AN EVALUATION OF STREPTOMYCES PATHOGENICITY IN TURNIP 

By 

Kerry Ann VerMeulen 

 

 

 

 

 

A THESIS 

Submitted to 

Michigan State University 

in partial fulfillment of the requirements 

for the degree of  

Plant Pathology—Master of Science 

 

2021 

 

 

 

 

 

 

ABSTRACT 

AN EVALUATION OF STREPTOMYCES PATHOGENICITY IN TURNIP 

 

By 

Kerry Ann VerMeulen 

Plant pathogenic Streptomyces spp. grow both inter- and intracellularly in host tissue 

and are good saprophytes. Thus, it is likely that these pathogens produce cell wall degrading 

enzymes in the process of infection and in saprotrophic growth. Streptomyces spp. isolated 

from scabby turnips were tested for the ability to grow and produce enzymes on media 

containing pectin, polygalacturonic acid, or cellulose as a sole carbon source. The production of 

cell wall degrading enzymes in vitro was tested. Disintegration of inoculated turnip root slices 

was used as an indicator of enzyme production. Most of the isolates with the highest tissue-

degrading tendencies were among the highest enzyme producers; however, many isolates that 

did not elicit symptoms produced high levels of enzymes, so cell wall degrading enzyme 

production is not a characteristic unique to pathogenic species. Several Streptomyces isolates 

that elicited symptoms in turnip either lacked the ability, or caused significantly fewer, 

symptoms on potato. Seedling inoculations with most Streptomyces isolates resulted in 

hypocotyl stunting in most varieties. Reduced hypocotyl lengths were also correlated with 

symptoms on root structures. Inoculated roots were stunted, lacked lateral roots, and exhibited 

necrosis as compared to controls. Purple Prince turnip showed the most resistance to 

inoculation with isolates compared to other turnip varieties. Application of the phytotoxins 

thaxtomin and coronatine also caused stunted hypocotyls and symptoms on roots. 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 

 

 
 

This thesis is dedicated to my children Katherine, John, Nathan, and Gordon, without 
whose loving support I would not have been able to discover the confidence, passion, and 
security I needed to embark on this journey. You have been my roots, anchoring me while I 

reached for the sun. 

To my dear friend Laura, and the entire Moher family, for warmly and generously welcoming 

me to their home full of comfort, friendship, and love. You have sustained me. 

“Love… 

…is an ever-fixed mark 

That looks on tempests and is never shaken;” 

Sonnet 116, William Shakespeare

iii 

 

 

 

 

ACKNOWLEDGMENTS 

 

I would like to thank Dr. Raymond Hammerschmidt for his leadership and guidance 

throughout this process. Dr. Hammerschmidt balances his searing intelligence with compassion 

and humor, which allows his students the confidence to try new things and the ability to 

explore. I will be forever grateful that he saw potential in an individual so long out of the 

classroom and gave me the opportunity to develop my skills and talents. New paths have 

opened to me which I could never have foreseen.  

Thank you to my guidance committee for providing review and oversight. Thank you to 

Chris Behrens, Celeste Dmytryszyn, and Dr. Pratima Devkota, for making every day in lab a joy. I 

benefitted so much from the help and expertise they provided in their own unique ways. I 

appreciate all the people in the basement labs of Plant Biology and owe a special debt to Dr. 

Carmen Medina-Mora and Doug Minier, for sharing equipment, time, and expertise. 

Thank you to Kyungmin Kim for the many hours spent on statistical analysis. Your 

assistance was invaluable, and I enjoyed our zoom sessions very much. Thank you for your 

patience answering my many questions. Thank you to the Michigan State University 

Histopathology Lab.  

 

 

 

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TABLE OF CONTENTS 

 

LIST OF TABLES .............................................................................................................................. viii 
 
LIST OF FIGURES ............................................................................................................................... x 
 
CHAPTER 1. LITERATURE REVIEW ................................................................................................... 1 
The Brassica Family ..................................................................................................................... 1 
Turnip as A Host .......................................................................................................................... 2 
Turnip Diseases ........................................................................................................................... 3 
Scab Disease ................................................................................................................................ 4 
Streptomyces Ecology ................................................................................................................. 9 
Streptomyces Life Cycle ......................................................................................................... 10 
Vegetative Mycelia ................................................................................................................ 10 
Aerial Mycelia ........................................................................................................................ 12 
Protein Secretion .................................................................................................................... 14 
Streptomyces as a Pathogen ..................................................................................................... 15 
Conserved Target-The Plant Cell Wall ....................................................................................... 19 
Disease Cycle ............................................................................................................................. 21 
Role of Toxins in Pathogenesis .................................................................................................. 23 
Thaxtomins ............................................................................................................................ 23 
Nec1 ....................................................................................................................................... 24 
Coronatine Homologs ............................................................................................................ 25 
Additional Toxins ................................................................................................................... 25 
Non-Toxin Virulence Factors .................................................................................................. 26 

 
CHAPTER 2. CELL WALL DEGRADING ENZYMES IN THE TURNIP–STREPTOMYCES HOST 
PATHOGEN INTERACTION ............................................................................................................. 27 
Primary Plant Cell Walls ............................................................................................................ 28 
Cellobiose-Mediated Signaling .................................................................................................. 30 
Virulence Mechanisms .............................................................................................................. 31 
Objectives .................................................................................................................................. 32 
MATERIALS AND METHODS .......................................................................................................... 33 
Petri Dish Assays for Enzyme Activity ....................................................................................... 33 
Pectolytic Activity Determination .......................................................................................... 34 
Isolates in Shake Culture ........................................................................................................... 35 
Assays of Culture Filtrates ..................................................................................................... 36 
Turnip Tissue Disintegration Assays .......................................................................................... 39 
Potato Tissue Disintegration Assays ...................................................................................... 40 
Turnip and Other Root Tissue Discs, Further Analysis ........................................................... 40 
Statistical Analyses .................................................................................................................... 41 
RESULTS......................................................................................................................................... 42 

 

v 

 

 

Petri Dish Assays for Enzyme Activity ....................................................................................... 42 
Isolate Growth in Liquid Culture ............................................................................................... 50 
Culture Filtrate Experiments ..................................................................................................... 52 
Turnip Tissue Disintegration Assays .......................................................................................... 54 

Thin Layer Chromatography (TLC) of Culture Filtrates from Shake Culture and Tissue 
Disintegration Assays ............................................................................................................ 60 
DISCUSSION ................................................................................................................................... 61 
Types of Enzymatic Activity Zones ............................................................................................ 64 
Effect of Yeast Extract on Enzyme Production ....................................................................... 65 
Cellulase Production and Virulence ....................................................................................... 65 
Growth in Various Media ....................................................................................................... 66 
Disc Disintegration and Host Specificity .................................................................................... 66 

 
CHAPTER 3. RESPONSE OF TURNIP SEEDLINGS TO INOCULATION WITH STREPTOMYCES AND 
TREATMENT WITH PHYTOTOXINS ................................................................................................ 68 
Objectives .................................................................................................................................. 69 
MATERIALS AND METHODS .......................................................................................................... 70 
Seedling Inoculation Studies ..................................................................................................... 70 
Toxin Study ................................................................................................................................ 73 
Turnip Seedling Histopathology ............................................................................................. 74 
Statistical Analyses ................................................................................................................ 75 
RESULTS......................................................................................................................................... 76 

Quantitative and Qualitative Results of Pathogen Inoculation of Purple Top White Globe 
(PTWG) ...................................................................................................................................... 76 
Quantitative Effects on PTWG ............................................................................................... 76 
Qualitative Effects PTWG ...................................................................................................... 77 
Quantitative and Qualitative Results of Pathogen Inoculation of Purple Prince Turnip (PPT) . 80 
Quantitative effects on PPT ................................................................................................... 80 
Qualitative effects on PPT...................................................................................................... 80 
Quantitative and Qualitative Results of Pathogen Inoculation of Golden Globe Turnip (GGT) 83 
Quantitative effects on GGT .................................................................................................. 83 
Qualitative Effects on GGT ..................................................................................................... 84 
Quantitative and Qualitative Results of Pathogen Inoculation of Japanese Baby Turnip (JBT) 85 
Quantitative effects on JBT .................................................................................................... 85 
Qualitative Effects on JBT ...................................................................................................... 87 

Quantitative and Qualitative Results of Pathogen Inoculation of Early Scarlet Globe Radish 
(ESGR) ........................................................................................................................................ 87 
Quantitative Effects on ESGR ................................................................................................. 87 
Qualitative Effects on ESGR ................................................................................................... 89 
Overall Comparison of Host Cultivar Against Streptomyces Isolates ........................................ 90 
PTWG Compared with PPT .................................................................................................... 90 
PTWG Compared with GGT ................................................................................................... 91 
PPT Compared with ESGR ...................................................................................................... 92 
PPT Compared with GGT ....................................................................................................... 92 

 

vi 

 

 

PPT Compared with JBT ......................................................................................................... 93 
Effect of Phytotoxins ................................................................................................................. 94 
Thaxtomin Effects on Hypocotyl Length ................................................................................ 94 
PTWG ..................................................................................................................................... 95 
Coronatine ............................................................................................................................. 99 
Effect of Both Toxins Combined ........................................................................................... 102 
Histological Examination of Toxin Effects ........................................................................... 104 
DISCUSSION ................................................................................................................................. 107 
Symptoms Caused by Streptomyces Isolates ....................................................................... 107 
Indications of Resistance in Turnip Varieties ....................................................................... 108 
Effect of Phytotoxins ............................................................................................................ 108 
Further Studies ..................................................................................................................... 109 

 
REFERENCES ................................................................................................................................ 110 
 

 

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LIST OF TABLES 

 

Table 1.1 Diseases and Pests of Brassica ........................................................................................ 3 
 
Table 1.2 List of Pathogenic Streptomyces Species ...................................................................... 18 
 
Table 2.1 Pectins of Eudicot Primary Cell Walls ........................................................................... 30 
 
Table 2.2 Rating Scale Applied to Tissue Discs ............................................................................. 40 
 
Table 2.3 Means and Standard Deviations of Activity Zones as a Measure of Enzyme Activity .. 47 
 
Table 2.4 Percentage of Isolates Producing Zones of Clearance, by Media and Size................... 48 
 
Table 2.5 Cellulase Production by Top Three Disc Degrading Isolates ......................................... 49 
 
Table 2.6. Activity Zone Diameters for Key Isolates in Pectin Media Containing Yeast Extract vs. 
Pectin Media Without Yeast Extract ............................................................................................. 50 
 
Table 2.7 Protein Concentration of CF Samples from Pectin Media #1 As Measured by Bradford 
Assay ............................................................................................................................................. 52 
 
Table 2.8 Reducing Sugar Released as a Measure of Pectinase Activity ...................................... 54 
 
Table 2.9 Turnip Tissue Disintegration Rating 7 Days Post Inoculation ....................................... 54 
 
Table 2.10 Percent Isolates per Disease Rating Category ............................................................ 55 
 
Table 2.11 Turnip Tissue Disintegration Rating 7 Days Post Treatment ...................................... 59 
 
Table 2.12 Fluorescing Bands Separated by TLC and Visible at 254 nm. Rf Values Were 
Calculated ..................................................................................................................................... 60 
 
Table 2.13 Summary of Top Ten Tissue Disc Macerating Isolates with Enzyme Zone of Clearance 
Values ............................................................................................................................................ 63 
 
Table 3.1 Root Rating Applied to Seedlings .................................................................................. 71 
 
Table 3.2 Seedling Cultivars Used in the Study ............................................................................. 72 
 
Table 3.3 Streptomyces Pathogenicity and Host Source .............................................................. 73 
 
Table 3.4 Toxin Treatment Per Filter Paper Strip (nmoles) .......................................................... 74 

 

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Table 3.5 Qualitative Symptoms on PTWG Caused by Streptomyces Spp. Isolates ..................... 78 
 
Table 3.6 Qualitative Symptoms on Purple Prince Turnip Caused by Streptomyces Spp. Isolates
....................................................................................................................................................... 81 
 
Table 3.7 Qualitative Symptoms on Golden Globe Turnip Caused by Streptomyces Spp. Isolates
....................................................................................................................................................... 85 
 
Table 3.8. Qualitative Symptoms on JBT Caused by Streptomyces Spp. Isolates ......................... 87 
 
Table 3.9 Qualitative Symptoms on ESGR Caused by Streptomyces Spp. Isolates ....................... 89 
 
Table 3.10 Hypocotyl Lengths as a Percent of Control for Each Variety and Isolate ................... 90 
 
Table 3.11 Hypocotyl Length as % Control for PTWG vs. PPT ...................................................... 91 
 
Table 3.12 Hypocotyl Length as % Control for PTWG vs. GGT ..................................................... 91 
 
Table 3.13 Hypocotyl Length as % Control for PPT vs. ESGR ........................................................ 92 
 
Table 3.14 Hypocotyl Length as % Control for PPT vs. GGT ......................................................... 92 
 
Table 3.15 Hypocotyl Length as % Control for PPT vs. JBT ........................................................... 93 
 
Table 3.16 Qualitative Effects of Thaxtomin Application to PTWG .............................................. 95 
 
Table 3.17 Hypocotyl Length as Percent of Control in Thaxtomin Treatments for All Varieties . 96 
 
Table 3.18 Pairwise Comparison of Different Varieties to GGT ................................................... 97 
 
Table 3.19 Qualitative Effects of Coronatine Application to PTWG ........................................... 102 
 
Table 3.20 Measurement of Representative Cell Lengths at 10X Magnification ....................... 105

 

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LIST OF FIGURES 

 

 

 

Figure 1.1 Streptomyces Symptoms on Turnips from Michigan Fields .......................................... 5 
 
Figure 1.2 Streptomyces Scabies Disease Cycle in Potato .............................................................. 5 
 
Figure 1.3 Streptomyces Periderm Penetration ............................................................................. 8 
 
Figure 1.4 Primary Plant Cell Wall Components in Eudicots ........................................................ 21 
 
Figure 2.1 Comparison of Streptomyces Growth on Oat Bran Agar vs. Glucose Media .............. 43 
 
Figure 2.2 Enzyme Activity in Pectin Media at pH 7 (no yeast extract in media) ......................... 45 
 
Figure 2.3 Growth Habits of Streptomyces Spp. Isolates in Pectin Media Shake Cultures .......... 51 
 
Figure 2.4 Agarose Well Assay Showing Pectolytic Degradation from Culture Filtrates .............. 53 
 
Figure 2.5 Turnip Disintegration Results by Rating Category ....................................................... 56 
 
Figure 2.6 Turnip Tissue Discs–Low Relative Activity ................................................................... 57 
 
Figure 2.7 Turnip Disintegration Rating of Key Isolates................................................................ 57 
 
Figure 2.8 Disc Disintegration Ratings on Turnip vs. Potato ......................................................... 58 
 
Figure 3.1 Purple Top White Globe Hypocotyl Length as % of Control for All Isolates ................ 77 
 
Figure 3.2 PTWG Control vs. NV4 Isolate ...................................................................................... 78 
 
Figure 3.3 PTWG Control vs. PBd1 Isolate .................................................................................... 78 
 
Figure 3.4 Purple Prince Turnip Hypocotyl Length as Percent of Control .................................... 80 
 
Figure 3.5 Treatment Effect on Hypocotyl Length of NV4, B9.8, and 1-1 on PPT ........................ 82 
 
Figure 3.6 Golden Globe Turnip Hypocotyl Length as Percent of Control ................................... 83 
 
Figure 3.7 Treatment Effect of B9.8 on Golden Globe Turnip ...................................................... 84 
 
Figure 3.8 Japanese Baby Turnip Hypocotyl Length ..................................................................... 85 
 

 

x 

 

 

Figure 3.9 Effect of 1-1 on JBT Hypocotyl Length ......................................................................... 86 
 
Figure 3.10 Effect of Isolates on ESGR Hypocotyl Length ............................................................. 88 
 
Figure 3.11 TB-CD Promotes Hypocotyl Growth in ESGR ............................................................. 89 
 
Figure 3.12 Effect of Different Concentrations of Thaxtomin on Hypocotyl Length in Seedlings of 
Five Varieties ................................................................................................................................. 95 
 
Figure 3.13 PTWG in 100 ng (18.3 nmoles) Thaxtomin ................................................................ 96 
 
Figure 3.14 Comparison of GGT, PPT, PTWG, and ESGR in 9.10 nmoles Thaxtomin ................... 98 
 
Figure 3.15 Hypocotyl Length as % of Control for all Varieties Treated with Coronatine ........... 99 
 
Figure 3.16 PTWG in Various Toxin Treatments ......................................................................... 101 
 
Figure 3.17 Effect of Both Toxins Combined on PTWG .............................................................. 103 
 
Figure 3.18 Comparison of Treated Tissue at 72-hpi in Thaxtomin, Coronatine, and Combined 
Toxins at 10X Magnification ........................................................................................................ 105 
 
Figure 3.19 Comparison of Treated Tissue at 144-hpi in Thaxtomin, Coronatine, and Combined 
Toxins at 10X Magnification ........................................................................................................ 106 
 

 

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The Brassica Family 

CHAPTER 1. LITERATURE REVIEW 

 

The Brassicaceae family (formerly Cruciferae) includes approximately 375 genera 

encompassing 3,200 species of plants and a large group of commercially and nutritionally 

valuable crops including arugula, bok choy, broccoli, Brussels sprout, cabbage, cauliflower, 

horseradish, kale, mustard, rapeseed, radish, rutabaga, turnip, and watercress (Ishida et al., 

2014). Three important genera within the family are the Armoracia, Brassica and Raphanus. 

