm C1 chlorine wash treatment always resulted in
yields as good as the captan-streptomycin treatment. The
500 ppm C1 wash was usually intermediate. Storing chlorine
treated seed for up to five days under various environmental

conditions did not adversely affect seed performance.

10.

11.

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