Final Directed Research

Determining the Effect of Select Pythium Isolates on the Soybean Growth Performance

Victor Brenk

JANUARY 25, 2016Word count: 5,573

University of Minnesota Plant Pathology

AbstractModern agriculture has a lot to fear from a number of pathogens that adversely affect crop

quality and yield, one of the more well know species being Pythium. This pathogen’s interaction with its soybean host results in lesions on roots and delays germination of seedlings. Until

recently, it was assumed that all Pythium species were equally detrimental to soybean health, and that soybean resistance breeding should target all identified Pythium species. Our findings

dispute this assumption. In experimenting with our two hypothesized growth promoting Pythium isolates, we found strong evidence indicating that Pythium pleroticum and Pythium minus can be beneficial to soybean growth performance across all three biometrics measured to determine the

effect of their interaction: root length, aboveground shoot length, and root dry weight. Furthermore, there is evidence that these isolates prevent more damaging pathogens such as Aspergilli and Fusarium, from infesting the soybeans. Our results find that not all Pythium

isolates are detrimental as previously thought, and that some may have the potential to be utilized as biocontrol measures to protect soybeans in certain concentrations. This finding is significant

to the agricultural industry, as implementing a natural biocontrol is less disrupting to ecosystems than introducing chemical oomycete suppressors and other toxic agents.

Introduction

Pythium spp. are a widespread soil-borne organism considered to be major pathogens of a

wide range of hosts including soybean (Glycine max) and corn (Zea mays). This ubiquitous

pathogen affects all areas of the continental United States (Hendrix and Campbell 1970);

however, investigations into its impact on agriculture are still in their infancy. Until recently, the

vast number of Pythium species that are components of agricultural ecosystems and influence

their outputs was poorly understood until molecular techniques enabled accurate identification of

species. Subsequently more than 20 species of Pythium have been confirmed as pathogens on

soybean in the United States (Lievens et al. 2006). Among the species found in the North Central

states, Pythium irregulare and Pythium ultimum have been considered to be the most important

seed and seedling pathogens. Research conducted in Minnesota during 2012, 2013, and 2014

identified 28 Pythium species infecting either corn or soybean (Sprague 1950). When

pathogenicity of Pythium spp. collected in this survey was evaluated, infection with individual

isolates of several species appeared to increase plant growth and root development. An additional

observation of this research was that growth performance was influenced by temperature and the

concentration of inoculum applied (Radmer et al. 2015). This experiment seeks to reproduce

these conditions, and answer whether increased soybean growth associated with infection by

Pythium spp. occurred, and whether some Pythium isolates are mutualists of soybeans at variable

concentrations. Our objectives are: 1) to determine the effect on plant development of

inoculation with Pythium pleroticum and Pythium minus, two putative mutualist Pythium

species, in contrast to inoculation with Pythium irregulare and Pythium ultimum, two species

considered to be aggressive pathogens and 2) to determine if inoculation rate influences the

outcome of the symbiosis between soybean and the four Pythium spp.

Materials and Methods

All Pythium isolates used (Table 1) in this experiment were each subjected to the same

inoculation protocol. Inoculum containing Pythium spp. was produced by infesting autoclaved,

parboiled rice with the required Pythium isolate. To prepare the inoculum for use in the

experiment, each Pythium isolate was transferred from their master plates (Table 1) onto two

corn meal agar plates per isolate and allowed to grow for a period of six days. After this growth

period, the presence of oospores was confirmed using a compound microscope, and each plate of

the first transfer series was transferred onto two new plates, creating the second transfer series