Horseradish, in the genus Armoracia, has been valued since ancient times as an herb for its 

pungent flavor and medicinal use in folk remedies (Agneta et al., 2013). Rich in bioactive 

glucosinolates and their derivatives, the crop has generated new interest as a preservative and 

medicinal additive (Agneta et al., 2013). The Raphanus genus includes radish and daikon which 

are cultivated as a vegetable crop around the world, but especially in China, Japan, and Korea 

(Kobayashi et al., 2020). This genus has been cultivated for more than 2,000 years and the 

evolution of wild radish into cultivated varieties is unclear (Wang et al., 2015). 

The largest, and most commercially important genus in the family, Brassica, includes 

broccoli, Brussels sprouts, cabbage, rapeseed, rutabaga, turnip and more. Global Brassica 

oilseed production lags behind only palm and soybean, and it provides the second leading 

source of vegetable oilseed worldwide (Sparks, 2012). Turnip, or Brassica rapa var. rapa, is one 

of the oldest known cultivated vegetables and has been used “for human consumption since 

prehistoric times” (Fernandes et al., 2007). A white-fleshed, large global root “develops at the 

base of the leaf petioles” and its edible greens and stems provide a food source in addition to 

the root (Fernandes et al., 2007). Turnips are also a valuable forage crop for livestock and 

 

1 

 

present a rich source of energy and highly digestible nutrients (Benedict, 2012). Turnips are 

particularly popular throughout Europe and Asia. This is demonstrated by the three leading 

producers, which are China, Uzbekistan and Russia (Burton, 2016), whose climates are ideal for 

 

production of this cold-weather vegetable.  

Turnip as A Host 

Turnip is a high-yielding crop that is well suited for Michigan’s climate. Its most vigorous 

root growth can occur at temperatures as low as 40–60 °F, which makes it a useful second-

harvest crop. The fall crop provides the largest root and seeds planted in July can be used for 

animal grazing from September through November (Undersander et al., 2012). In 2017, the 

USDA National Agriculture Statistics Service Census stated that 3,916 acres of turnip were 

harvested nationwide, with an estimated 3,000 acres for the fresh market (NASS, 2017). 

Michigan ranks in the top four states for turnip production and cultivates about 770 acres per 

year: 20% of the nationwide total according to the Michigan Vegetable Council ("Vegetable 

Production in Michigan").  

Home gardeners can easily grow these minimal-maintenance, fast-growing vegetables. 

Although Purple Top White Globe is the most common variety found in the U.S., there are many 

varieties and hybrids with assorted flavors, root textures, and color combinations. The Market 

Express turnip root matures in just 38 days, and the Scarlet Queen matures in 45 days and 

yields a bright red root with white flesh. The White Lady produces succulent green tops, and 

roots with a sweet flavor, tender texture, and white skin (University of Illinois Extension, 2020). 

 

2 

 

Turnip Diseases  

 

The following diseases and pests affect Brassicas and may require crop rotation and 

fungicide or insecticide applications (Table 1.1). Some fields may not be suitable for turnip 

production for more than two years in a row, especially when clubroot or another soil borne 

disease has occurred. Insect pests include the flea, cabbage flea and striped flea beetle, as well 

as the turnip aphid/louse (https://hort.purdue.edu/newcrop/afcm/turnip.html). The turnip 

aphid/louse is especially problematic to Brassicas since it also vectors diseases like cabbage 

black ring spot, cauliflower mosaic, radish mosaic, and turnip mosaic virus 

(https://link.springer.com/referenceworkentry/10.1007%2F978-1-4020-6359-6_2594). 

Table 1.1 Diseases and Pests of Brassica  

 

 

 

 

Bacterial, Fungal and Viral Pathogens 

Common Name 

White Rust 

Pathogen 

Symptom 

Albugo candida 

white blister-like pustules, 

Leaf Spot 

Alternaria brassicae 

Anthracnose 

Colletotrichum 
higginsianum 

 

Clubroot 

Plasmodiophora brassicae 

white spots on leaf 

undersides 

Small dark brown or black 
spots with a yellow halo. 

Target-like rings and 
necrotic leaves appear 

small dry, circular, pale gray 

to straw-colored lesions 

lead to yellowing, leaf death 
Galls form on roots, results 
in yellowing, stunting, and 

wilting on leaves 

Root Rot 

Rhizoctonia Solani 

Damping off, wirestem, and 

Scab 

Streptomyces spp. 

 

 

3 

rot of bottom, root, and 

head 

lesions, scabs, and pitting 
on roots that may include 

raised lesions 

 

 

 

Table 1.1 (cont’d.) 

 

 

Turnip Mosaic Virus 

TuMV; family Potyviridae 

Dark and light mosaic 

 

 

 

pattern on leaves, 

especially around main 

veins. Stunting and 

yellowing of plant. Aphid 

vectored 

Insect and Nematode Pathogens 

 

Beetles; cabbage flea and 

Chrysomelidae family 

Shallow pits and rounded 

striped 

Turnip Louse/Aphid 

Lipaphis erysimi 

holes in leaves, stems 
Stem and leaf damage, 

 

stunting, vectoring of TuMV 

Root-knot nematode 

Meloidogyne hapla 

stunted growth, leaf 
yellowing and wilting 

Scab Disease 

Streptomyces scabies is the primary organism that causes the disease commonly 

referred to as scab. The disease is usually characterized by superficial or raised lesions (scabs), 

pitting, or russeting on the surface of the root or tuber (Loria et al., 1997). On turnips the 

disease usually presents as “crackling” (personal communication, Ben Werling, MSU Extension, 

Oceana County). Crackling is a description given to the uneven mottling or cracks that form in 

the underground, white area of the vegetable, often near the main root emergence (Figure 

1.1). The disease cycle in Figure 1.2 depicts the formation of scab in potato, and the 

reproductive process of the pathogen in the soil matrix.

 

4 

 

 

 

Figure 1.1 Streptomyces Symptoms on Turnips from Michigan Fields  
Note. White arrow indicates crackling symptom. 
 
 

Figure 1.2 Streptomyces Scabies Disease Cycle in Potato 
Note. Courtesy Michigan State Extension Bulletin E-2990 (Wharton et al., 2007).

 

5 

 

 

 

 Although Streptomyces can attack a wide range of host crops, the symptoms are a 

problem primarily on tuber-forming vegetables such as potato and taproots including beet, 

carrot, radish, and turnip (Hooker, 1949; Joshi et al., 2007; Loria et al., 1997).  

Scab symptoms and disease development have been well studied in potato. Only limited 

studies exist for turnip, beet, radish, parsnip, and other crops, but the relationship between the 

scab caused by “actinomycetes species” on potatoes and that of scab appearing on other hosts 

grown in soils infested with potato scab has long been known (KenKnight, 1939). The type of 

lesion formed is thought to be determined by the combined effects of pathogen strain, 

environmental conditions, timing of infection, and host (Wharton et al., 2007). For example, 

russeting or crackling in turnip is a more prevalent symptom than scabbing, particularly on the 

white area of flesh that remains underground.   

A study from 1937 examined the host range of S. scabies by growing a wide range of 

vegetables in fields known to be heavily infested by the pathogen. The 1937 study included 

cabbage, carrots, eggplant, onion, peanut, red pepper, spinach, and tomato (KenKnight, 1939). 

Only eggplant and peanut scab were noted, with lesions on eggplant similar to those found on 

the roots of turnip and rutabaga and small, raised lesions on peanut (KenKnight, 1939). 

Although the commercial losses due to disease in these other hosts may not be significant, their 

role in maintaining pathogenic Streptomyces spp. as inoculum in the soil is important 

(KenKnight, 1939).  

Another study to determine crop rotation as an approach to control potato scab 

examined the host range of pathogenic Streptomyces spp. in beet, corn, cucumber, pea, radish, 

soybean, and wheat (Hooker, 1949). In this study, the effect of pathogenic Streptomyces spp. 

 

6 

 

 

applied to the roots of seedlings grown in soil agar was examined. Severe root necrosis was 

noted in all the plants except cucumber. Considerable resistance was seen in 18–dpi cucumber 

seedlings (Hooker, 1949). In pea, radish, and soybean, the fresh root weights were half of those 

obtained in controls and the aerial portions of the plants were both stunted and hypertrophied, 

possibly as a result of the necrotic condition of the roots and systemic invasion by the bacteria 

(Hooker, 1949). Beet had a more severe response than radish with even greater necrosis and 

almost complete inhibition of lateral root formation. Necrosis of wheat tips and lateral root 

emergence failure was also evident (Hooker, 1949). Although browning and some necrosis 

appeared on corn roots, mature corn plants did not seem susceptible to the pathogenic 

Streptomyces spp. Of all the plants tested, cucumber demonstrated the most resistance with no 

necrosis in the roots and little difference in fresh root weight compared to controls (Hooker, 

1949). These experiments were duplicated in other small-seeded vegetables and legumes such 

as carrot, eggplant, lettuce, onion, and tomato, most of which exhibited some susceptibility. 

Alfalfa is noted as having more resistance than clover (Hooker, 1949).  

The vast majority of studies have been performed on potatoes since they are the most 

commercially valuable, and susceptible (Loria et al., 1997). Tuber or root symptoms such as 

raised or pitted lesions versus superficial ones may indicate a difference between the 

Streptomyces spp. causing disease (Loria et al., 1997). Infections on sweet potato caused by S. 

ipomoeae are markedly different from infections caused by Streptomyces scabies, since S. 

ipomoeae will cause root rot, and reduced plant growth and yields, but not pitted or scabbed 

lesions (Loria et al., 1997). Crops that form large, fleshy taproots like turnip, and tubers like 

potato, are most susceptible to Streptomyces when lenticels are immature, which coincides 

 

7 

 

with the rapid growth and expansion of the tissue (Loria et al., 1997). S. ipomoeae has been 

shown to directly penetrate the root tissue of sweet potato (Clark & Matthews, 1987) and 

potato. This can be seen in Figure 1.3 where a Streptomyces filament is shown directly 

penetrating a potato periderm (Hammerschmidt, unpublished). 

 

Figure 1.3 Streptomyces Periderm Penetration 
Note. Photo courtesy of Dr. Raymond Hammerschmidt. 
 

 

Streptomyces can survive indefinitely as a saprophyte in the absence of a host, and 

therefore is nearly impossible to eradicate from soil. The target of the main toxin produced by 

pathogenic Streptomyces, thaxtomin, is the plant cell wall, which may give this pathogen an 

unlimited host and tissue range (Joshi et al., 2007).  

The pathogen does not kill the plant, nor does it cause systemic infection, but the 

damage on potato tubers or fleshy roots causes economic loss due to decreased marketability 

from the deep pitting (Loria, 1993). Decreased crop marketability can occur across multiple 

distribution channels including the fresh market, processed, and seed production (Loria et al., 

1997). In the case of S. ipomoeae infections of sweet potato, significant losses result from 

 

8 

 

decreased yield and decreased marketability of the storage roots (Loria et al., 1997). 

Streptomyces has been ranked as the 4th most important disease of potato in its economic 

impact. S. ipomoeae is the most significant disease of sweet potatoes, but some resistant 

 

cultivars have been identified (Clark & Moyer, 1988; Ristaino, 1993). 

Streptomyces Ecology 

Streptomyces is the largest genus in the in the Actinobacteria phylum and one of the 

most commercially important. Actinobacteria contain at least 500 species of Gram positive, 

high G+C content (>55%), and primarily saprophytic bacteria (Barka et al., 2016). The seasonal 

abundance of Actinobacteria relative to other bacteria in the soil fluctuates from 20% during 

spring to over 30% in the fall when “large amounts of crop residues” are available (Barka et al., 

2016). The phylum is reduced to just 13% of all bacteria in winter due to frost (Barka et al., 

2016). 

An ability to scavenge nutrients from diverse macromolecules leads to carbon cycling of 

relatively insoluble organic debris and makes Streptomyces a key player in soil ecology (Barka et 

al., 2016). Streptomyces can use chitin as both a C and N source, unusual among bacteria 

(Chater et al., 2010). Hyphal filaments adhere to and penetrate the insoluble “remains of fungi, 

plants, and other soil organisms,” breaking them down with hydrolytic, excreted enzymes 

(Chater et al., 2010).  The production of endo- and exo-chitinases has been shown to destroy 

and deform fungal hyphal complexes in co-culture studies against Sclerotinia sclerotinium 

(Fróes et al., 2012) and Aspergillus parasiticus is susceptible to thermophilic endochitinases 

produced by Streptomyces varieties (Gomes et al, 2001).   

 

9 

 

Although Streptomyces are found in marine and other aquatic environments, they are 

most prevalent in soils and comprise over 95% of all Actinomycetales found in the soil column 

(Barka et al., 2016). They prefer organic material and neutral to alkaline soils at a pH between 

6–9, but some species like the phytopathogenic S. acidiscabies thrive at pH values below 5.2 

 

(Loria et al., 1997).   

Streptomyces Life Cycle  

Streptomyces are known for their prolific secretion of cell wall degrading enzymes, and 

varied secondary metabolite production, including many lifesaving and commercially valuable 

antibiotics. To understand why this remarkable organism performs such complex extracellular 

protein secretion and transportation it is useful to examine how its life cycle, marked by 

morphological differentiation, leads to the production of chemically distinct inhibitors including 

antibiotics, fungicides, cytostatics, and a vast array of other secondary metabolites (Chater et 

al., 2010). 

Vegetative Mycelia  

Streptomyces do not form colonial clusters protected by a thick biofilm like many 

bacteria but grow hyphal mycelia that network and differentiate similar to fungi. These hyphal 

filaments grow exponentially through tip extension into a branched, vegetative mycelium when 

nutrients are plentiful (Chater, 2001). Another rare characteristic among bacteria is that this 

branching does not lead to cell fission but to a multicellular bacterium with hyphal 

compartments that contain multiple chromosome copies (Barka et al., 2016). When nutrients 

become depleted and starvation occurs, the vegetative mycelium is eventually cannibalized in a 

nutrient-recycling process that transforms the mycelia into sporogenic structures called aerial 

 

10 

 

 

hyphae (Barka et al., 2016). Through the production of cell wall and other polymer-degrading 

enzymes, Streptomyces metabolizes external nutrients and its own hyphae using a programmed 

cell death (PCD)-like mechanism that is tightly linked to and controlled by metabolic regulators 

(Barka et al., 2016; Miguélez et al., 1999). The evidence indicates strict metabolic links between 

nutrient metabolism, aerial hyphae formation, and antibiotic production (Rigali et al., 2006). 

One metabolic link is the N-acetylglucosamine (GlcNAc) byproduct of chitin breakdown. GlcNAc 

can both suppress or stimulate reproductive development depending on the source from which 

the GlcNAc is derived. This has been elucidated by studies of Streptomyces grown in rich media. 

When nutrients are plentiful because GlcNAc is coming from externally derived chitin sources, 

the sugar transporters activated by the bacteria are different than if the GlcNAc comes as a 

byproduct of the breakdown of its own cell wall as a result of PCD (Barka et al., 2016; Rigali et 

al., 2006). Streptomyces differentiates between monomeric and dimeric GlcNAc and responds 

accordingly to signal the onset of aerial development in starvation and cause the global 

elicitation of antibiotic production (Barka et al., 2016; Nothaft et al., 2010). 

The autolytic degradation of the vegetative mycelia through PCD results in a pool of 

nutrients, and evidence suggests antibiotic production occurs to protect these amino acids, 

sugars, lipids, and nucleotides from motile organisms attracted to them (Barka et al., 2016; 

Rigali et al., 2006). The varied transcription factors that signal Streptomyces to stimulate or 

suppress antibiotic production have not been completely determined but are of continued 

interest to researchers, especially in light of recent studies indicating that Streptomyces may 

contain even greater antibiotic production capabilities than are currently recognized (Barka et 

al., 2016).  

 

11 

 

 

The prolific ability of Streptomyces to produce antibiotics has long been known, but 

their capacities continue to surprise researchers, as new studies indicate some species may 

harbor more than “50 different secondary metabolite gene clusters” (Barka et al., 2016). These 

clusters were suspected when the genome sequence of S. coelicolor, already a well-known 

producer of antibiotics, was found to contain over 20 biosynthetic clusters that had been 

previously unknown (Barka et al., 2016). Referred to as cryptic or “sleeping” antibiotics, the 

discovery of these sequences has prompted applied genomics research into activating the 

biosynthesis of these silent metabolites (Barka et al., 2016). The importance of already 

discovered Streptomyces products to humans including antibiotic, anticancer, anthelmintic, 

antifungal, antiviral, and cytostatic compounds, cannot be overstated. They are the originators 

of more than two-thirds of all naturally derived antibiotics, including polyketides which are 

used as both antimicrobials and immunosuppressants, the aminoglycosides of which 

Streptomycin is a member, and glycopeptides like Vancomycin to treat C-diff, MRSA and 

pneumonia, among other diseases (Barka et al., 2016).  