(of four plates for each isolate) for the inoculum of the rice. These plates were allowed to grow

for a period of six days until oospore presence was observed. The Pythium inoculum was then

prepared by adding 500mL of enriched long grain white rice and 310mL Di water to mushroom

culture bags. These bags sat at room temperature for a period of an hour to allow full saturation

of the rice. Each of the eight bags, containing the rice and deionized water mixture, was then

autoclaved for a 30 minute interval, and allowed to cool overnight. The bags were autoclaved

again for an hour the next day, while hand separating any rice clumping that occurred between

autoclaving. These prepared bags were allowed to cool overnight in preparation for inoculation

with the Pythium isolates chosen to be tested. Rice inoculum preparation involved subdividing of

the Pythium isolate plates into eighths, and gently placing each section into the bag, using one

full plate of a chosen species per each of the eight bags. The bags were shaken to spread the

infested agar slices, and double sealed using an impulse sealer to ensure no contaminants entered

the bag while the Pythium infested the rice. The rice bags infested with the Pythium isolates

grew out at room temperature for a period of seven days in a dark room. One bag containing

each of the four isolates was selected for vermiculite inoculation in the following experiment,

using the more heavily infested bag for each isolate.

Each Pythium isolate and rice mixture from the inoculated bags was added to clean

vermiculite in the following ratios: 0:1 (900mL vermiculite), 1:10 (90mL rice + 810mL

vermiculite), 1:20 (45mL rice + 855mL vermiculite), and 1:30 (30mL rice + 870mL

vermiculite). The concentrations were labeled using blue, green, yellow, and white labels

respectively. The four replicates of this experiment were conducted simultaneously in order to

keep variables in growing condition as uniform as possible. To prepare the seed of soybean

cultivar McCall variety for planting, the seeds were surface sterilized. The sterilization required

first submerging each seed in a bleach- sterile DI water solution (1:10) for one minute, followed

by two DI water baths consecutively, for one minute each. The seeds were examined for any

physical deformities and poor quality seeds were excluded from use in planting. Each rep

consisted of sixteen 900mL jumbo junior pots, each of which was inoculated with one of the

Pythium isolates at one of the four inoculum concentrations. A total of 64 pots represented the

four reps for this trial, where each pot contained 3 McCall sterilized seeds planted one inch

below the surface. Following planting, the pots containing the rice inoculum and vermiculite

mixture were moved to the growth chamber. Light intensity was maintained in the growth

chamber at 1500 lumens (Figure 21-23) provided by a combination of incandescent and

fluorescent lighting, set to a 14 hours on, 10 hours off cycle. The pots were watered to field

capacity, and the growth chamber temperature was maintained at 25oC, with both temperature

and light measurements recorded by a HOBO for analysis. The plants were left to grow for a

period of fourteen days, watering every other day, at which point they were removed for data

collection.

After the fourteen day growth period, pots containing the soybean plants were removed

from the growth chamber, complete plants removed from the pots, and roots cleaned of soil

using a gentle stream of water. The root lengths, measured from the highest below ground point

to the end of the tap root, as well as the aboveground shoot length, determined as the lowest

aboveground point to the apical meristem, were recorded, and each pot’s three intact plants were

photographed for reference. The roots were then separated from the rest of the plant at the soil

line using a razor, and rolled into paper towels in preparation for the root dry weight analysis.

The roots were dried in a 95oF oven for a period of three days to remove moisture. Following this

drying process, root dry weights were obtained by measuring each individual root mass using an

Accuris scale, model WE100A-120. This data was compiled along with the root and shoot length

measurements.

In analyzing the data, the ratio used to determine growth performance relative to control

plants was calculated using: inoculated value

uninoculated value x 100, with a ratio greater than 1.0 indicating a

growth factor performance above the control. The data from the calculated root length, shoot

length, and root dry weights were analyzed using SAS analysis software to generate means,

ratios, and significance values for the obtained data sets.

To ensure that the rice inoculum didn’t have an intrinsic effect on soybean growth,

another control group was prepared using the same protocols for rice production as for the

inoculation treatments, except no pathogen was allowed to infest this rice. This clean rice was

added in a 1:20 rice/vermiculite ratio to four jumbo junior pots, and seeded following established

protocol. This secondary control group was evaluated across all three biometrics as well.

Results

The variables root length, shoot length, root weight and the ratios of dry weight, shoot

length, and root length were affected significantly by the interaction of isolate with

concentration, and the main effects concentration and isolate (Table 1), with the single exception

of the variable root length, which was found not to be significant (Pr>F value >0.05) in the

isolate*concentration interaction.