Aerial Mycelia 

Aerial hyphae are less branched and more than twice as wide as their vegetative 

mycelial counterparts. A sheath that forms on the exterior of the aerial hyphae lends structure 

and eventually becomes part of the spore coat (Barka et al., 2016). An additional function of the 

sheath may be to facilitate nutrient transport along the outer hyphal wall by providing a way for 

metabolites to diffuse from the basal colony up to the growing tips of the aerial hyphae (Chater 

et al., 2010). This sheath is hydrophobic on the air-facing side, and with assistance from high 

turgor pressure generated inside the hyphae, breaks through the moist soil-air surface (Barka et 

 

12 

 

al., 2016). The high turgor pressure, combined with the release of a powerful surfactant called a 

chaplin, provides a reduction in surface tension from 72 to 24 mJ m-2, and enables the sensitive, 

fragile tips to reach into the air (Barka et al., 2016). An additional hydrophobic surfactant 

protein, SapB, is also widespread in Streptomyces and plays a role in the developmental growth 

 

of aerial hyphae (Rigali et al., 2006).  

The aerial hyphae that lend Streptomyces colonies their signature fluffy appearance 

develop as a result of signaling events controlled by Bld regulators. Named after the bald or 

“hairless” aerial hyphae that develop in bld mutants, Bld regulators are required for exospores 

to fully differentiate through a “synchronized septation event” (Schumacher et al., 2017). One 

regulator in particular, BldD, sits atop a signaling cascade, repressing the transcription of an 

estimated 170 sporulation genes with targets that encode “critical components of the cell 

division and chromosome segregation” that eventually result in spore formation (Schumacher 

et al., 2017). The activity of BldD is regulated by a second messenger called 3’,5’-cyclic 

diguanylic acid (c-di-GMP), “one of the most widespread second messengers in bacteria” 

(Schumacher et al., 2017).   

Desiccation-resistant spores form on the chains of aerial mycelia and can endure in the 

soil matrix until nutrient availability stimulates them to germinate and form new vegetative 

hyphae (Chater, 2001). The morphology of these chains is often spiral resembling corkscrews, 

or wavy, and this structural differentiation allows for taxonomic distinction (Loria et al., 1997). 

The spores may be smooth, hairy, spiky or warty (Barka et al., 2016). The spore length on the 

long axis has been measured in S. coelicolor and is about 1.1 µm (Bennett et al., 2018).  

 

 

13 

 

Protein Secretion 

 

  

Streptomyces’ ability to secrete proteins and proteases extracellularly may reside in 

their extraordinary and numerous signal transduction systems, including about 50 “extra-

cytoplasmic sigma functions,” which is unusual for bacteria and for comparison, E. coli has just 

one. These secretions enhance their ability to interact with and respond to the soil environment 

(Chater et al., 2010). The capability of microbes to produce secondary metabolites, which has 

evolved separately from primary, survival metabolism (O’Brien & Wright, 2011) is thought to 

provide physiological and defensive benefits, and may be involved in establishing mutualistic 

relationships. Secondary metabolites are intrinsically linked to the ecological niche of the 

microbe. In the case of Streptomyces, secondary metabolites such as antibiotics are likely 

microbial agents of warfare, the production of which is stimulated by complex inter- and intra-

cellular communication and signaling (O’Brien & Wright, 2011). Secondary metabolites are the 

likely reason why Streptomyces have formed mutualistic relationships with eukaryotes like ants. 

Attine ant species harbor Streptomyces species because the antifungal compounds they 

produce provide protection for the ant’s farms (O’Brien & Wright, 2011).   

Melanoid pigments provide crucial protection from radiation and temperature extremes 

and are critical to survival and competitiveness of bacteria in the soil matrix (Srinivasan et al., 

2016). Melanin compounds produced by Streptomyces have proven valuable for 

pharmaceutical and cosmetic use, and supply broad protection as antiviral, antibiotic, 

antitumor and immunosuppressive compounds and enzymes (Srinivasan et al., 2016). The 

pigment produced usually varies by species and may even differ within strains based on growth 

 

14 

 

 

medium, and age of colony. Streptomyces most commonly produce light to dark grey melanin, 

but a range of colors from pink, to red and orange have been characterized (Barka et al., 2016).  

The Streptomyces genome encodes at least three different types of protein secretion 

systems—the Sec translocase pathway, the Esx secretion system, and the TAT pathway. The Sec 

translocase pathway is the main route by which proteins are secreted, as is common in most 

prokaryotes (Driessen & Nouwen, 2008). The Gram positive-specific Esx or Type VII secretion 

system is usually associated with virulence, but since this transport machinery is also found in 

non-virulent Streptomyces it likely serves many roles (Chater et al., 2010). Although the 

function of this transport machinery is unknown it likely plays a significant role based on 

analogy with similar systems in other organisms (Chater et al., 2010).  

The TAT pathway differs from the other protein secretion systems because it 

“transports pre-folded proteins across the cytoplasmic membrane” and Streptomyces rely on 

this system for the excretion of the large amounts of proteins they produce (Chater et al., 

2010). Knockout of the TAT pathway in S. scabies affects the production of melanin and these 

mutants show defects in sporulation (Chater et al., 2010; Schaerlaekens et al., 2004). Multiple 

proteomic studies indicate a lack of virulence in TAT mutants unable to secrete novel proteins, 

particularly enzymes like glycosyl hydrolases (Planckaert et al., 2018).  

Streptomyces as a Pathogen 

Streptomyces that are pathogenic to root vegetables include a growing number of 

species because of the transfer of a pathogenicity island or PAI—examples of which are rare in 

Gram-positive bacteria (Kers et al., 2005). These mobile gene clusters are commonly 

transferred across genera and are recognized by variant G+C content (Kers et al., 2005). The 

 

15 

 

 

pathogenicity island that transforms Streptomyces turgidiscabies is also an integrative and 

conjugative element, or ICE, which can mobilize and insert at site-specific locations during 

conjugation (Kers et al., 2005). In the study by Kers, this PAI was shown to be capable of 

integrating into the chromosome of nonpathogenic Streptomyces spp. and in one species, S. 

diastatochromogenes, confer pathogenicity (Kers et al., 2005). The PAI insertion occurs at a 

bacitracin resistance gene called bacA, and it encodes at least four virulence factors: thaxtomin, 

or txtA, phytotoxin proteins; a saponinase, tomA, that encodes for α-tomatinase (common in 

many fungal genomes); a necrogenic protein, nec1; and the Fas operon (Kers et al., 2005). 

These modules possess a G+C content of 68% compared to the genome’s usual 72% 

(Huguet-Tapia et al., 2011). The new protein expression capabilities conferred by a PAI often 

transform the host range or phenotypic interactions of the bacteria, which may cause a 

saprophytic species like Streptomyces to be transformed, under certain inducing conditions, 

and depending on the genetic background of the recipient strain, into a pathogen (Kers et al., 

2005).  

These inducing conditions likely involve the carbon catabolite repressor (CCR) regulatory 

operon CebR, which is constitutive in Streptomyces. CebR functions similar to the Lac operon 

and represses transcription of β-galactosidase and other glucosyl hydrolases to prohibit 

utilization of secondary carbon sources except in the absence of a preferred substrate like 

glucose or chitin (Marushima et al., 2009). CebR includes Ceb genes-E, F and G that are thought 

to encode ABC transporters, and bglC, a β-glucosidase that hydrolyzes cellobiose (CB) and 

cellotriose. Cellobiose is the most effective inhibitor of the DNA binding of CebR and likely 

serves as a pathogenicity signal in S. scabies to postpone starch and glucose utilization 

 

16 

 

 

(Planckaert et al., 2018). Cellobiose has been proposed as a signal that indicates expanding 

plant cell growth and expanding tissue where cellulose synthesis is taking place (Bignell et al., 

2014). Research indicates cellobiose also represses the saprophytic breakdown of lignocellulose 

into glucose through a 1.91-fold decrease in glucose kinase enzymes, although this interaction 

occurs independent of carbon catabolite repression signals like CebR (Planckaert et al., 2018).  

CB utilization becomes an important regulator in Streptomyces strains where the PAI has 

been acquired, because its metabolism leads to the loss of genetic cofactors that inhibit txtR 

(Planckaert et al., 2018). The repression of the CebR operon through the loss of cis-acting 

elements leads to the allosteric activation of the binding of txtR (Planckaert et al., 2018) and the 

production of thaxtomin. This pathway has been elucidated through various studies 

demonstrating an absence of thaxtomin production in common growth media, including luria 

and tryptic soy broths, and its production in living host tissue or plant-based media such as 

potato, oatmeal and oat bran broths (Kers et al., 2005). Suberin and cellobiose in combination 

will stimulate much higher production of thaxtomin than the addition of either carbon source 

(Bignell et al., 2014) on its own. 

S. scabies is more closely related to several benign, saprophytic Streptomyces than to 

other pathogenic ones (Loria et al., 1997) and it has not been determined what characteristics 

make some Streptomyces more likely to become pathogenic. A few of the most studied, like S. 

acidiscabies,  S. ipomoeae, S. scabies, and S. turgidiscabies, have a similar host range and 

produce similar symptoms, but are not closely related according to DNA-DNA homology, 16S 

rRNA sequences and ribosomal DNA sequence comparisons (Bukhalid et al., 1998). DNA-DNA 

hybridization data suggests that S. ipomoeae is not closely related to either S. scabies (39% 

 

17 

 

homology) or S. acidiscabies (17% homology) which supports the idea that plant pathogenicity 

in Streptomyces is the result of genes conferred through lateral transfer (Loria et al., 1997). In 

Table 1.2 the known strains of pathogenic Streptomyces spp. that have been characterized to 

 

date are listed. 

Table 1.2 List of Pathogenic Streptomyces Species 

Potato is Primary Host 

Streptomyces acidiscabies** 

Streptomyces spp. GK18 

Streptomyces cheloniumii 

Streptomyces spp. IdahoX** 

Streptomyces europaeiscabiei** 

Streptomyces spp. MAFF225003 

Streptomyces luridiscabiei 

Streptomyces spp. MAFF225004 

Streptomyces niveiscabiei 

Streptomyces spp. MAFF225005 

Streptomyces puniciscabiei 

Streptomyces spp. MAFF225006 

Streptomyces reticuliscabiei 

Streptomyces stelliscabiei** 

Streptomyces scabies (scabiei)** 

Streptomyces turgidiscabies** 

Streptomyces spp. DS3024** 

 

Alternate Host or Disease 

Streptomyces ipomoeae** 

Soil Rot of Sweet Potato  

Streptomyces scabies (scabiei)** 

Pod Wart of Peanut 

Note. **Known to produce thaxtomins Bignell et al., 2014). 

 

More recently characterized species include Streptomyces europaeiscabiei, commonly 

found in Europe, and S. turgidiscabies, first discovered in Japan but now found in China, North 

America, Korea and the UK (Dees et al., 2013). The genome of S. turgidiscabies has been 

 

18 

 

studied in detail. The study of this genome led to the discovery of the pathogenicity island that 

 

causes scab disease (Kers et al., 2005). 

This species variety and lack of commonality can be seen in the range of morphological 

differences. For instance, the gray, flexuous spore chains produced by S. scabies are distinct 

from the pink to white spores produced by S. acidiscabies, which lacks melanin. As the name 

implies, S. acidiscabies can cause disease in soils with a pH below 5.0, which hampers the scab 

avoidance technique of lowering soil pH (Loria et al., 1997).  

S. ipomoea differs the most because it attacks sweet potato and produces quite 

different symptoms from common potato scab. S. ipomoeae causes necrosis on fleshy sweet 

potato roots, and cankerous growths (Loria et al., 1997).  

Conserved Target-The Plant Cell Wall 

Saprophytic organisms like Streptomyces are well known for producing cell wall 

degrading enzymes at levels equal to those produced by phytopathogenic organisms, either 

fungal or bacterial (Dean & Timberlake, 1989). Plant pathogenic Streptomyces grow both inter- 

and intracellularly, and it is possible that the cell wall degrading enzymes made constitutively by 

Streptomyces in their role as non-pathogenic saprophytes are also used in pathogenic 

interactions. These interactions involve the plant cell wall (PCW), the cellulolytic and pectolytic 

enzymes capable of its degradation, and the chemical stimuli that govern bacterial signaling 

mechanisms. The primary PCW is composed of three major polysaccharide types: cellulose, 

pectin, and cross-linking glycans sometimes referred to as hemicelluloses. The amount and type 

of each polysaccharide changes over time as plants grow, develop, and respond to 

environmental and biological stimuli (Voiniciuc et al., 2018). Growing tissue requires not just 

 

19 

 

expansion but additional material deposition and recycling, coordinated to loosen and 

restructure the polysaccharides as dynamic changes occur in the structure and function of the 

PCW (Buchanan et al., 2011). The plant uses modifying enzymes continuously to alter the 

composition of the cell wall, ripen reproductive structures, promote organ growth, and for 

 

defense (Buchanan et al., 2011). 

Cellulose is a linear, simple glucose polysaccharide and is the first cell wall component to 

be synthesized in the primary cell wall (Buchanan et al., 2011). Cross-linking glycans that attach 

to these microfibrils are branched and highly substituted with post-translational modifications 

that incorporate methyl, acetyl, and phenol groups. Abundant cross-linking glycans include 

xyloglucan (XyG), xylans, mannans, with XyG by far the most abundant in eudicots. XyG is 

believed to be important in tip elongation because of its abundance at the interface between 

the plant and its environment (Voiniciuc et al., 2018).  

Pectins are rich in α-1,4-linked galacturonic subunits like homogalacturonan (HG), 

Rhamnogalacturonan I (RGI), and Rhamnogalacturonan II (Voiniciuc et al., 2018). Pectins can 

incorporate 12 different sugars with as many as 25 various glycosidic linkages, but the most 

abundant pectin in primary walls is HG (Voiniciuc et al., 2018).  

Figure 1.4 shows a broad overview of primary plant cell wall components, and the 

relative abundance of each type, when known. 

 

20 

 

 

 

Cellulose Microfibrils

β-1,4-linked glucose (Glc)

(15-30%)*

Pectins

Homogalacturonan HG

Rhamnogalacturonan I (RGI)

Rhamnogalacturonan I (RGI)

(35%)***

Cross-linking glycans 

(hemicelluloses)

xyloglucans-XyG**

(20%)*

Proteins

hydroxyproline rich glycoproteins-HRGP's

proline-rich proteins-PRP's

glycine-rich proteins-GRP's

(amount varies by tissue and species)

Figure 1.4 Primary Plant Cell Wall Components in Eudicots 
Note: *Percent of dry mass weight in primary plant cell walls (Buchanan et al., 2011). **XyG is the major 
polysaccharide in eudicots. Other hemicellulosic polymers are xylan, glucuronoxylan, arabinoxylan, mannan, 
glucomannan and galactoglucomannan (Buchanan et al., 2011). ***Pectin content is highly variable by plant type. 
35% is estimated for dicots, but some grasses could have as little as 2-10% (Kameshwar & Qin, 2018). 
 

Disease Cycle 

 Common scab is most prevalent in dry soils with a pH between 5.2–7.0 (Loria et al., 

1997). Strategies to reduce disease symptoms include raising and maintaining soil moisture 

when tubers or roots are in rapid growth stages and planting resistant cultivars when possible. 

Some known resistant cultivars of potato are Russet Burbank and Superior (Loria et al., 1997). 

Scab resistance in turnips has not yet been reported.  

 

21 

 

 

The optimum temperature for disease formation is 68–72 °F but the pathogen is active 

at range of temperatures from 50 to 88 °F (Wharton et al., 2007). The mode of entry of 

Streptomyces spp. is primarily through lenticels or wounds, (Wharton et al., 2007) although 

evidence exists for direct penetration (Hammerschmidt unpublished). The pathogen can spread 

easily from root to root, or through contaminated soil, but is prevalent in most potato growing 

fields throughout Europe, North America, South Africa, New Zealand, Australia, and Israel (Loria 

et al., 1997).   

The early secretion of the necrosis-inducing peptide nec1 may be the first step in 

pathogenic Streptomyces’ attack on plant host tissue. It is believed that nec1 expression is not 

regulated by the carbon catabolite repression signal that governs thaxtomin and coronafacic 

acid production (Joshi et al., 2007).  The small amount of glucose that is released around root 

tissue during cell wall expansion and modification may initiate colonization attempts by 

upregulating production of Nec1 (Joshi et al., 2007).  Streptomyces turgidiscabies colonizes the 

exterior, intra- and inter-cellular portions of Arabidopsis root tips within seven days (Joshi et al., 

2007). Studies show that Nec1 secretion is an important first step in colonization of host root 

tissue, since ∆nec1 mutants exhibit only mild symptoms of infection with limited or no 

colonization of the epiphytic zone, and no intra- or inter-cellular growth observed compared to 

wild type (Joshi et al., 2007).  

Thaxtomin causes a hypertrophic response in onion, radish, and tobacco seedling tissue 

(Scheible et al., 2003). The pathogen may also secrete a coronafacic acid derivative that induces 

tissue hypertrophy (Planckaert et al., 2018), and this phytotoxin, along with thaxtomin, is likely 

what leads to the comprised cell wall integrity seen in the disease (Loria et al., 1997). The roots 

 

22 

 

are especially vulnerable during rapid expansion, which takes place in turnips around 49–56 

days (Gupta et al., 2001). The stimulation of thaxtomin synthesis may aid the penetration of the 

pathogen into the expanding tissue (Bignell et al., 2014). Hypertrophy, also induced by 

thaxtomin, likely causes the erumpent, or raised lesions, while pitted tissue is thought to be the 

 

result of toxin-induced cell death (Fry & Loria, 2002).  