Root Length

Root length was not affected significantly by the interaction of isolate with concentration

(Table 1 and Figure 7). None of the Pythium infested soybeans outperform the control group at

any of the concentrations. This data finds P. pleroticum and P. minus reducing root length half

that observed in P. irregulare and P. ultimum infested plants. Root length was significantly

affected by the main effect of isolate (Table 1 and Figure 1). When inoculated with either P.

minus or P. pleroticum, root length was 40 to 60 percent greater than root length of plants

inoculated with P. irregulare and P. ultimum. We find that P. irregulare and P. ultimum reduced

soybean root growth to less than 50% of the root growth of P. pleroticum and P. minus

inoculated soybeans. P. pleroticum had the highest average root length of the two putative

mutualists, outperforming P. minus by 4.8%. The effect of concentration on root length was

highly significant (Table 1 and Figure 4). Inoculation with the Pythium isolates reduced root

length by 57% or more at all concentrations (Figure 4). The response of root length does not

appear to be correlated with inoculum concentration since root length was 30% greater when

inoculated at a 1:20 ratio than root length at either a higher or lower concentration (1:10 and

1:30).

Shoot Length

Shoot length was significantly influenced by the interaction of isolate with inoculum

concentration and the main effects, isolate and inoculum concentration (Table 1). Shoot length

was reduced 60% and 80% for P. irregulare and P. ultimum respectively at all inoculation rates

when inoculated shoot lengths were compared to the shoot length of the uninoculated controls

(Figure 8). Isolate effect finds inoculation with P. ultimum or P. irregulare reduced shoot length

by 50% when compared to inoculation at all rates with P. pleroticum and P. minus (Figure 2).

Inoculation with P. ultimum is the most detrimental, reducing growth to only 38% that of either

P. pleroticum or P. minus. Our other effect, concentration, is also highly significant in its

influence on shoot length (Table 1 and Figure 5). On average, all three concentrations reduced

shoot length by approximately 48%. The 1:20 concentration is the least damaging, reducing

shoot length by 43% when compared to shoot length of the uninoculated control.

Root Dry Weight

Root dry weight is affected significantly by the interaction of inoculum concentration

with isolate and the main effects of isolate and concentration (Table 1). The isolate and

concentration interaction finds inoculation with P. pleroticum and P. minus reduced root weight

by less than 20% on average while inoculation with P. irregulare and P. ultimum results in

greater than 60% and 70% weight reduction respectively (Figure 9). Isolate effect finds P.

pleroticum and P. minus inoculation as half as damaging to root weight as P. irregulare and P.

ultimum (Figure 3). The concentration effect finds the 1:30 inoculation rate is the least damaging,

reducing root weight by 34% compared to control root weight measurements (Figure 6). This

concentration effect also finds the 1:20 and 1:10 rate is slightly more detrimental, with root

weight reduced by 38% and 48% respectively compared to the control.

Root Length Ratio

Root length ratio score is not affected significantly by the interaction of isolate with

concentration (Table 1 and Figure 16). This data does however support P. irregulare and P.

ultimum as detrimental to root growth, with each experiencing a root length reduction of

approximately 80%. P. minus infestation in a 1:30 concentration increases root growth 8.7%

beyond control lengths, while the other two inoculation concentrations are less the 10% smaller.

P. pleroticum has a similar result, with the 1:20 concentration exceeding control benchmark

values by 3.4% (Figure 16). The concentrations for each of our putative mutualists that did not

outperform uninoculated plants are still within standard error, indicating they fail to cause

significant harm to root length even at their more damaging inoculation concentrations. The

isolate effect is significant (Table 1), and finds P. minus inoculated soybeans with the longest

root growth, followed by P. pleroticum. P. irregulare and P. ultimum respectively. Our putative

mutualists have twice the root length ratio values as our more pathogenic isolates (Figure 10).

Concentration of inoculation is also significant (Table 1), and finds that the 1:20 concentration is

the least damaging, reducing root length by 50% against controls (Figure 13).