Role of Toxins in Pathogenesis 

Thaxtomins 

Thaxtomins are cyclic dipeptides derived from phenylalanine and L-4-nitrotryptophan 

entities of which at least eleven forms have been identified and which differ only in hydroxyl 

and N-methyl groups at particular sites (Bignell et al., 2014; King & Calhoun, 2009). One step of 

the biosynthesis of thaxtomin is catalyzed by nitric oxide synthase produced by nitric oxide (NO) 

derived from arginine (Kers et al., 2005; King & Calhoun, 2009). An intermediate called 

thaxtomin D is N-methylated on both the nitrotryptophyl and phenylalanine moieties (King et 

al., 2003). The last step in thaxtomin biosynthesis is the additional of hydroxyl groups to the 

phenylalanyl moiety. The thaxtomin phytotoxin appears to be unique to Streptomyces 

infections and its activity is disrupted by movement or replacement of any of the nitro groups, 

phenyl side chains or D,L configurations, indicating that these chemical features are required 

for phytotoxicity (King & Calhoun, 2009; Loria et al., 1997). Thaxtomin A is the most prevalently 

identified toxin and is produced by S. scabies, S. acidiscabies, S. turgidiscabies and many other 

species (Bignell et al., 2014). Thaxtomin C is the primary kind produced by S. ipomoea. 

Thaxtomin  inhibits crystalline cellulose biosynthesis in the cell wall (King et al., 2003; 

Loria et al., 1997) with a similar, but not identical, mode of action as the herbicide isoxaben and 

 

23 

 

 

appears to increase lignification and callose deposition (Scheible et al., 2003). This leads to a 

reduction in seedling shoot and root length, and radial swelling due to hypertrophy (Fry & Loria, 

2002; Loria et al., 1997). This hypertrophy occurs even at thaxtomin concentrations below 1.0 

µM, indicating that thaxtomin affects plant growth like a hormone (Loria et al., 1997).  

Thaxtomin affects both monocots and dicots and so the target is thought to be 

conserved (Loria et al., 1997). The cell hypertrophy produced in seedlings mimics the erumpent 

lesions produced on the more mature tuber tissue, but there is evidence that both tubers and 

seedlings gain some resistance to thaxtomin A as they mature (Loria et al., 1997).  

A non-hydroxylated member, thaxtomin C, is the primary form produced by the sweet 

potato pathogen S. ipomoea, although it is also made by S. acidiscabies and S. scabies (Loria et 

al., 1997). Mutants lacking thaxtomin C are unable to penetrate the adventitious root structure 

of sweet potatoes to cause infection (Guan et al., 2012). Thaxtomin C production is not 

stimulated by the cello-oligosaccharides that induce production of thaxtomin A, and the exact 

interaction of TxtR to induce thaxtomin production in S. ipomoeae remains to be discovered 

(King et al., 2003 and Guan, 2012). 

Nec1  
 

Nec1 is a necrosis-causing, water soluble phytotoxin with no known homologs that 

possesses a low G+C content (54%) indicative of lateral genus transfer (Bukhalid et al., 1998). 

Nec1 has a strong influence on root infection, is highly conserved among plant pathogenic 

Streptomyces, and also has a conserved plant cell target (Joshi et al., 2007). Unlike thaxtomin, 

which is “catabolically repressed,” Nec1 is produced in a rich growth medium early in the plant-

pathogen interaction (Joshi et al., 2007). Studies indicate the presence of Nec1 just 20 hours 

 

24 

 

post infection (hpi) whereas thaxtomin is not apparent until 48 hpi (Joshi et al., 2007). Studies 

on Arabidopsis to isolate the effect of the Nec1 protein showed that the peptide causes “gall-

like structures at the tips of lateral roots,” with eventual necrosis of the roots (Joshi et al., 

 

2007).    

Coronatine Homologs 
 

Another biosynthetic gene cluster discovered in S. scabies is highly similar to the 

coronafacic-acid (CFA) biosynthetic gene cluster (Bignell et al., 2014). CFA is the polyketide 

component of the nonhost-specific toxin coronatine (COR) that is produced by many pathovars 

of Pseudomonas Syringae and Pectobacterium atrosepticum (Bignell et al., 2014). The COR toxin 

in Ps. syringae causes hypertrophy and may function as a jasmonic acid (JA) mimic to suppress 

plant defense responses. Although S. scabies lacks biosynthetic genes that would enable the 

production of COR, it is likely to be producing similar COR-like toxins possible of enhancing 

virulence (Bignell et al., 2014). The CFA biosynthetic cluster has been identified in S. scabies but 

not in S. turgidiscabies or S. acidiscabies (Bignell et al., 2014).  

Additional Toxins 
 

 Additional phytotoxins have been elucidated from select pathogenic Streptomyces 

species. In addition to thaxtomin, S. scabies has been reported to produce at least two types of 

concanamycin secondary metabolites (Bignell et al., 2014). First isolated from S. 

diastatochromogenes, the concanamycin family are polyketide macrolides characterized to 

exhibit antifungal, anti-neoplastic qualities, and to cause root growth inhibition in seedlings 

(Natsume et al., 1996).  

 

 

25 

 

 

Non-Toxin Virulence Factors 
 

 The third putative virulence factor identified as part of the Car8 PAI in S. turgidiscabies 

is encoded by genes with homology to a saponinase-encoding gene called tomA (Huguet-Tapia 

et al., 2011). Although deletion studies did not show a direct effect on virulence, its 

conservation in the PAI indicates a role that has not been determined. TomA has known 

homologs in Clavibacter michiganensis subsp michiganensis (CMM) and the coding regions 

flanking tomA “are predicted to encode 2 β-glycosidases, an ABC sugar transporter and TetR 

regulator”. The tomA gene is syntenous in CMM and plant pathogenic Streptomyces may to 

share a virulence mechanism with CMM that involves carbohydrate metabolism (Huguet-Tapia 

et al., 2011). The research carried out in the present study seeks to yield additional information 

about the process of Streptomyces pathogenicity in turnip, the importance of cell wall 

degrading enzymes, the disease symptoms caused by isolated toxins compared to virulent 

Streptomyces strains, and whether there is resistance within turnip cultivars to the pathogen. 

 

 

 

26 

 

CHAPTER 2. CELL WALL DEGRADING ENZYMES IN THE TURNIP–STREPTOMYCES HOST 

 

PATHOGEN INTERACTION 

Plant-pathogenic Streptomyces spp. grow inter- and intracellularly (Joshi et al., 2007) 

suggesting an important role for cell wall degrading enzymes in pathogenesis. The plant cell 

wall (PCW) is the most abundant source of organic carbon on the planet, a primary defensive 

barrier against many bacterial and fungal pathogens (Rai et al., 2015), and a primary 

phytopathogenic target. The ability to degrade plant cell walls has repeatedly and 

independently evolved in countless bacterial and fungal pathogens (Kubicek et al., 2014). 

Higher order animals like nematodes are believed to have acquired PCW degrading abilities 

from fungal and bacterial pathogens (Kikuchi et al., 2017). The production of cell wall degrading 

enzymes capable of penetrating roots, stems, leaves, or reproductive structures is well studied 

in various fungi (Mendgen & Deising, 1993) and is a hallmark of soft-rot bacteria. Soft rot 

diseases caused by Dickeya spp. and various Pectobacterium spp. occur primarily through the 

production of extracellular pectolytic enzymes and a wide range of cell wall degrading enzymes 

(Pérombelon, 2002). Pectin-degrading enzymes, in particular, are the first to be produced by 

many microorganisms when attacking plants or growing on purified cell walls. 

Depolymerization of pectin may be a prerequisite for further cell wall breakdown and pathogen 

colonization (Dean & Timberlake, 1989).

 

27 

 

Primary Plant Cell Walls 

 

Primary plant cell walls are mainly composed of cellulose (15-30%), cross-linking glycans 

(20%), and pectin biopolymers (35%). The specific amount and composition of each varies 

among groups of plants and cell function (Buchanan et al., 2011). Primary cell walls are 

important because they allow for cell expansion during growth phases and provide the shape of 

the cell and tissues. When cell division occurs and more cells are formed, the intermediate 

space, or middle lamella, is filled with a pectin-rich substance that aids in cell to cell adhesion, 

nutrient flow, and defense. Lignin and other proteins are formed when the secondary cell wall, 

a more rigid and defensible structure, is formed after cell differentiation (Buchanan et al., 

2011). The composition of the wall changes throughout the life of the plant in response to 

growth, reproductive development, defense, and abiotic stimuli (Kubicek et al., 2014). 

Differences exist between monocot and dicot plants. Dicots such as turnip are rich in xyloglucan 

and pectin  (Kubicek et al., 2014).  

Although the relative amount of each compound varies, the cell wall is comprised 

mostly of cellulose, a substantially crystalline, β-1,4-linked glucose polymer. Attached to this 

fibril platform, and connecting them, are cross-linking glycans that order the direction of 

growth and expansion of the cell while maintaining the inherent strength of the linear cellulose 

chains (Buchanan et al., 2011). This aggregate of cellulose and cross-linking glycans is 

embedded in a porous network of pectin (Buchanan et al., 2011). Hydroxyproline-rich 

glycoproteins (HGRPs), proline-rich proteins (PRPs) and glycine-rich proteins (GRPs) are added 

to the structure over time. Extensin, an HGRP, is believed to be part of the mechanism that 

 

28 

 

locks the cell wall into place after the process of elongation is completed (Buchanan et al., 

 

2011). 

Cellulose utilization has long been believed to require the synergy of multiple cell wall 

degrading enzymes. The crystalline microfibrils of cellulose prevent access and degradation by 

cellulases, requiring the synergy of both endo- and exo-acting enzymes, and the action of 

loosening compounds to increase the reactivity of the tightly-packed microfibrils before 

degradation can occur (Arantes & Saddler, 2010; de Souza Moreira et al., 2016). Working in 

coordination on the hydrolysis of the 1,4-β-glycosidic bonds that link cellulose, endo-acting 

cellulase enzymes create new ends for exo-acting cellobiohydrolases to attack. In addition, the 

presence of compounds that perform what is called “amorphogenesis” may be required 

(Arantes & Saddler, 2010). Amorphogenesis enhances the reactivity of cellulose fibers by 

reducing the degree of crystallinity and increasing the surface area of cellulose available to the 

enzymes (Arantes & Saddler, 2010). Biochemical and structural data suggest cellobiohydrolases 

may not be exclusively exo-acting. A single endo-processive cellulase in the GH9 family of 

enzymes is essential in the successful degradation of Clostridium phytofermentans (Gilbert, 

2010). 

Although there are three major forms of pectin, homogalacturonan (HG) is most 

commonly found in nature ( about 65%;) (Keggi & Doran-Peterson, 2020). The degree of 

methylation or acetylation of HG can require organisms to remove these side groups before 

breaking down the pectin (Keggi & Doran-Peterson, 2020) complicating the elucidation of 

various pathways for HG deconstruction by microorganisms.  

 

29 

 

 

Table 2.1 Pectins of Eudicot Primary Cell Walls 

Pectin Polysaccharide 

Backbone Chains 

Homogalacturonan (HG) 

Polygalacturonic acid backbone 

Rhamnogalacturonan I-alternating 

α-1,4-D-GalA and α-1,2-L-Rha 

disaccharides 

Rhamnogalacturonan II 

13 different sugars with over 20 different 

linkages 

 

Cellobiose-Mediated Signaling 

Streptomyces are known saprophytes and prolific cell wall degrading enzyme producers. 

Their ability to switch from a saprophyte to a pathogen may result from their ability to colonize 

a living host versus dead organic matter (Planckaert et al., 2018). This ability to be a pathogen is 

likely to reside in the combined effect from CWD enzymes and toxins working synergistically. 

This ability to colonize enables pathogenic Streptomyces to occupy a niche for survival and 

reproduction when a saprophytic lifestyle is energetically unsuitable (Planckaert et al., 2018). 

The production of cellulose-degradation byproducts is a key step since cellobiose disables the 

repression of the thaxtomin biosynthesis activator gene txtR that is present in the pathogenicity 

island (Planckaert et al., 2018). The utilization of cellobiose as a virulence determinant is not 

well understood since cellobiose is also the most abundant carbon source in the pathways that 

provide glucose in saprophytic interactions. In the pathogenic interaction, cellobiose and 

cellotriose transported into the cell lead to signaling effects that stimulate thaxtomin 

production. In pathogenic interactions the presence of cellobiose downregulates the glucose 

kinase pathways, and instead of using glucose as a carbon source, alternative and more 

complex carbon sources are prioritized (Planckaert et al., 2018). Other plant materials that 

seem to trigger thaxtomin A biosynthesis come from xylan and suberin degradation products. 

 

30 

 

 

The role of these materials has not been as well characterized as the cellobiose interaction with 

the cellulose utilization regulator CebR (Planckaert et al., 2018). Proteomics studies show a link 

between the presence of suberin and the production of gylcosyl hydrolases and other 

extracellular enzymes that are likely needed in the degradation of cellulose and suberin 

(Planckaert et al., 2018). The importance of enzyme production is also indicated by the 

impaired virulence in ∆tatC mutants unable to produce glycosyl hydrolases (Planckaert et al., 

2018). 

Virulence Mechanisms 
 

The variety of symptoms observed with Streptomyces spp. suggests a range of virulence 

mechanisms, which may differ by host and be relevant to the process of pathogenesis (Loria et 

al., 1997). For example, the superficial lesions with a netted pattern that have been called 

netted scab produce necrotic lesions not just on expanding plant tissue but on all underground 

parts (Loria et al., 1997). Netted scab is also more severe in high soil moisture and low 

temperature conditions, the opposite of conditions that suppress common scab (Loria et al., 

1997). Some potato cultivars such as ‘Green Mountain’ are highly susceptible to common scab 

but resistant to netted scab, suggesting differences in virulence pathways (Loria et al., 1997). 

Streptomyces ipomoeae, the cause of soil rot of sweet potato, infects both the fleshy and 

fibrous root parts. The infected tissues become necrotic with evidence that S. ipomoeae causes 

rot by directly penetrating fibrous roots (Loria et al., 1997). The necrosis and rot caused by 

netted scab and S. ipomoeae indicate a soft-rot type of disease with a potential virulence role 

played by cell wall degrading enzymes. 

 

31 

 

Objectives 

 

Pathogenic Streptomyces may penetrate potato tubers via immature lenticels (Loria et 

al., 1997) or via direct cell wall penetration (Figure 1.3; Hammerschmidt) indicating cell wall 

degrading enzymes. The role that cell wall degrading enzymes play in the virulence of 

pathogenic Streptomyces spp. isolates on turnip are of interest. Streptomyces produce cell wall 

degrading enzymes in vitro, but the role these enzymes play in pathogenesis is unknown. The 

first objective of this research was to determine whether there is a correlation between the 

pathogenic tendency of Streptomyces spp. isolated from diseased turnips and the ability of 

these isolates to degrade polysaccharide components found in plant cell walls such as pectin 

and cellulose. Secondly, the ability of isolates to grow on media containing pectin, 

carboxymethyl cellulose, or glucose as the sole carbon source was determined. Also, the ability 

of Streptomyces spp. isolates to produce cell wall degrading enzymes in these media and the 

type of enzyme produced was evaluated. Finally, the ability of Streptomyces spp. isolates to 

cause disease in turnip tissue discs was used to determine whether pathogenesis is correlated 

with pectolytic and cellulolytic enzyme production.   

 

32 

 

 

 

MATERIALS AND METHODS 

The reference isolates used in this study were provided by the lab of Dr. Raymond 

Hammerschmidt from Michigan-grown turnips (except for 1-1 which was isolated from potato) 

exhibiting symptoms of scab. The isolate colonies were grown for 7-10 days and were 

maintained at 29 °C on oat bran media (Schaad et al., 2001). A 5-mm plug from a culture was 

used in the growth measurements on media containing pectin, cellulose, or glucose as the sole 

carbon source, as described below. 

Petri Dish Assays for Enzyme Activity 
 

A total of 47 isolates were placed on each of four different media: 

carboxymethylcellulose (CMC), and pectin at three pH levels (5, 7, or 8). A total of 33 isolates 

were grown on media containing glucose as the sole carbon source. All media contained 500 mL 

of a mineral salts solution (MSS) consisting of the following (per liter): 2 g (NH4)2S04; 4 g  

KH2PO4; 6 g Na2HPO4; 0.2 mg FeSO4*7H20; 0.2 g MgSO4 ; 1 mg CaCl2; 10 µg HBO3; 10 µg MnSO4; 

70 µg ZnSO4; 50 µg CuS04; pH 7.4 (Hankin et al., 1971). In addition to MSS, the pectin media 

contained, per liter: 15 g agar, 1 g yeast extract, and 5 g citrus pectin (Hankin et al., 1971). 

Additional tests using select isolates 1-1, B98, NV1, NV4, PBd1, TB-CD, V4 and V5 were run in 

pectin media without yeast extract. The media were homogenized in a blender to mix the 

pectin thoroughly and the pH was adjusted to either 5, 7, or 8. The media containing CMC was 

made as described for the pectin mixture with the substitution of 40 g CMC, just 5 g agar and 

no yeast extract. The same ingredients and protocols were also followed to make the glucose 

media, substituting 5 g glucose for pectin or CMC, 15 g agar, and no yeast extract. Isolates were 

also grown in oat bran media for growth comparisons.  

 

33 

 

 

All media were autoclaved and cooled before being dispensed. Using a sterile 

serological pipet, five mL of test media were added to each 60 x 15 mm Petri dish. One plug 

containing bacterial growth was applied (bacteria side down) to the center of each Petri dish. 

Three replicates of each isolate were made. Plates were incubated at 29 °C for 3 to 5 days, or 

until growth reached 5–10 mm. Some plates exhibited condensation after a few days of growth 

and the moisture that collected on the lids was removed periodically. 

Pectolytic Activity Determination  
 

To detect pectolytic activity, the inoculated plates containing pectin were flooded with a 

1% hexadecyltrimethylammonium bromide (CTAB) solution dissolved in 15% ethanol in water 

(EtOH) (Hankin et al., 1971). This solution “precipitates polysaccharides” that have not been 

degraded. Thus, pectolytic enzyme activity is seen as a clear zone around the colony (Hankin et 

al., 1971). The clear zone, including the area containing the plug of bacteria, was measured to 

estimate the level of enzyme activity. For photographic recording of pectolytic activity, selected 

plates were stained with a 0.1% (w/v) ruthenium red solution (Spooner, 1994). Areas of 

undegraded pectin become pink, resulting in clear “halos” where enzymatic activity has 

occurred (Spooner, 1994). Growth in glucose media was measured, but enzymatic activity was 

not. 

The plates containing CMC were flooded with a 0.25% (w/v) solution of Congo Red 

(Carder, 1986). The area degraded by cellulase does not stain as red as the areas where 

cellulose is intact (Carder, 1986). The area indicated as degraded by cellulase was measured 

and select isolates photographed. 

 

34 

 

 

Isolates in Shake Culture 
 

Select isolates were chosen for further study in shake cultures after the plate assays 

were completed. The isolates were selected for their relative ability to produce low, mid, or 

high levels of enzymatic activity according to the ring size produced in the various pectin and 

CMC media. The isolates selected were 1-1 (S. scabies) from potato, and from turnip; 3-2, 4TA, 

5TB, 7T, 7 Pit, 11 Pit, B9.8, Hud1, NV1, and V5. The isolates were placed in several different 

types of media to see if growth or enzymatic activity was enhanced by media type. The media 

contained, per liter: (1) Simple pectin broth contained 5g pectin, 500 mL MSS. (2) 

Polygalacturonic acid (PGA) media contained 3g yeast, 5g PGA, 2g K2HPO4, 1g MgSO4*7H20, and 

2g (NH4)2SO4 at pH 5.0 (Jacob et al., 2008). (3) Pectin media containing 3g yeast, 5g pectin, 2g 

K2HPO4, 1g MgSO4*7H20, and 2g (NH4)2SO4 at pH 7.0 (Jacob et al., 2008). (4)High pectin media 

contained 0.5g yeast, 10g pectin, 1g K2HPO4, 0.88 g MgSO4*7H20, and 1g NaNo3, and 1g KCl at 

pH 7.0 (Mohandas). (5)High CMC media contained 0.5g yeast, 5g CMC, 1g K2HPO4, 0.88 g 

MgSO4*7H20, and 1g NaNo3, and 1g KCl at pH 7.0 (Mohandas). (6) Oat bran broth containing 

20g oat bran, 0.1g FeSO4*7H20, 0.1g MnCl2*4H2O, and 0.1g ZnSO4 *7 H2O (Schaad et al., 2001). 

Each media type was mixed and sterilized in the same manner as the agar Petri dish 

assays, and 50 mL aliquots were measured into sterile 250 ml Erlenmeyer flasks. A loopful of 

selected bacterial spores was added to the media and placed on a shaker at room temperature 

and 103 rpm. After 6 days, another loopful was added to some flasks from cultures grown in oat 

bran broth on shaker if significant bacterial growth was not visible. 

Cultures were placed on the shaker at 103 rpm and maintained at room temperature for 

approximately 28 days before being filtered through two layers of cheesecloth, and then 

 

35 

 

 

centrifuged at 6000 rpm for 10–15 min at 4°C. The supernatant was decanted into large falcon 

tubes, frozen at -20°C and then lyophilized. Samples were lyophilized in an attempt to 

concentrate proteins at close to 1mg/mL for enzyme assays. Two isolates (B9.8 and 5TB) were 

grown as described above in media number 3, 4, and 5 and then placed in incubated shakers at 

29 °C to see if elevated temperature enhance enzymatic activity. From selected isolates, 1 mL 

samples were removed for the agarose well assays described below. These samples are 

described as culture filtrates (CF) in the biochemical analyses.  

Assays of Culture Filtrates 
 

Enzyme activity produced in shake culture filtrates was detected and quantified using 

the Bradford assay for total protein, (Bradford, 1976), the DNS reducing sugar (Miller) assay for 

enzyme activity, and agarose well enzyme assays. 

Protein Determination 

Using the Bio-Rad kit based on the Bradford protein assay, select culture filtrates (CF) 

were analyzed following lyophilization. Sample CF were reconstituted in 1 mL of a 60 mM tris 

buffer at pH 6.6. This buffer has negligible interference. A standard curve was prepared using 

bovine serum albumin (BSA) at concentrations from 0.11 to 0.90 mg/mL. For comparison, a 10% 

pectinase standard (PS) in tris buffer was also measured and a standard curve prepared. The 

absorption of samples was compared to the standard curve to estimate protein levels. 

 

36 

 

 

Agarose Well Assays 
 
CF fractions were applied to the following buffered, pectin-agarose media wells containing: 1) 

1.5 g agarose, 100 mL tris buffer at pH 6.6, and 0.5 g pectin, 2) 2.0g agarose, 50 mL acetate 

buffer (pH 3.9), 50 mL dH20, and 0.2g pectin, 3) 0.5% (w/v) ammonium oxalate, 0.2% sodium 

azide, 1.0g agarose, in 100 mL 0.2 phosphate buffer adjusted to pH 5.3 with sodium salt of PGA 

(0.1%), 4) 1g pectin, 2g agarose, 0.05g MgSO4, and 5) 1g pectin, 2g agarose, 0.08g MgSO4. 

Each medium was boiled, cooled, and poured to completely cover a glass Petri dish with a thin 

layer. Following media solidification, 5-mm wells were created with a cork borer. Into these 

wells 50 µL of CF from select isolates was applied and the plate incubated at 29°C for 24 h. 

Some CF samples were applied to the wells without centrifugation. Following the addition of 

1% CTAB solution (Hankin et al., 1971) a 0.1% ruthenium red solution was added before the 

Petri dish was placed back in the incubator for 1 day. Results were photographed.  

The procedure steps were carried out in several different media. Applying pectinase 

standards (PS) in concentrations from 10–90% achieved a standard of ring degradation in media 

#1. The PS concentrations showed ring degradation areas ranging from 1.9 to 2.5 cm and were 

used as a comparison for CF samples applied to the media. 

DNS Reducing Sugar Assay to Detect Pectolytic Enzyme Activity 
 

Following the DNS reducing sugar method modified by Miller (1959), CF samples from 

all isolates grown in media #3 were analyzed for their ability to release reducing sugars from 

pectin. Following lyophilization, CF samples were reconstituted in 2 mL of a 0.1M phosphate 

buffer at pH 7.3 (Arijit et al, 2013) and then centrifuged at 3000 rpm for 10 min. The 

supernatant was decanted, and the pellet discarded. For a zero-time measurement, each CF 

 

37 

 

 

sample was added to a 1.0 % citrus pectin solution in a 1:1 ratio, using 0.25 mL of each. The 

DNS reagent solution was added, and the mixture boiled for 5 min before absorption was 

measured at 540 nm for Value 1. To determine enzyme activity, the same procedure was 

repeated except the solution was incubated 30 min at room temperature before the DNS 

reagents were added. The mixture was boiled for 5 min. Some CF samples had precipitants 

following heating in water bath and were centrifuged and the supernatant decanted. 

Absorption was measured at 540 nm. Subtracting the zero-time value from the 30-minute value  

yields an estimate of the reducing sugars released as a result of pectolytic enzyme activity. A 

0.1% (w/v) mono-D-galacturonic acid (mDGLA) solution was diluted to concentrations from 0.01 

to 0.71 mg/mL for the standard curve.  

Thaxtomin Analysis by Thin Layer Chromatography (TLC) 

Streptomyces spp. isolates 1-1, 5TB, B9.8, NV1, PBd1, and V4 were grown in oat bran 

broth (media #6) for 30 days. Broth cultures were filtered through two layers of cheesecloth 

and extracted in equal volumes ethyl acetate (Doumbou et al., 1998). The ethyl acetate extract 

was concentrated through rotary evaporation at 35°C under vacuum. The remaining residue 

was redissolved in ethanol in two 0.5 mL portions and these samples were applied to silica gel G 

plates with UV254 indicator and developed with chloroform-methanol (95:5, v:v). Some samples 

were separated in 85% chloroform, 15% methanol. Bands were visualized with UV light (254 

nm) and Rf values were calculated from visible bands.

 

38 

 

Turnip Tissue Disintegration Assays 

 

Purple Top White Globe (PTWG) turnip tissue discs were prepared according to the 

method used by Hammerschmidt (1984) for potato tissue discs. Turnips were maintained at 

room temperature for 24 hours prior to use, washed with Alconox, rinsed in deionized water, 

and patted dry. The surface of each turnip was sterilized in 70% ethanol and flamed. This 

sterilization was repeated twice per turnip. Working with ethanol and flame sterilized 

instruments, the ends of the turnip were removed, and a cork borer was inserted through the 

center to remove a 2-cm core (Hammerschmidt, 1984). From this core, discs were made by 

slicing through the tissue at 5-mm intervals to create uniform, sterile pieces of tissue 

(Hammerschmidt, 1984). These pieces were washed in sterile water and rinsed three times to 

remove starch. Four pieces were added to pre-sterilized glass Petri dishes containing Whatman 

#1 filter paper. A few drops of sterile water were added to each Petri dish to moisten the filter 

paper before placing the tissue discs inside (Hammerschmidt, 1984). A 5-mm plug of 7 to 10-

day-old culture from a Streptomyces spp. isolate was placed in the center of each tissue disk. A 

plug of sterile, non-inoculated oat bran media (Schaad et al., 2001) was added to the center of 

the control. Three replicates were made per isolate.  

 Turnip tissues were also tested for a reaction to enzymes and toxins. Filter paper discs 

of 5-mm diameter were dipped in solutions of 0.1% and 0.01% pectinase, and 0.1% cellulase 

and placed at the center of the tissue discs. A set of filter paper disks were made with 150 µg 

thaxtomin per disk, and each disk was placed on the center of a turnip tissue disk. A total of 46 

sets of tissue discs were made. These were stored in the dark at room temperature for 7 days. 

Following rating according to   

 

39 

 

Table 2.2, the discs were weighed, select isolates were photographed, and tissue 

samples were frozen at -20°C. 34 Streptomyces spp. isolates were tested on turnip tissue discs.  

 

Table 2.2 Rating Scale Applied to Tissue Discs 
0 

“No browning or disintegration compared to control” 

1 

“Tissue discoloration at disc edges with no disintegration” 

“Uniform discoloration with some disintegration” 
“Brown tissue with moderate, consistent disintegration” 
“Brown or black tissue with significant disintegration” 

2 
3 
4 
Note. (Spooner, 1994) 

Potato Tissue Disintegration Assays 

A select group of isolates was tested on ‘Manistee’ potatoes using the same method. 

Seedling assays (unpublished data) indicate that some turnip varieties with resistance to the 

Streptomyces spp. strain isolated from potato were susceptible to turnip isolates. Additional 

tests were initiated on potato discs for evaluation. The isolates tested on potato were 1-1, B9.8, 

NV4, PBd1, TB-CD, and V5. Two isolates (1-1, B9.8) were applied to beet, radish, and rutabaga. 

Turnip was the host for the experiment unless otherwise stated. 

Turnip and Other Root Tissue Discs, Further Analysis 

Compounds produced in infected tissues were extracted in ethanol by macerating 

frozen tissue discs using a frozen mortar and pestle. The tissue samples were separated into 

two equal portions. One portion was lyophilized and stored at -20°C for future studies. The 

other portion was extracted overnight in 10 mL ethanol/g tissue. Samples were covered with 

parafilm and heated in a water bath for 1 hr., then centrifuged and the supernatant reserved as 

fraction 1. The process was repeated with the remaining pellet. The second supernatant 

fraction was added to fraction 1 and the ethanol was removed using a rotary evaporator at 

80°C. Sticky residue remaining in vials was washed with dH2O and then extracted in ethyl 

 

40 

 

 

acetate following the method of Doumbou (Doumbou et al., 1998). Extracts obtained from 

these discs were redissolved in ethanol at a rate of 0.5 mL/g tissue and these samples were 

applied to Silica Gel G 254 silica plates and developed with chloroform–methanol (95:5). Bands 

were detected with UV (254nm) light and Rf values were calculated for each band. 

Statistical Analyses 
 

Statistical model for the rating of turnip tissue disintegration included treatment 

(isolate) as a fixed factor and Petri dish nested within the treatment as a random factor. Since 

the effect of treatment was significant (p < .05), the mean separation was performed using 

Fisher’s least significant difference.  

 

41 

 

Petri Dish Assays for Enzyme Activity 

RESULTS 

 

Although most isolates grew in pectin media at a pH of 5, 7, or 8, a few isolates showed 

little to no visible growth and no measurable pectolytic enzyme production. It was determined 

that 44 out of 47 isolates grew on media containing carboxymethylcellulose (CMC) as a sole 

carbon source suggesting that they could produce cellulases. All but four out of 33 isolates were 

able to grow on glucose as the sole carbon source. Several strains failed to survive in storage 

and could not be included. 

On glucose as the sole carbon source, growth was not robust compared to oat bran 

(Figure 2.1). In glucose, Streptomyces spp. failed to form grey colonies with sporulating hyphae, 

although some pigment was produced by some isolates (Figure 2.1). The colonies grown in 

glucose also did not spread as they did in oat bran, pectin, or CMC media, where spores 

scattered new colonies away from the starting plug. Pectolytic and cellulolytic enzyme activity 

by isolates grown on glucose were not measured. 

 

42 

 

 

Figure 2.1 Comparison of Streptomyces Growth on Oat Bran Agar vs. Glucose Media 
Note. White arrows indicate aerial hyphae formation by sporulating colonies. Blue arrow indicates melanin 
formation. Black arrow indicates sparse growth compared to oat bran media. 

 

 

Zones of clearing, or activity, around the growing culture on media containing pectin or 

CMC were detected in most isolates indicating their ability to produce cell wall degrading 

enzymes. However, a few showed no measurable enzyme production in some media (Table 

2.3).

 

43 

 

Isolate 10T showed neither measurable pectinase production at all three pH levels nor 

cellulase production in CMC. Isolates 1T and 2T also produced no pectinase at pH 5 and little 

pectinase at 8; however, 1T produced some pectinase at pH 7, and 2T showed some cellulase 

 

production. 

In addition to differences in the measurable size or diameter of the activity zone around 

the bacterial plug, there was also variation in the type of activity produced. For example, some 

isolates produced completely clear zones around the bacterial plug, even before the addition of 

CTAB, where it also appeared the agar had been degraded by the bacteria. These isolates were 

apparently capable of degrading both agar and pectin. Other isolates produced more hazy or 

indistinct zones around the plug where the top layer of agar appeared to have thinned but the 

enzymes had not diffused as readily. A few isolates produced areas where the media was 

dissolved in a uniform circle around the bacterial plug, and some isolates produced areas that 

spread out from the plug unevenly.  

The production of crystal-clear areas of degradation with all media dissolved was rare 

and observed in isolates exhibiting both pathogenic and non-pathogenic characteristics. For 

example, the pathogenic isolate TB-CD in pectin media at pH 7 (with yeast extract) produced 

small zones where all media was disintegrated in the area of activity. In the same media (pectin 

at pH 7 with yeast extract) two isolates exhibiting saprophytic characteristics, Sup A3 and 

SupV3, also produced clear activity with disintegrated media. Isolate B9.8 by contrast, showed 

some of the largest zones of activity measured across all isolates and in all media, but with 

widespread and uneven areas without the complete dissolution of media seen in TB-CD, Sup A3 

 

44 

 

and SupV3. Isolate NV4 made smaller zones than B9.8, but they were more circular and distinct 

 

(Figure 2 a-b). 

 

Figure 2.2 Enzyme Activity in Pectin Media at pH 7 (no yeast extract in media) 
Note. a. B9.8 b. NV4 
 

Some isolates produced “mottling” where the enzymatic activity was spread throughout 

the dish in an uneven manner and was difficult to measure visually. The cultures that contained 

mottling were measured as 0.0 cm due to an inability to visualize and measure a zone. These 

isolates often produced a large, visible area of activity in one replicate but mottling in one or 

two replicates that resulted in a high standard deviation for that isolate. 

 

45 

 

Table 2.3 lists the isolates tested and the average and standard deviation of their zone 

 

of activity.  