Shoot Length Ratio

Shoot length is significantly influenced by the interaction of isolate with inoculum

concentration and the main effects, isolate and inoculum concentration (Table 1). The isolate and

concentration interaction (Figure 17) finds P. irregulare and P. ultimum as limiting shoot length

at all concentrations, failing to achieve even 50% of their control group’s growth. P. minus

strongly outperforms the control benchmark in a 1:30 inoculation, with 20% better shoot growth

against control performance. P. minus inoculated soybeans at the 1:10 and 1:30 concentration are

within standard error of control values as well. P. pleroticum have positive effects as well,

reaching control plant growth performance within reasonable error across all three inoculation

concentrations. Isolate effect shows similar results, indicating P. pleroticum and P. minus fall

slightly short (<7%) of the composite control values (Figure 11). Concentration effect finds the

1:20 is the least damaging concentration, followed by 1:30 and 1:10 respectively (Figure 14).

Root Weight Ratio

Root dry weight is also significantly influenced by the interaction of inoculum

concentration with isolate and the main effects of isolate and concentration (Table 1). The isolate

and concentration interaction (Table 18) finds P. pleroticum and P. minus inoculated plants

receiving growth promoting effects at 1:20 and 1:30 concentrations, while approaching control

values within standard error at other concentrations. This interaction shows our putative growth

promoters failing to cause significant reduction in root weight at any applied concentration to

their host plant. P. irregulare and P. ultimum inoculation is again shown to be very harmful to

root development, failing to produce even 50% of the root weight observed in control samples.

The isolate effect finds P. pleroticum and P. minus inoculation half as damaging to root weight

values as P. irregulare and P. ultimum inoculation (Figure 12). The concentration effect supports

these results, and finds the 1:30 inoculation concentration as the least harmful (Figure 15).

Rice Effect on Soybean Performance

To ensure that the controls used to determine baseline growth performance of uninfested

soybeans was valid, their measured values were measured against another control group, which

had uninfested rice added in a 1:20 concentration to the vermiculite to determine whether the rice

in the treated groups may have influenced soybean growth performance (Figure 19). The

uninfested rice control group experienced detrimental soybean growth effects, which was due to

other pathogens infesting the rice and infecting the plant. From this analysis we find using

controls grown with no rice is the better benchmark for uninfested soybean growth than those

grown with uninfested rice.

Seedling Emergence

In analyzing the effect of each isolate on emergence, Figure 20 shows our hypothesized

growth enhancers, P. pleroticum and P. minus emerging at rates close to the control varieties.

These two isolates have very little impact on final emergence scores, with neither of the three

inoculation concentrations showing a significant impact of emergence. From this Figure, it is

also clear that P. irregulare and P. ultimum infestation of soybeans significantly influenced the

emergence scores at all three points of data collection. Emergence time is greatly extended at all

three concentrations for these isolates, and final emergence scores show only a 50% emergence

relative to their respective control group.

Growth Chamber Conditions

A HOBO Environmental Data Logger (Onset Computer Co., Bourne MA) measured light

intensity and temperature in the growth chamber throughout the experiment. An average of 1200

lumens was produced, and temperature was maintained at an average of 26±10C (Appendix 1, 2,

and 3). This data confirms that the target conditions for this experiment were met, and

representative of the conditions in the Radmer et al. experiment.

Discussion

The findings of this experiment are significant since most agricultural interest in Pythium

focuses on its removal and mitigation. This strategy is illustrated in current research focusing on

indiscriminate resistance breeding to all catalogued Pythium species (Rosso et. al. 2008) under

the assumption that all isolate varieties are detrimental to plant growth. The wide differences in

pathogenicity of P. irregulare, P. ultimum, P. pleroticum and P. minus at the three inoculation

rates indicates that a reevaluation of our current approach toward breeding for resistance to

Pythium species is needed. Unless species selected for resistance phenotyping are carefully

selected, species identity confirmed, and inoculum applied at uniform and identical rates, the

results of resistance screening may be useless or at best misleading. In addition, because two of

the Pythium species, P. pleroticum and P. minus failed to significantly damage soybean and at

some inoculum concentrations promoted growth, this indicates that some isolates may be

candidates for use as biocontrol agents in agriculture.