 

 

46 

 

Table 2.3 Means and Standard Deviations of Activity Zones as a Measure of Enzyme Activity 

pH 5 

pH 7 

pH 8 

CMC 

Isolate 

Mean 
(cm) 

std dev 

Mean 
(cm) 

std dev  Mean 
(cm) 

std dev 

Avg 
(cm) 

std dev 

 

1-1 

1 T 

10 T 

11 Pit 

1-9 

2T  

3 surfB 

3 T 

3-2 

4 T 

4 TA 

4 TB 

5 surfA 

5 surfB 

5 surfC 

5 surfD 

5 T 

5 TA 

5 TB 

6 surfA 

6 surfB 

6 surfC 

6 T 

7 T 

7 Pit 

8 T 

B9.8 

Hud1 

NV1 

NV2 

NV3 

NV4 

PBd1 

PitA1 

PitV5 

SupA1 

SupA2 

SupA3 

 

0.2 

0.0 

0.0 

1.7 

1.8 

0.0 

1.8 

3.1 

1.2 

0.0 

3.2 

3.4 

2.3 

0.0 

0.0 

0.0 

0.0 

2.0 

2.2 

1.7 

0.7 

4.0 

0.5 

4.0 

4.2 

0.0 

3.7 

4.0 

2.3 

3.0 

1.0 

3.8 

4.4 

2.8 

1.4 

0.1 

3.0 

0.0 

0.1 

0.0 

0.0 

0.2 

0.1 

0.0 

0.2 

0.7 

0.3 

0.0 

0.7 

0.4 

0.2 

0.0 

0.0 

0.0 

0.0 

0.0 

0.4 

0.3 

1.2 

0.0 

0.5 

0.0 

0.3 

0.0 

0.3 

0.0 

0.6 

1.0 

1.2 

0.3 

0.2 

1.6 

0.2 

0.2 

0.1 

0.0 

2.8 

1.6 

0.0 

3.2 

3.6 

0.0 

1.9 

0.2 

1.4 

1.0 

4.4 

2.7 

0.0 

2.7 

3.7 

1.8 

2.4 

2.8 

2.8 

2.3 

1.9 

0.0 

0.0 

0.0 

1.6 

2.5 

3.6 

2.6 

2.7 

1.2 

2.8 

1.4 

1.7 

3.3 

1.5 

1.5 

1.3 

2.8 

3.6 

0.7 

0.0 

2.6 

2.6 

0.1 

0.2 

1.8 

1.4 

0.0 

2.8 

0.2 

0.0 

0.0 

0.5 

0.0 

2.1 

0.8 

2.9 

0.4 

0.4 

0.4 

0.9 

0.2 

0.2 

0.0 

4.3 

1.1 

3.3 

3.0 

3.8 

3.8 

3.2 

1.6 

1.6 

3.2 

2.3 

2.6 

1.2 

0.1 

0.0 

0.6 

1.4 

0.0 

0.4 

0.3 

0.4 

1.0 

0.3 

0.6 

0.0 

2.4 

0.7 

1.6 

0.5 

0.8 

1.0 

1.0 

0.4 

0.0 

0.0 

0.0 

1.4 

0.5 

0.1 

1.2 

0.9 

1.0 

1.3 

0.1 

1.4 

0.6 

2.6 

0.5 

1.2 

0.3 

47 

0.2 

1.2 

0.0 

0.6 

0.5 

0.0 

0.2 

1.5 

0.3 

0.0 

0.2 

0.3 

0.0 

0.0 

0.9 

0.0 

0.4 

1.4 

0.3 

0.2 

0.2 

0.5 

0.6 

0.1 

0.1 

0.0 

0.3 

0.9 

0.4 

0.6 

0.3 

0.3 

0.6 

0.2 

2.1 

0.7 

1.1 

2.3 

3.4 

0.0 

0.0 

2.4 

2.0 

1.2 

2.7 

1.7 

2.1 

2.3 

2.2 

2.6 

3.4 

3.5 

2.8 

2.3 

2.1 

2.0 

2.2 

3.3 

2.3 

2.1 

0.0 

2.7 

1.6 

1.0 

3.2 

1.7 

1.8 

2.8 

0.2 

0.3 

0.4 

0.4 

2.8 

0.3 

0.7 

0.4 

1.1 

0.0 

0.0 

0.2 

0.0 

0.2 

0.3 

0.3 

0.4 

0.2 

0.3 

1.6 

0.2 

0.5 

0.3 

1.3 

0.4 

0.6 

0.9 

0.8 

0.3 

0.5 

0.0 

0.3 

0.8 

0.8 

0.7 

0.2 

1.8 

1.0 

0.1 

0.0 

0.5 

0.4 

0.8 

0.1 

0.5 

0.4 

 

 

Table 2.3 (cont’d.) 
SupV3 

1.8 

SupV6 

SupV7 

TB-CD 

TBd2 

Tsup2 

V2 

V4 

V5 

1.4 

0.0 

0.0 

0.0 

0.0 

0.0 

1.1 

1.0 

 

0.0 

0.1 

0.0 

0.0 

0.0 

0.0 

0.0 

1.2 

1.7 

 

2.6 

1.7 

0.0 

0.5 

2.2 

0.8 

1.7 

0.7 

1.3 

 

0.5 

1.5 

0.0 

0.5 

0.6 

1.4 

0.1 

1.2 

1.3 

 

0.9 

2.9 

1.5 

3.5 

2.2 

3.0 

2.5 

3.1 

2.3 

 

0.4 

1.9 

2.0 

1.7 

1.9 

0.9 

1.3 

1.1 

2.1 

 

0.6 

0.2 

0.8 

0.2 

0.6 

0.1 

3.1 

4.2 

3.3 

 

0.4 

0.1 

0.7 

0.3 

0.4 

0.1 

1.8 

0.8 

1.6 

Note. Zone of clearance (activity) was measured across the widest area of visible media degradation for each 
replicate. 
 

Although fewer isolates produced zones of activity larger than 2 or 3 cm in pectin at pH 

5 than at pH 7 or 8, or in CMC, this was the pH at which the highest percentage (11%) of zones 

larger than 4 cm were produced. The production of zones of activity larger than 4 cm was 

usually rare and was only seen in 2% of the isolates grown in CMC, or in pectin at pH 7 or 8 

(Table 2.4). 

Table 2.4 Percentage of Isolates Producing Zones of Clearance, by Media and Size 

Isolate 

% > 2cm 

% > 3cm 

% > 4cm 

36 

43 

47 

51 

Pectin pH 5 

Pectin pH 7 

Pectin pH 8 

CMC 

 

26 

13 

21 

17 

11 

2 

2 

2 

Although 94% of all isolates tested produced at least a small zone of activity in CMC (the 

highest percentage for any media type) with an average of 1.75 cm, the top three disc-

degrading isolates produced almost no enzymatic activity in CMC (Table 2.5). 

 

48 

 

Table 2.5 Cellulase Production by Top Three Disc Degrading Isolates 

 

Isolate 

Cellulase 
Activity 
0.17 
0.40 
0.23 
1.75 

TB-CD 
PBd1 
NV4 
Mean*  
Note. *Mean value of all isolates 
 

Turnip 

Disintegration 

Rating 

4.0 
4.0 
3.9 
1.9 

Impact of Yeast Extract on Enzyme Activity for Select Isolates 

Yeast extract also changed pectinase activity in many isolates. Although most 

Streptomyces spp. isolates were capable of growth on media limited to pectin, yeast extract 

may have provided an alternative source of carbon and so the tests were repeated on key 

isolates without yeast extract. The absence of yeast extract greatly diminished the ability of 

almost all the isolates to produce pectinase at pH 5. Although isolate 1-1 increased from 0.2 cm 

to 2.2 cm the high variability lessens confidence in this finding (Table 2.6). The ability of to 

produce pectinase at pH 5 was greatly diminished in PBd1 (from 4.4 cm to 0.5 cm) and NV4 (3.8 

cm to 0.7; Table 2.6).  

Isolates showed more ability to produce pectinase at pH 7 in media not supplemented 

with yeast extract. For example, five out of eight isolates showed increased enzymatic activity, 

with V4 showing an increase in average activity zone from 0.7 cm (yeast) to 4.2 cm (no yeast). 

V5 showed a similar result, going from a 1.3 cm activity zone with yeast extract to a 3.3 cm 

activity zone with no yeast extract (Table 2.6).  

Although the activity zones were generally smaller at pH 8, most isolates were still 

capable of producing pectinase. The ability of isolate TB-CD to produce enzymes was the most 

reduced as shown by the decrease in activity zone from 3.5 cm to 0.2 cm (Table 2.6). 

 

49 

 

 

Table 2.6. Activity Zone Diameters for Key Isolates in Pectin Media Containing Yeast Extract vs. 
Pectin Media Without Yeast Extract 

Pectin Media 

 

Isolate 

1-1* 

B9.8 

NV1 

NV4 

PBd1 

TB-CD 

V4 

pH 5 Yeast 

pH 5 NY 

pH 7 Yeast 

pH 7 NY 

pH 8 Yeast 

pH 8 NY 

Avg  

sd 

Avg 

sd 

Avg 

sd 

Avg 

sd 

Avg 

sd  

Avg 

sd 

0.2 

3.7 

2.3 

3.8 

4.4 

0.0 

1.1 

0.1 

0.3 

0.6 

0.3 

0.2 

0.0 

1.2 

2.2 

4.0 

2.9 

0.7 

0.5 

0.0 

0.0 

2.0  2.8 

1.2  1.8 

0.9  3.6 

0.1  3.1 

2.1  2.7 

0.9  2.2 

0.1  1.4 

0.1  2.4 

0.6  1.7 

1.4  2.7 

0.0  0.5 

0.5  1.7 

0.0  0.7 

1.2  4.2 

0.2 

1.6 

0.4 

0.5 

1.6 

2.4 

0.6 

3.6 

4.3 

3.3 

3.8 

3.2 

3.5 

3.1 

0.2 

0.3 

0.4 

0.3 

0.6 

1.7 

1.1 

2.4 

2.1 

2.2 

2.4 

3.2 

0.2 

1.0 

0.7 

0.1 

0.4 

0.6 

0.6 

0.3 

1.3 

Note: No yeast extract present (NY). Average values are zones of clearance measured in cm. * Strain isolated from 
potato. 
 

Isolate Growth in Liquid Culture 

Cultures in simple pectin broth #1 were grown for 31 days with constant shaking. It was 

noted that NV1 appeared to have the most growth compared to NV4, PBd1 and V4. The isolates 

grown in media #1-3 appeared to grow well based on visual observations. In media #3 

containing pectin, yeast, and various minerals, some isolates produced the grey and white 

growth indicative of sporulation that is seen when grown in oat bran agar. Some isolates 

formed globular balls (Figure 2.3). The media containing low quantities of yeast and high 

quantities of pectin (10 g pectin-media #4) produced bacterial colonies of yellow, ribbon-like 

filaments (Figure 2.3) with no globular balls visible. The isolates in media #5 containing CMC 

remained mostly clear with no visible balls, grey filaments, or other bacterial signs. These also 

did not yield bacterial pellets when centrifuged, unlike all the other media. It is possible the 

bacteria did not grow in the CMC broth at all.   

 

50 

 

 

Figure 2.3 Growth Habits of Streptomyces Spp. Isolates in Pectin Media Shake Cultures 
Note. Clockwise from top left: NV4 in Media #3, PBd1 in Media #3, PBd1 in Media #4, and NV4 in Media #4. Yellow 
arrow indicates regular, grey colonies typically seen in oat bran agar. Red arrow indicates globular growth in 
PBd1.White arrows indicate growth habit with yellow filaments.

 

 

51 

 

Culture Filtrate Experiments 

Total Protein Determination in Culture Filtrates 

 

The Bradford protein assay was used to measure protein concentrations in CF samples 

prior to running agarose well and DNS reducing sugar assays. The sample CF used were from 

media #1 from isolates 1-1, 7T, 7 Pit, 11 Pit, B9.8, and a 10% pectinase standard (PS). The 

results indicated that the culture filtrates had low levels of protein, between 0.11 and 0.25 

mg/mL (Table 2.7). 

Table 2.7 Protein Concentration of CF Samples from Pectin Media #1 As Measured by Bradford 
Assay 

Sample 

10% PS 

1-1 

Protein mg/mL  >0.90 

0.19 

7T 

0.19 

7Pit 

0.22 

11Pit 

0.17 

B9.8 

0.21 

 

Agarose Well Assay for the Determination of Pectinase  

A radial diffusion assay in agarose was attempted to quantify enzymatic activity in the 

culture filtrates. After many attempts in various media, CF samples in agarose #1 containing tris 

buffer at 6.6 with 0.5g pectin showed zones of dissolved media in from CFs of isolates 1-1 

(turnip disc CF), and PBd1 and V4 (pectin shake CF). Although further attempts were made with 

culture filtrates from the same experiments, this result was not replicated (Figure 2.4). It is 

possible that the bacterial colonies had remained too long in the incubation period with the 

available nutrients and reached starvation. A shorter incubation time of 7–14 days is 

recommended for future attempts.  

 

52 

 

 

 

Figure 2.4 Agarose Well Assay Showing Pectolytic Degradation from Culture Filtrates 
Note. Black arrow indicates 1.6 cm ring of pectolytic activity from CF of V4 isolate in pectin shake culture media #1 
and green arrow indicates small ring from PBd1 CF from the same media. White arrow indicates 2.5 cm ring from 
CF of isolate 1-1 (turnip disc). 
 

Measurement of Total Pectinase Activity Using DNS 

Culture filtrate (CF) fractions B9.8 and 5TB from media #2 (PGA) and 5TB from media #1 

(pectin) formed precipitates when heated while obtaining Value 2. An attempt was made to 

remove the precipitant through mechanical filtration (cheesecloth) and centrifugation, but the 

results may have been compromised by the formation of these precipitants. Enzymatic activity 

as measured by reducing sugars is listed below (Table 2.8).

 

53 

 

Table 2.8 Reducing Sugar Released as a Measure of Pectinase Activity 

Isolate 

Media 
Type 

Estimated mg/mL 

Sugar 

 

1 
2 
4 
2 
1 
3 
3 

0.0450 
0.0525 
0.0150 
0.0475 
0.0200 
0.0400 
0.0200 

5TB 
5TB 
5TB 
B9.8 
NV4 
PBd1 
V4 
Note. Media no. 1 contains 5 g pectin, 500 mL MSS. Media no. 2 contains 3 g yeast, 5 g PGA, 2 g K2HPO4, 1 g 
MgSO4*7H20, and 2 g (NH4)2SO4 at pH 5.0 (Jacob et al., 2008). Media no. 3 contains 3 g yeast, 5 g pectin, 2 g 
K2HPO4, 1 g MgSO4*7H20, and 2 g (NH4)2SO4 at pH 7.0 (Jacob et al., 2008). Media no. 4 contains 0.5 g yeast, 10 g 
pectin, 1 g K2HPO4, 0.88 g MgSO4*7H20, and 1 g NaNo3, and 1 g KCl at pH 7.0 (Mohandas) 
 
Turnip Tissue Disintegration Assays 
 
Table 2.9 Turnip Tissue Disintegration Rating 7 Days Post Inoculation 
Isolate 

Rating (0-4)*g 

4 
4 
4 
3 
3 
3 
3 
3 
3 
3 
3 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
1 

PBd1 
TB-CD 
NV4 
V2 
V5 
1-1 
NV2 
6 surfC 
B9.8 
6 surfA 
V4 
7 T 
1 T 
Hud1 
NV3 
1-9 
5 SD 
PitA1 
SupA1 
TBd2 
3-2 
SupA3 
5 SC 

 

Std. 
dev. 
0.0 
0.0 
0.2 
1.1 
1.0 
1.3 
1.1 
0.8 
0.8 
1.2 
0.9 
0.8 
1.2 
1.4 
0.6 
0.9 
0.9 
1.6 
0.5 
0.9 
0.7 
0.5 
1.0 

54 

 

 

Table 2.9 (cont’d.) 
5 SB 
10 T 
4TA 
2 T 
4 T 
5TB 
7 pit 
4TB 
5 TB 
5 TA 
NV1 
Avg 
sd 
 ≥ 1 sd 
Note. *Rating scale as follows: 0 is no browning, 1 is browning with no disintegration, 2 is browning with some 
disintegration, 3 is browning with consistent disintegration, and 4 is necrotic tissue with significant disintegration. 
 

1 
1 
1 
1 
1 
1 
1 
1 
0 
0 
0 
2 
1 
3.0 

1.1 
0.3 
0.5 
0.6 
0.3 
0.6 
0.6 
0.4 
0.7 
0.3 
0.3 

 
 
 

Table 2.10 Percent Isolates per Disease Rating Category 

Rating category 

Descriptions 

Percent 

0 
1 
2 

3 

4 

No browning  

Browning with no disintegration 

Browning with some disintegration 

Browning with consistent disintegration 

Necrotic tissue with significant disintegration 

9 
26 
32 

24 

9 

 

 

 

 

 

Most of the 34 isolates applied to turnip tissue discs had the ability to cause browning 

with no disintegration with 65% of the Streptomyces spp. isolates showing a rating of 2 or 

higher, indicating at or above browning with some disintegration (Table 2.10). Those rated ≥ 

3.0, or +1sd, showed browning with consistent disintegration, and three isolates with a rating of 

4 showed necrotic tissue with significant disintegration. 

 

55 

 

 

 A closeup of turnip tissue discs representing each rating category is seen in Figure 2.5. 

In Figure 2.6, the disintegration caused by three of the lowest overall enzyme producers 

compared to control is seen. Statistical analyses of the disintegration ratings of key isolates 

indicated significantly greater disintegration from the control in the ratings of isolates 1-1, B9.8, 

NV4, PBd1, TB-CD and V4 (Figure 2.7). The isolate NV1 was not different from control. 

Figure 2.5 Turnip Disintegration Results by Rating Category 
Note. A closeup of one of three replicates per isolate is shown with tissue disintegration rates in parentheses. a. 
NV1 (0) b. 4TB (1) c. 1-9 (2) d. B9.8 (3) e. NV4 (4)  
 

 

 

56 

 

 

 

Figure 2.6 Turnip Tissue Discs–Low Relative Activity 
Note. Red arrow indicates turnip disc disintegration by isolate I T.  
 

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

)
4
-
0
(
 
g
n
i
t
a
R

B

B

B

B

B

B

B

A

A

Control

NV1

V4

B9.8

1-1

V5

NV4

PBd1

TB-CD

Isolate

 

Figure 2.7 Turnip Disintegration Rating of Key Isolates  
Note. Different letters indicate statistically significant differences among the isolates (p < .05).  

 

57 

 

Potato Tissue Disintegration 

 

Isolates 1-1 (from potato), B9.8, NV4, PBd1, TB-CD, and V5 were selected for study on 

potato to see if differences in turnip variety susceptibility as indicated by seedlings assays in 

this study (Chapter 2) carried over into differences on tissue discs with potato as host. With the 

exception of isolate B9.8, which showed no difference in virulence between potato and turnip, 

there was a significant difference among all isolates tested. The most striking result is that 

PBd1, rated 4 on turnip tissue discs, was completely non-pathogenic on potato tissue discs 

(Figure 2.8). 

Isolates NV4, TB-CD and V5 had some ability to degrade potato but they were not as 

virulent on this host as turnip. The strain isolated from potato, 1-1, was the only isolate that 

was more virulent on potato than turnip.   

**

**

**

**

**

)
4
-
0
(
 
g
n
i
t
a
R

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

1-1

B9.8

NV4

Turnip

PBd1

Potato

TB-CD

V5

 

Figure 2.8 Disc Disintegration Ratings on Turnip vs. Potato 
Note. A symbol ** indicates statistically significant differences between turnip and potato in the same isolate (p < 
.05).  

 

 

58 

 

Toxin Disintegration Assay 

 

The discs treated with pectinase and cellulase standard solutions showed moderate 

tissue discoloration, but thaxtomin application did not result in tissue discoloration or 

disintegration (Table 2.11). 

Table 2.11 Turnip Tissue Disintegration Rating 7 Days Post Treatment 
Treatment 
0.10 % PS 
0.01% PS 
0.10 % CMC 
150 µg/mL thaxtomin 

0.8 
0.6 
0.5 
0.4 

1 
2 
1 
0 

Rating (0-4)*g 

Std. dev. 

Note. *Rating scale as follows: 0-No browning or disintegration compared to control; 1, Tissue discoloration at 
edges with no disintegration; 2, Uniform discoloration with some disintegration; 3, Brown tissue with moderate, 
consistent disintegration; 4, Brown or black tissue with significant disintegration

 

59 

 

 

Thin Layer Chromatography (TLC) of Culture Filtrates from Shake Culture and Tissue 
Disintegration Assays 

 

Several bands were visualized for most CF when examined under visible and UV light 

(365 nm). The separated components of the CF were visible in the formation of bands, 

especially thaxtomin which appears yellow under visible light. The band colors ranged from 

blue, to white/blue, to purple, to yellow (Table 2.12).  

Table 2.12 Fluorescing Bands Separated by TLC and Visible at 254 nm. Rf Values Were 
Calculated 
Culture Filtrate 

0.16* 

0.39* 

0.44  0.51 

Rf Value 
0.61 

0.70* 

0.86 

0.97 

1-1 (Oat Bran 

Shake) 

5TB (Oat Bran 
Shake) 
B9.8 (Oat Bran 
Shake) 
NV1 (Oat Bran 
Shake) 
NV1 (Turnip) 

PBd1 (Turnip) 

X 

X 

X 

X 

  

X 

1-1 (Potato) 

 (0.19) X 

1-1 (Rutabaga) 

1-1 (Beet) 

  

X 

  

  

  

X 

  

  

  

  

  

  

(0.32) 

X 

  

  

  

X (Y) 

  

X (Y) 

  

  

  

X 

  

  

  

  

  

  

  

  

  

X 

X 

  

  

  

  

  

  

X 

X 

X 

X 

 (0.75) 

  

  

  

X 

  

X 

X 

X 

X 

  

  

  

  

  

  

X 

X 

X 

X 

X 

 
Note. *Indicates some variation in exact Rf value among CF. Exact values, where different, in parenthesis. (Y) 
indicates yellow band. Yellow band is likely thaxtomin. Thaxtomin A and B, and their derivatives, have been 
previously identified by Rf values 0.27, 0.29, 0.35, 0.41, 0.45 (King et al., 1992).  
 

 

60 

 

DISCUSSION 

 

This research to uncover the role of pectolytic and/or cellulolytic enzymes in Streptomyces 

pathogenesis concluded with several findings. Most Streptomyces spp. isolates are capable of 

making some pectolytic and/or cellulolytic enzymes and some isolates are far more robust 

producers of pectolytic and/or cellulolytic enzymes than others. There is a lot of variation, by 

Streptomyces spp. isolate, as to which pectolytic and/or cellulolytic enzymes is produced. Some 

non-pathogenic Streptomyces spp. isolates are highly robust producers of pectolytic and/or 

cellulolytic enzymes. There was no single kind of pectolytic and/or cellulolytic enzymes required 

for pathogenesis, but the most pathogenic strains are robust producers of at least one kind of 

pectolytic and/or cellulolytic enzymes. There is host specificity in some pathogenic 

Streptomyces spp. isolates.  

In general, there was a high degree of variation in enzyme production by Streptomyces spp. 

isolate, and within each isolate by media type. For example, isolate V4 produced an enzyme 

ring of 4.2 cm in CMC but just 1.1 cm in pectin media at pH 5, and 0.7 cm in pectin media at pH 

8 (both with yeast extract). These variations are likely due to the complex regulated carbon 

catabolism of Streptomyces that parse nutrient sources and initiate metabolic pathways not 

just on the type of nutrient but also the sugar transporter and molecular form in which it 

arrives through the cell membrane.   

There were trends in which higher enzymatic activity correlated with necrosis in turnip 

tissue discs. Producers of pectinase at pH 8 showed a tendency to be correlated with virulence 

indicated by the result that five of the isolates that caused the most symptoms in turnip tissue 

discs produced their highest pectinase activity at pH 8. Three of the isolates that caused the 

 

61 

 

most symptoms in turnip tissue discs produced their highest pectinase activity at pH 5 and two 

in cellulase. None of the isolates that caused the most symptoms in turnip tissue discs produced 

their highest pectinase activity at pH 7. Seventy-one percent of the isolates that were rated 3.0 

or higher (+1 standard deviation) in turnip tissue-degrading tendency produced zones of activity 

 

in pectin media at pH 8 that were ≥ 1 standard deviation. Only forty-three percent of the 

isolates that were rated 3.0 or higher (+1 standard deviation) in turnip tissue-degrading 

tendency produced activity zones ≥ 1 standard deviation at pH 5 or in cellulose. None of the 

isolates that were rated 3.0 or higher (+1 standard deviation) in turnip tissue-degrading 

tendency produced activity zones ≥ 1 standard deviation in pectin at pH 7. 

 

62 

 

Table 2.13 Summary of Top Ten Tissue Disc Macerating Isolates with Enzyme Zone of Clearance 
Values 

 

 

Isolate 

TB-CD 
PBd1 
NV4 
V2 
V5 
1-1* 
NV2 
B9.8 
6 surfC 
6 surfA 

Zone of Clearance (cm) 

Pectin 
pH 5 

Pectin 
pH 7  

Pectin 
pH 8  

0.0 
4.4 
3.8 
0.0 
1.0 
0.2 
3.0 
3.7 
4.0 
1.7 

0.5 
1.7 
1.4 
1.7 
1.3 
2.8 
1.2 
3.6 
0.0 
2.3 

3.5 a 
3.2 
3.8 a 
2.5 
2.3 
3.6 a 
3.0 a 
4.3a 
0.4 
0.4 

Cellulose 
0.2 
0.4 
0.3 
3.1 
3.3 
3.4 
2.8 
3.2 
2.1 
3.3 

Turnip Tissue 
Disc Rating  
0-4 (4 most 
degraded) 

4.0 
4.0 
3.9 
3.2 
3.2 
3.1 
3.1 
2.9 
2.9 
2.6 

Note. *Isolated from potato. All pectin media contained yeast extract. a Highest enzyme activity for this isolate was 
in pectin at pH 8. Turnip tissue disc rating score: 0 is no browning, 1 is browning with no disintegration, 2 is 
browning with some disintegration, 3 is browning with consistent disintegration, and 4 is necrotic tissue with 
significant disintegration. 
 

The ability of an isolate to cause tissue disintegration on turnip discs is not solely due to 

the production of CWDE since several isolates that produced higher levels of CWDE in vitro 

were not able to cause disease in turnip. For example, NV1, a strong cellulase and pectinase 

producer, caused no disintegration on turnip tissue in comparison to isolate 1T that did not 

show evidence of much pectinase or cellulase production, yet still caused some disintegration 

on turnip tissue discs. 

Toxins play an important role in pathogenicity and it would be interesting to repeat 

these enzyme assays only on those isolates known to possess the pathogenicity island and see 

if CWDE production is correlated with increased virulence, and if so, if virulence is correlated 

with a particular substrate.

 

63 

 

Types of Enzymatic Activity Zones 

 

It was observed that in addition to more typical zones of activity observed around 

bacterial growth on pectin agar, some other patterns of apparent degradation were observed. 

For example, some of the Streptomyces spp. isolates were capable of hydrolyzing agar, which is 

usually typically quite resistant to microbial degradation. While it is not particularly surprising 

for Streptomyces spp. to possess these enzymatic capabilities, it was interesting that this ability 

did not seem tied to higher overall pectinase or cellulase production. In fact, some zones of 

activity produced by the Streptomyces spp. that degraded agar as a nutritional source were 

smaller than average. It was a rare occurrence to see the media completely degraded to the 

bottom of the plastic Petri dish, but it did occur with some isolates. This tendency seemed to be 

associated neither with pathogenicity nor higher overall enzyme-producing capacity.  

 

It may be important that the largest areas of degradation appeared far more frequently 

in pectin media at pH 5. Two possibilities are that either polygalacturonases were more 

diffusible in the pectin media or they were produced in greater quantities in some isolates. 

Diffusion of enzymes in plant tissue could be an important characteristic of plant cell wall 

penetration considering that pectin plays an important role in regulating the porosity of the 

plant cell wall, including the diffusion of enzymes that regulate growth and development. 

Enzymes that are more readily diffused may enable pathogens to inflict more damage on the 

plant cell wall than enzymes that are produced at a higher rate but are not able to spread to 

their target through the pectin layer, or middle lamella of more developed plant cell walls. 

 

64 

 

Effect of Yeast Extract on Enzyme Production 

 

Yeast extract is often added as a nutrient for the degradation of pectin in commercial 

industries like fruit juice production, so it is not surprising that yeast extract would enhance 

pectin degradation. What is surprising is that enzyme production declined in the presence of 

yeast extract in isolates NV4, PBd1, TB-CD, V4, and V5 at pH 7. Yeast could be triggering a 

catabolite repressor in these isolates. 

There was a high degree of variability in 1-1 with no yeast extract present that makes 

the results difficult to interpret. This isolate in pectin media at pH 5 with no yeast extract  

consistently demonstrated a wide range of variability in producing zones of clearing, which may 

indicate that yeast components are more susceptible to carbon catabolite regulation in this 

strain (known to be Streptomyces scabies) than to other strains. 

Cellulase Production and Virulence 

The production of cellulase by pathogenic isolates might also merit further study since 

cellulase was barely produced in the three most virulent isolates on turnip tissue discs. This was  

a surprising result when considering that overall, cellulase was the most consistently produced 

enzyme of the four types tested.  

One of the low cellulase-producing isolates was PBd1, which was also unique as it was 

pathogenic on turnip but not potato. This might indicate that while cellulase production is not a 

strong predictor of pathogenicity in turnip, it may play a factor in pathogenicity against other 

hosts. 

 

65 

 

Growth in Various Media 
Glucose 

 

The lack of sporulation in glucose media is not surprising since it is well known that 

Streptomyces have repressors to block reproductive development such as aerial hyphae when 

glucose is available. This result demonstrates how effective and complete the carbon catabolite 

repression mechanisms that block development can be for this organism.  

Disc Disintegration and Host Specificity 
 
The three top-rated tissue disc degraders, PBd1, TB-CD, and NV4, were all originally isolated 

from turnip. Although the potato isolate, 1-1, was capable of degrading turnip tissue it was not 

as virulent as some of the turnip-isolated strains on turnip. Isolate 1-1 was significantly more 

virulent on potato discs than on turnip, indicating host specificity. 

When these isolates were applied to potato discs, PBd1, the most virulent isolate on 

turnip tissue discs, had no ability to degrade potato. This may imply that some Streptomyces 

spp. isolates have developed adaptions uniquely suited to pathogenicity on turnip and may be 

worth further study.  

One of these adaptations may be an ability to detoxify glucosinolates commonly 

produced by Brassicas like turnip. These compounds break down to isothiocyanates (Gouws-

Meyer et al., 2018) which have been indicated to possess biostatic effects on Streptomyces. 

Isolates that have developed a capability to resist these compounds may be interesting for 

future study. 

Although there was not a clear-cut and statistically significant indicator for CWDE 

production and virulence, a correlation seems likely based upon how many of the highest 

CWDE producers were also capable of causing disease on turnip. This result underscores the 

 

66 

 

 

importance to pathogenic Streptomyces spp. of possessing factors other than CWDE. Some of 

these factors, like toxins, have been explored further in Chapter 2 of this study.  

The extent of host specificity by this pathogen has not been widely explored, and most 

literature points to a lack of host specificity by the toxin thaxtomin to conclude that host 

specificity is not likely. This conclusion may underestimate the importance of other factors in 

disease establishment by the pathogen. The results herein indicate that further study may be 

indicated. 

 

 

 

67 

 

 

CHAPTER 3. RESPONSE OF TURNIP SEEDLINGS TO INOCULATION WITH STREPTOMYCES 

AND TREATMENT WITH PHYTOTOXINS 

Seedling pathogenicity assays are often performed as a quick and effective way to 

understand in vivo the dynamic interaction between plants and pathogens, and how these 

interactions affect long-term growth and survival (Maskell et al., 1999). Seedling inoculations 

have several advantages over soil-grown plant inoculation assays such as decreased growth and 

incubation periods, reduced greenhouse or growth chamber requirements, and ease of 

inoculation and handling (Ishiga et al., 2011). The pathosystem Arabidopsis thaliana-

Pseudomonas syringae has been studied as a model of microbial pathogenesis and plant innate 

immunity, and the strain which causes bacterial speck of tomato Pst DC3000, has been used as 

a model for studying the molecular basis of plant-pathogen interactions. A more rapid and 

reliable assay for this model was delivered by switching from soil-grown plants to 2-week-old 

seedlings for easier inoculation with a bacterial suspension (Ishiga et al., 2011).  

In a study by Maskell et al. (1999), the effects of turnip mosaic potyvirus (TuMV) and 

turnip yellow mosaic tymovirus (TYMV) in Brassica oleracea, wild cabbage, were investigated 

through the inoculation of seedlings germinated from five populations of wild cabbage.  

Seedlings at the three to five leaf stage were inoculated with TuMV, TYMV or water and the 

ability of the virus to cause reduced survival, reproduction and growth was measured (Maskell 

et al., 1999).  

For Streptomyces bacteria, seedling inoculations provide a realistic simulation of 

pathogen entry through the immature tissues of host plants. In a 1949 study by Hooker, the 

growth responses of beet, corn, cucumber, pea, radish, and soybean seedlings grown in agar 

 

68 

 

 

inoculated with Streptomyces were evaluated. The root weights of soybean and pea seedlings 

inoculated with S. scabies were less than half of those grown in either water controls or with 

nonpathogenic Streptomyces cultures. Both Purple Top White Globe (PTWG) turnip and Early 

Scarlet Globe radish seedlings were susceptible to S. scabies (Hooker, 1949). This seedling study 

indicated that reduced root growth, severe impairment of lateral root development, and 

premature death could be predicted from the early effects of Streptomyces inoculation 

(Hooker, 1949).  

Phytotoxins play a role in virulence and/or pathogenicity in several plant-pathogens 

systems. Thaxtomin has been used to determine relative resistance or susceptibility of potato 

to scab. However, the results of these studies suggest that screening with thaxtomin may not 

correlated 100% with resistance to the pathogen (Kaiser et al., 2020; Tegg et al., 2010). Part of 

this may be that pathogenic Streptomyces produces other toxins, including a coronafacic acid 

derivative that has properties similar to the well-studied toxin coronatine (Bignell, 2014). Thus, 

it is likely that any effective toxin screen for resistance may need to use more than one toxin. 

Objectives 

The purpose this study was to determine: 1) Disease reactions on the root, hypocotyl, 

and cotyledons of various turnip variety seedlings resulting from inoculations with selected 

Streptomyces isolates, and 2) The effect of the phytotoxins thaxtomin and coronatine on turnip 

and one radish cultivar. The overall objective was to determine if either pathogen inoculations 

and/or toxin application could be used to determine the relative resistance or susceptibility of 

turnip to pathogenic Streptomyces. 

 

69 

 

 

 
Seedling Inoculation Studies 
 

MATERIALS AND METHODS 

Purple Top White Globe (PTWG) turnip seeds were surface sterilized by washing for 5 

minutes in a 70% ethanol solution, rinsed with sterile water, and then washed for 10 minutes in 

a 10% chlorine bleach solution (0.525% hypochlorite). Seeds were rinsed in sterile water and 

allowed to dry completely. Glass Petri dishes containing a disc of #1 Whatman filter paper were 

autoclave sterilized, moistened with sterile water, and surface-sterilized seeds were germinated 

in the dark. Nine Petri dishes with water agar had a line drawn across the middle of each plate. 