This experiment was conducted at approximately 250C, which was identified by earlier

research (Radmer et al. 2015) as most likely to permit P. pleroticum and P. minus to have a

positive growth effect on their host soybeans. However, research by Radmer et al. also identified

other isolates that exhibit growth enhancing effects at the other two temperatures investigated

(150C and 200C). From our data, it seems likely that different temperatures create environments

suitable for different Pythium isolates, some of these isolate temperature combinations have

benign or even a growth promoting effects on soybean growth. In particular P. pleroticum and P.

minus in some inoculation concentrations promoted increase soybean growth. This indicates that

an investigation into the possibility of using select Pythium isolates as a biocontrol would require

determination of growing requirements, temperatures, inoculation rates, and soil moisture in

order to exploit their biocontrol potential.

More specifically our results confirm that P. irregulare and P. ultimum are in fact

soybean pathogens and that their effect on growth at all concentrations is fully supported. They

also exhibited this effect over the range of temperatures maintained in these experiments. These

two isolates of these two species used in this experiment killed a large number of their host

plants (Figure 20), and delayed emergence of those plants not killed outright. These isolates also

greatly reduced root length, shoot length, and root dry weight across all three inoculation

concentrations, further confirming their negative impact on soybean growth performance.

A potentially valuable follow-up to this experiment would be to determine the effect of

inoculating soybeans with both our growth promoting isolates and growth inhibiting isolates in

differing sequences of application times. If this testing found that some growth promoting

Pythium species could outcompete the established detrimental varieties, these isolates would be

very attractive candidates for further research into their potential as biocontrol agents.

Another finding is that concentration of inoculate has a significant effect on soybean

growth performance. We found that when averaged across all four isolate varieties tested, the

1:20 inoculation ratio was the least damaging; an interesting result as this is the intermediate rate.

A possible explanation for this finding is that the inclusion of both the growth promoters and

inhibitors in determining average values for each of the three biometrics produced average

values that are intermediary between the two isolate interactions. Since both P. irregulare and P.

ultimum are damaging to soybean survival and growth, the inoculation rate must have mattered

less as soybean survival was randomly determined. The two growth promoting Pythium isolates

were beneficial at some concentrations, but did not have significant detrimental effects from that

of the controls. The best growth promoter was P. minus at a 1:30 concentration, followed by P.

pleroticum in a 1:20 inoculation ratio. These two isolates each have shown potential to have a

positive effect on soybean growth performance at at least one inoculation rate.

The experiment investigating control group conditions suggests that Pythium spp. act as

biocontrols. The control treatment inoculated with rice that had no initial infestation of Pythium

suffered massive damage from other contaminant fungi (likely Aspergilli spp. and Fusarium

spp.), likely present in the growth chamber environment, while the control group grown without

rice had none of the pathogen damage. A likely cause of this is that uninfested rice is an

excellent nutrition source for other pathogens, and provided an easy source of initial

colonization. It is significant that our soybeans inoculated with Pythium spp. in this experiment

remained undamaged, as this indicates that these Pythium isolates can suppress other more

damaging pathogens if introduced first, while having limited or no effect on the host plant

applied as inoculants themselves. Further research into these findings would be very useful,

ideally involving repetition of our experimental protocol in inoculating seedlings with our benign

pathogen, but additionally introducing a secondary pathogen and observing how the soybean

plant performed across our three biometric measurements. Such an experiment could clarify

whether Pythium could be used to counter damaging pathogen varieties of other species, as some

recent research has identified in Pythium-Pythium interactions (Vallance et al. 2015).

Conclusion

Our results find that both P. irregulare and P. ultimum are detrimental to soybean growth

at all variables measured; root length, shoot length, and root dry weight, when applied at the

three concentrations used in this experiment. In contrast we observed that our putative growth

promoting isolates P. pleroticum and P. minus both enhance growth of soybean plants at selected

concentrations, and fail to do significant damage to soybean growth performance outside this

range. These two isolates did not harm the host plant significantly at any of the three

experimental inoculation rates. We also found that inoculation of soybeans with P. pleroticum

and P. minus prevented more damaging pathogens in the environment from adversely effecting

soybean growth performance. The ability of P. pleroticum and P. minus to promote soybean

growth and repress outside pathogen damage indicates that these isolates are good candidates for

further research as a biocontrol agents. Further testing is needed to confirm this effect along with

an examination of temperature as contributing variable influencing Pythium-host interaction.

Finally, the most important observation in this study is that pathogenicity differs widely among

Pythium species. The species chosen as pathogens in phenotyping experiments could elicit

widely different responses from soybean varieties included in phenotyping experiments. Careful

attention is necessary to assure the identity and purity of Pythium isolates chosen as inoculum.

References

Bart Lievens, Margreet Brouwer, Alfons C.R.C. Vanachter, Bruno P.A. Cammue, and Bart P.H.J. Thomma. 2006. Real-time PCR for detection and quantification of fungal and oomycete tomato pathogens in plant and soil samples. Plant Science. Volume 171, Issue 1, Pages 155–165.

Floyd F. Hendrix Jr. and W. A. Campbell. 1970. Distribution of Phytophthora and Pythium species in soils in the Continental United States. Canadian Journal of Botany. Pages 377-384.

Jessica Vallance, Gaétan Le Floch, Franck Déniel, Georges Barbier, C. André Lévesque, and Patrice Rey. 2015. Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere. Applied and Environmental Microbiology. Volume 81, issue 24.

Radmer, L., Anderson, G., Malvick, D.K., and Kurle, J.E. 2015. Pythium species from Minnesota soybean fields, their relative pathogenicity to soybeans and corn, and their sensitivity to seed treatment fungicides. Plant Disease (In Review).

R Sprague. 1950. Diseases of cereals and grasses in North America.

Tables and Figures

Table 1Species Name Isolate Identifier Isolation Method Location Obtained

Pythium pleroticum Kramer 1-3 Obtained from Plant Material MinnesotaPythium minus 1.38.2h Obtained from Plant Material MinnesotaPythium irregulare Kloss8 Obtained from Plant Material MinnesotaPythium ultimum Clay 1-1 Obtained from Plant Material Minnesota

Pythium isolates used as inoculum in pathogenicity study, identifiers, and sources. All isolate varieties were obtained from plant samples harvested within Minnesota.

Table 2 Variable

Source Dry Weight

Shoot Length

Root Length

Dry Weight Ratio

Shoot Length Ratio

Root Length Ratio

Rep NS NS NS NS NS NSIsolate <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0003Concentration <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001Isolate*Concentration 0.0257 0.0111 0.0688 0.0189 0.0096 0.2545

Probability values obtained in Analysis of Variance on measured and calculated variables in pathogenicity study. The dependent variables shown are those that were determined to be significant at value of P<=0.05. Not shown are the Rep*Isolate, and Rep*Concentration significance values, which were not significant. NS = Not Significant.

Figure 1

P. irregulare P. minus P. pleroticum P. ultimum0

2

4

6

8

10

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Root Length by Isolate

Isolate Species

Leng

th (c

m)

Root lengths averaged over four inoculum concentrations (1:10, 1:20, and 1:30) resulting after inoculation with four Pythium species. The control groups for each isolate were not included in the computation of these scores.

Figure 2


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8

9

Shoot Length by Isolate

Isolate Species

Leng

th (c

m)

Shoot lengths averaged over four inoculum concentrations (1:10, 1:20, and 1:30) resulting after inoculation with four Pythium species.. The control groups for each isolate were not included in the computation of these scores.

Figure 3


0.02

0.04

0.06

0.08

0.1

0.12

Dry Weight by Isolate

Isolate Species

Wei

ght (

g)

Root dry weight values resulting after inoculation with four Pythium species.averaged over three inoculum concentrations (1:10, 1:20, and 1:30). The control groups for each isolate were not included in the computation of these scores. Figure 4

1:10 1:20 1:30 control0

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Root Length by Concentration

Isolate Concentration

Leng

th (c

m)

Root length values resulting from inoculation with Pythium isolates at three different inoculum concentrations and as an untreated control. The control group has no Pythium inoculum present. The concentration series are the composite averages from across all four Pythium isolates tested.

Figure 5

1:10 1:20 1:30 control0

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Shoot Length by Concentration


Leng

th (c

m)

Shoot length values resulting from inoculation with Pythium isolates at three different inoculum concentrations and as an untreated control. The control group has no Pythium inoculum present. The concentration series are the composite averages from across all four Pythium isolates tested.

Figure 6

1:10 1:20 1:30 control0

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Dry Weight by Concentration


Wei

ght (

g)

Shoot length values resulting from inoculation with Pythium isolates at three different inoculum concentrations and as an untreated control. The control group has no Pythium inoculum present. The concentration series are the composite averages from across all four Pythium isolates tested.

Figure 7


101214161820

Average Root Length Concentration*Isolate

1:101:201:30control

Isolate Species

Leng

th (c

m)

Root lengths resulting from interaction of Pythium species and four inoculum concentrations (1:10, 1:20, 1:30, and untreated control). Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Control group variation is due to each rep having specific controls associated with that particular sample, which were subject to minor differences of environment due to edge effect and growth chamber light output differentials.

Figure 8


2

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Average Shoot Length Concentration*Isolate

1:101:201:30control

Isolate Species

Leng

th (c

m)

Shoot lengths resulting from interaction of Pythium species and four inoculum concentrations (1:10, 1:20, 1:30, and untreated control). Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Control group variation is due to each rep having specific controls associated with that particular sample, which were subject to minor differences of environment due to edge effect and growth chamber light output differentials.

Figure 9


0.02

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Average Dry Weight Concentration*Isolate

1:101:201:30control

Isolate Species

Wei

ght (

g)

Root dry weights resulting from interaction of Pythium species and four inoculum concentrations (1:10, 1:20, 1:30, and untreated control). Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Control group variation is due to each rep having specific controls associated with that particular sample, which were subject to minor differences of environment due to edge effect and growth chamber light output differentials.

Figure 10


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Root Length Ratio by Isolate

Isolate Species

Leng

th (c

m)

Ratio values calculated for root dry weight resulting from inoculation with the four Pythium

species, where the ratio was determined using: inoculated value

uninoculated value x 100, encompassing all three

infestation concentrations (1:10, 1:20, and 1:30). The control groups for each isolate were not included in the computation of these scores.

Figure 11


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Shoot Length Ratio by Isolate

Isolate Species

Leng

th (c

m)

Ratio values calculated for aboveground shoot length resulting from inoculation with the four

Pythium species, where the ratio was determined using: inoculated value

uninoculated value x 100, averaged over

three inoculum concentrations (1:10, 1:20, and 1:30). The control groups for each isolate were not included in the computation of these scores.

Figure 12


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1

1.2

Dry Weight Ratio by Isolate

Isolate Species

Wei

ght (

g)

Ratio values calculated for root dry weight resulting from inoculation with the four Pythium

species, where the ratio was determined using: inoculated value

uninoculated value x 100, encompassing all three

infestation concentrations (1:10, 1:20, and 1:30). The control groups for each isolate were not included in the computation of these scores.

Figure 13

1:10 1:20 1:30 control0

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Root Length Ratio by Concentration

Concentration

Leng

th (c

m)

Root length ratio values, resulting from inoculation with the four Pythium species at three inoculum concentrations and an untreated control determined using the formula:

inoculated valueuninoculated value x 100 to compare each inoculation concentration to the performance of the

uninoculated soybeans. The control group is the benchmark measurement, and represents 100% growth performance.

Figure 14

1:10 1:20 1:30 control0

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0.4

0.6

0.8

1

1.2

Shoot Length Ratio by Concentration

Concentration

Leng

th (c

m)

Shoot length ratios, resulting from inoculation with the four Pythium species at three inoculum

concentrations and an untreated control determined using the formula: inoculated value

uninoculated value x 100

to compare each inoculation concentration to the performance of the uninoculated soybeans. The control group is the benchmark measurement, and represents 100% growth performance.

Figure 15

1:10 1:20 1:30 control0

0.2

0.4

0.6

0.8

1

1.2

Dry Weight Ratio by Concentration

Concentration

Wei

gth

(g)

Root dry weight ratios resulting from inoculation with the four Pythium species at three inoculum concentrations and an untreated control, determined using the formula:

inoculated valueuninoculated value x 100 to compare each inoculation concentration to the performance of the

uninoculated soybeans. The control group is the benchmark measurement, and represents 100% growth performance.

Figure 16


0.2

0.4

0.6

0.8

1

1.2

Root Length Ratio by Isolate

1:101:201:30control

Isolate Species

Leng

th (c

m)

Root length ratios resulting from inoculation with the four Pythium species at three inoculum concentrations and an untreated control at four different inoculum concentrations, Ratio

determined using the formula: inoculated value

uninoculated value x 100, segregated by both isolate variety and

concentration. Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Each control group value is the 100% growth benchmark for each isolate series.

Figure 17


0.2

0.4

0.6

0.8

1

1.2

1.4

Shoot length Ratio by Isolate

1:101:201:30control

Isolate Species

Leng

th (c

m)

Shoot length ratios resulting from inoculation with the four Pythium species at three inoculum concentrations and an untreated control at four different inoculum concentrations,, Ratio

determined using the formula: inoculated value

uninoculated value x 100, segregated by both isolate variety and

concentration. Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Each control group value is the 100% growth benchmark for each isolate series.Figure 18


0.2

0.4

0.6

0.8

1

1.2

1.4

Dry Weight Ratio by Isolate

1:101:201:30control

Isolate Species

Wei

ght (

g)

Root dry weight ratios resulting from inoculation with the four Pythium species at three inoculum concentrations and an untreated control at four different inoculum concentrations,,

Ratio determined using the formula: inoculated value

uninoculated value x 100, segregated by both isolate variety

and concentration. Standard error was calculated using σm= σ/√N, where σm is standard error of the mean, σ is standard deviation of the original distribution, and N is sample size. Each control group value is the 100% growth benchmark for each isolate series, though the control values used to produce the ratios had different values as a result of minor differences in growth performance between reps.

Figure 19

root length shoot length dry weight0

5

10

15

20

25

30

35

Controls Grown with and without Rice

without rice with rice

Variables, root length, shoot length, and dry weight resulting when soybean are grown in growth media and with rice in a 1:20 inoculation ratio.

Figure 20Time Measured Concentration P. pleroticum P. minus P. irregulare P. ultimum

+ 5 days control 10/12 10/12 11/12 10/12

1:30 9/12 12/12 1/12 0/12

1:20 9/12 8/12 1/12 0/12

1:10 10/12 9/12 0/12 1/12

+10 days Concentration P. pleroticum P. minus P. irregulare P. ultimum

control 10/12 10/12 11/12 10/12

1:30 10/12 12/12 3/12 2/12

1:20 10/12 10/12 5/12 1/12

1:10 10/12 12/12 2/12 3/12

+14 days Concentration P. pleroticum P. minus P. irregulare P. ultimum

control 11/12 11/12 12/12 11/12

1:30 10/12 12/12 6/12 4/12

1:20 9/12 11/12 8/12 2/12

1:10 10/12 12/12 3/12 3/12

Emergence resulting when soybean is planted into media inoculated with four Pythium isolates at three inoculum concentrations and an untreated control. Emergence was categorized as a fully emerged cotyledon. Dead seedlings were counted as nonemerging.

Appendix 1

Data from the HOBO data logger, recording light intensity and temperature. This data is from the first five days the plants were in the growth chamber.

Appendix 2

Data from the HOBO data logger, recording light intensity and temperature. This data is from the fifth through the tenth day the plants were in the growth chamber.

Appendix 3

Data from the HOBO data logger, recording light intensity and temperature. This data is from the last four days the plants were in the growth chamber.

Final Directed Research

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