Five uniformly germinated seedling were place above that line and then Streptomyces isolates 

were streaked on the section of the agar media directly below the line. Each isolate was 

replicated two times. As a control, turnip seedlings were placed onto non-inoculated water agar 

media plates. The Petri dishes containing seedlings were placed upright to encourage roots to 

grow through the pathogen. The seedlings were grown under fluorescent lights with 12-hour 

photoperiod for 7 days. After 7 days, the hypocotyl length of each seedling from root juncture 

to just below the cotyledon juncture was measured. To compare the effects across different 

seedling varieties, each seedling hypocotyl length (HL) was divided by the average HL of the 

control for that variety. This normalized the observations since different varieties of seedlings 

naturally produce hypocotyls of different lengths. The Streptomyces isolates used in this study 

were 1-1, B9.8, NV1, NV4, PBd1, TB-CD, and V4. These were provided by the lab of Dr. Raymond 

Hammerschmidt from Michigan-grown turnips (except for 1-1 which was isolated from potato) 

showing symptoms of scab. 

 

70 

 

 

After HL measurement, qualitative ratings of necrosis, stunting and other visible signs of 

stress were made. The purpose of the qualitative ratings was to determine the symptoms 

correlated to a treatment effect on hypocotyl length. The qualitative observations were also 

used to evaluate the effect of the application of the toxins thaxtomin and coronatine. 

Qualitative observations included an evaluation of the following characteristics: necrosis 

(darkened or black tissue on any part of hypocotyl or root), stunting (how many hypocotyls 

were affected), hypertrophy (thickening), curling, and a root rating. The roots of each seedling 

were rated for the following symptoms: 

•  Thinning- decrease in root thickness, roots are weak and break 
•  Missing laterals-roots lack side branching and consist of one, singular root 
•  Stunting-roots were shorter than controls 
•  Necrotic or absent roots-some roots failed to grow, or a small root formed but 

was necrotic. 

 

The root rating consisted of counting how many symptoms were visible and assigning a 

higher number with each additional symptom present. The higher the score from 0 (no effect) 

to 4 (the most effect), the more detrimental the pathogen/toxin was to the seedling (Table 3.1).   

Table 3.1 Root Rating Applied to Seedlings 

Symptom 

Thinning 

Missing laterals 

Stunted 

Necrotic/absent 

Root rating 

 

 

 

 

0 

Symptoms Present  

x 

  

  

  

1 

x 

x 

 

 
2 

x 

x 

x 

 
3 

x 

x 

x 

x 

4 

Note. The presence of none of the symptoms resulted in a root rating of 0. One symptom resulted in a 1, and so 
forth. Roots showed fairly predictable progression of symptoms with thinning usually occurring first, followed by 
missing laterals, etc. so the table is designed to show the progression from 0, no symptoms, to 4, where necrosis or 
missing roots typically showed up in only the most affected seedlings. 

 

71 

 

After completing this experiment on PTWG, the other turnip and radish cultivars were 

tested using the same methods. The turnip cultivars used were Golden Globe (GGT), Japanese 

Baby (JBT), and Purple Prince (PPT) (Table 3.2). The radish cultivar used was Early Scarlet Globe 

 

(ESGR).  

Table 3.2 Seedling Cultivars Used in the Study 

Cultivar Name 
Golden Globe 
Japanese Baby  
Purple Prince  

Abbrev. 

GGT 
JBT 
PPT 

Purple Top White 

PTWG 

Attributes 

Golden skin, firm, crisp flesh, tasty tops, 55 days to harvest* 
Pearly-white flesh, mild flavor, tasty tops, quick growing** 
Crisp, tasty white flesh, delicious tops, fast maturing*** 
Pink to purple skin, white flesh, sweet, tender flavor * 

Globe 

Early Scarlet 
Globe  (radish) 

ESGR 

Popular variety, bright red, crisp, tender, juicy flesh* 

Note. *Burpee Seeds (www.burpee.com). **Renee’s Garden Seeds (www.reneesgarden.com). ***West Coast 
Seeds (www.westcoastseeds.com). 
 

This study was performed the same as the experiment on PTWG except the media was 

water agar instead of OBA. Seed of some cultivars required more than 1 day to germinate prior 

to placement in sterile Petri dish with inoculum, and some seed benefitted from 12 hours of 

light for germination.  

The isolates used in the study (Table 3.3) were selected for their ability to cause 

disintegration on turnip discs (Chapter 1) and for their ability to produce cell wall degrading 

enzymes. The NV1 isolate was selected because it produces high levels of cell wall degrading 

enzyme activity but does not appear to be pathogenic to either turnip, beet, or radish 

seedlings. 1-1 was isolated from potato and has been confirmed as Streptomyces scabies by 

PCR amplification. 

 

72 

 

Table 3.3 Streptomyces Pathogenicity and Host Source 

 

Source 

Potato 

Turnip 

Turnip 

Turnip 

Turnip 

Turnip 

Turnip 

Pathogenic 

Yes 

Yes 

No 

Yes 

Yes 

Yes 

Yes 

Isolate 

1-1* 

B9.8 

NV1 

NV4 

PBd1 

TB-CD 

V4 

 

Toxin Study 

Seed was washed and germinated in the same manner and then placed in a row across 

the middle of the Petri dish. The phytotoxins thaxtomin A and coronatine were applied at the 

concentrations in Table 3.4 to pre-autoclaved, 4 x 0.5 cm strips of #1 Whatman filter paper. The 

toxin strips were air dried in a laminar flow hood before being placed into the Petri dish 

immediately below the seeds, covering the roots if present.

 

73 

 

Table 3.4 Toxin Treatment Per Filter Paper Strip (nmoles) 

Thaxtomin A 

Coronatine 

Toxins Combined 

Thaxtomin A 

Coronatine 

 

0.18 
1.83 
9.13 
18.26 
91.32 
182.65 

1.83* 
3.65* 
9.13 
18.26 
91.32 
** 

2.50* 
5.01* 
12.52 
25.05 
125.23 
** 
Note. *Combined treatment amount differed from single treatment. ** Toxin level too high to permit growth 
 

0.25 
2.50 
12.52 
25.05 
125.23 
250.47 

The Petri dishes containing seedlings were placed upright to encourage roots to grow 

through the toxin(s), under fluorescent lights with 12-hour photoperiod for 7 days. After 7 days, 

the effect of toxins applied to the cultivars was first determined by comparing hypocotyl 

lengths (HL) of treated seedlings. The hypocotyl length of each seedling from root juncture to 

just below the cotyledon juncture was measured, and examined for integrity, discoloration, 

necrosis, and other signs of stress. Following measurement of HL, qualitative observations of 

each seedling were made that included a rating of root health. 

Turnip Seedling Histopathology  

Tissue from the hypocotyl and root was cut from each of three PTWG seedlings grown in 

the presence of the following treatments: control, 50 ng thaxtomin, 50 ng coronatine and 50 ng 

of each toxin. Using a sterile razor, a 4-cm section was cut that included 2 cm of the hypocotyl 

and 2 cm of radical root with the hypocotyl/radical junction in the center. Tissue sections were 

immediately placed in formalin–acetic alcohol (FAA; formaldehyde: acetic acid: 70% ethanol, 

5:5:9 v/v/v) fixative. After 48 hours, the FAA was decanted, and the samples were vacuum 

infiltrated with paraffin (melting point: 54‐55°C) on the tissue processor (Sakura VIP 2000) and 

embedded in paraffin with an embedder (Thermo Scientific™ HistoCentre™ III). Paraffin‐

 

74 

 

 

embedded samples were cut into sections (6-µm thick) using a rotary microtome (model 

RM2245, Leica Biosystems) according to the methods of Devkota and Hammerschmidt (2019), 

with the embedding and sectioning done by the Michigan State University histology lab 

(Biomedical Physical Sciences, 567 Wilson Rd. Rm 2133).  

Three sections were prepared from each tissue segment and were mounted on 

positively charged glass slides, processed through ethanol: xylene series, and stained with 0.1% 

T-blue by the histology lab. The toxin treatment effects and host responses in the sections were 

visualized using a light microscope at 10X magnification (DM2500, Leica). 

Statistical Analyses 

Hypocotyl length was normalized as a percent of the control to enable comparisons 

across a variety of seedling cultivars. Normality assumption was tested by using normal 

probability plots, and the dataset satisfied the assumption. Homogeneity of variance 

assumption was checked using manual Levene’s test. Since the result indicated heterogeneous 

variance, possible models were compared based on the Akaike’s Information Criteria and 

Bayesian Information Criteria to select the final model. Statistical model for hypocotyl length 

consisted of fixed effects of variety, treatment (isolate), and their interaction. Petri dish nested 

within the treatment and variety was included as a random factor. Heterogeneous variance by 

treatments was selected as a final model. Differences among isolates were presented 

separately by variety. When the effect of isolate was significant (p < .05), the mean separation 

was performed by using Fisher’s Least Square Difference. The analysis was performed by using 

PROC MIXED procedure in SAS 9.4 (SAS Institute, USA).  

 

75 

 

 

 

RESULTS 

Disease severity in all radish or turnip cultivars inoculated with Streptomyces spp. 

isolates was first determined by comparing hypocotyl lengths (HL) of inoculated seedlings.  

Following measurement of hypocotyl length, qualitative observations were made that included 

a rating of root health. Root health, based on Table 3.1, seemed to be the most important 

qualitative factor in predicting detrimental effect of pathogen inoculation. 

Quantitative and Qualitative Results of Pathogen Inoculation of Purple Top White Globe 
(PTWG) 
Quantitative Effects on PTWG 

All of the isolates except NV1 showed a significant treatment effect in hypocotyl length 

from the control on PTWG (Figure 3.1). Pairwise comparisons indicated a significant degree of 

separation between the isolates PBd1, TB-CD, and V4 compared to the isolates B9.8 and NV4 

(Figure 3.1). 

As a percentage of the control, in seedlings inoculated with NV4, hypocotyl length was 

reduced to just 35% of the control and was statistically similar to isolates 1-1 (42%) and B9.8 

(39%; Figure 3.1). 

 

76 

 

A

A

B

BC

BC

CD

D

D

 

120%

l

 

 

o
r
t
n
o
C
 
f
o
%
 
s
a
h
t
g
n
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L
 
l
y
t
o
c
o
p
y
H

100%

80%

60%

40%

20%

0%

NV1

Control

PBd1

V4

TB-CD

1-1

B9.8

NV4

Isolates

 

Figure 3.1 Purple Top White Globe Hypocotyl Length as % of Control for All Isolates 
Note. Different letters indicate significant differences among isolates at .05 significance level. 
 

Qualitative Effects PTWG  

PTWG was susceptible to necrosis and stunting in 1-1 and some hypertrophy was seen 

(Table 3.5). Hypertrophy, or a thickening in the hypocotyl, was most often seen in the thaxtomin 

treatments. Hypertrophy was less common with the turnip pathogen inoculations.  

In addition to stunted growth, the seedlings grown in B9.8 disintegrated readily, had 

stunted roots with no lateral roots, some black hypocotyls, small cotyledons, and browning 

(Table 3.5). B9.8 inoculation resulted in the shortest hypocotyls but did not present as many 

visible symptoms as 1-1. In Figure 3.2 the effect of pathogen inoculation between the control 

and NV4 (NV4, B9.8, and 1-1 were statistically similar) is shown. The healthy roots of the 

control contain many lateral roots with root hairs, and all are white and of normal size. NV4 

stunted the hypocotyls and roots of the seedlings and caused some hypocotyls to curl. The 

roots were thinned, broken and disintegrating, and some roots have not formed. 

 

77 

 

 

The characteristics evaluated in the qualitative rating of inoculation with PBd1, the least 

detrimental pathogen to PTWG, can be seen in Figure 3.3. There is a difference in hypocotyl 

length, root thinning, lack of lateral roots, and root stunting. The control had hypocotyls 

exceeding 5 cm in length and the roots had lateral roots emerging with smaller root hairs. This 

contrasts with the poor growth of the seedlings in PBd1, where the tallest hypocotyl reaches 

about 3 cm and the roots extend through one, often truncated, main shoot. PBd1 was 

statistically similar to TB-CD and V4 (Figure 3.1). 

Table 3.5 Qualitative Symptoms on PTWG Caused by Streptomyces Spp. Isolates 

Symptom 

necrosis 

stunting 

hypertrophy 

curling 

root rate* 

1-1 

B9.8 

4 

8 

4 

1 

Isolate 

NV4 

2 

9 

0 

3 

1 

5 

2 

3 

PBd1 

V4 

0 

2 

0 

1 

1 

5 

2 

2 

3.0 

3.0 

2.7 

2.0 

1.6 

Note. *Refer to root rating characteristics in Table 3.1 

 

Figure 3.2 PTWG Control vs. NV4 Isolate  
Note. Control on left and NV4 on right 

 

78 

 

 

Figure 3.3 PTWG Control vs. PBd1 Isolate 
Note. Control on left and PBd1 on right

 

 

 

79 

 

 

Quantitative and Qualitative Results of Pathogen Inoculation of Purple Prince Turnip (PPT) 
Quantitative effects on PPT 
 

The application of isolates to PPT resulted in a growth stimulation effect from NV1. 

There was a significant difference in HL produced when inoculated with this isolate that 

exceeded the control HL by 24%. There was no treatment effect of TB-CD on PPT. There was a 

statistical similarity in the treatment effect of isolates NV4, PBd1, B9.8, and 1-1, and between 

PBd1, B9.8, 1-1, and V4 (Figure 3.4). 

A

B

B

l

o
r
t
n
o
C
%
 
s
a

 

 
 

h
t
g
n
e
L
 
l
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t
o
c
o
p
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H

140%

120%

100%

80%

60%

40%

20%

0%

C

CD

CD

CD

D

NV1

Control

TB-CD

NV4

PBd1

B9.8

1-1

V4

 

Figure 3.4 Purple Prince Turnip Hypocotyl Length as Percent of Control 
Note. Different letters indicate significant differences among isolates at .05 significance level. 
 
Qualitative effects on PPT 
 

The most distinctive qualitative effect in PPT was the root growth (Table 3.6). Although 

the HL of PPT were not as markedly stunted as in the other isolates, the turnip showed 

significant symptoms of distress (Figure 3.5 and Table 3.6), severe root stunting and lack of 

lateral roots. 

 

80 

 

 

A side-by-side comparison of the treatment effect of NV4, B9.8 and 1-1 can be seen in 

Figure 3.5. The hypocotyls of the control were longer and healthier than in either B9.8 or 1-1. 

The hypocotyls in Petri dish B. inoculated with NV4 also appear more robust and healthier 

when compared to the other two pathogens, B9.8 and 1-1. The roots of the seedlings in Petri 

dish B. (root rating 2) show some lateral root development. The roots of the seedlings in both C. 

and D. show severely stunted, thin roots lacking lateral root formation (Figure 3.5). 

Table 3.6 Qualitative Symptoms on Purple Prince Turnip Caused by Streptomyces Spp. Isolates 

PPT 

HL as % Control 

Stunting 

Necrosis 

Root rate 

1-1 

57% 

2 

3 

3.3 

B9.8 

60% 

1 

1 

3.3 

Note. *For root rating see Table 3.1 

Isolate 

NV4 

72% 

1 

0.5 

2.0 

PBd1 

60% 

4 

0 

2.3 

V4 

56% 

3 

1 

1.7 

 

81 

 

 

Figure 3.5 Treatment Effect on Hypocotyl Length of NV4, B9.8, and 1-1 on PPT 
Note. A. control, B. NV4, C. B9.8, D. 1-1 

 

 

82 

 

 

Quantitative and Qualitative Results of Pathogen Inoculation of Golden Globe Turnip (GGT) 
Quantitative effects on GGT  
 

All of the isolate inoculations, even NV1, resulted in a significant difference in treatment 

effect to hypocotyl length from the control (Figure 3.6). Post-hoc tests indicated a significant 

degree of separation among the isolates NV1, TB-CD, NV4, and V4 and B9.8 and 1-1 (Figure 3.6), 

with similarities among TB-CD, NV4, V4, and PBd1, and similarities between PBd1 and 1-1. As a 

percentage of control, seedlings inoculated with B9.8 achieved a hypocotyl length of just 27%, 

with isolate 1-1 (36%) also having a profound effect. The hypocotyl stunting of B9.8 on GGT was 

the highest across all seedling varieties and isolates. 

A

 

l

o
r
t
n
o
C
%
 
s
a

 

 
 

h
t
g
n
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L
 
l
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t
o
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y
H

120%

100%

80%

60%

40%

20%

0%

B

BC

BC

BC

CD

DE

E

Control

NV1

TB-CD

NV4

V4

PBd1

1-1

B9.8

Isolate

Figure 3.6 Golden Globe Turnip Hypocotyl Length as Percent of Control 
Note. Different letters indicate significant differences among isolates at .05 significance level.

 

 

83 

 

 

Qualitative Effects on GGT 
 

The seedlings in this treatment group had very thin roots that were not stunted but 

were necrotic, weak, and lacking lateral root formation (Figure 3.7). Hypertrophy was seen in 

the application of thaxtomin but was rare in the treatments containing pathogens. GGT had the 

highest occurrence of hypertrophy in response to the application of pathogens compared to all 

the other isolates, with nearly twice as much as PTWG. The high rate of hypocotyl stunting in 

GGT correlated with a root rating of 4 (Table 3.7). 

Figure 3.7 Treatment Effect of B9.8 on Golden Globe Turnip 
Note. Orange arrow indicates enlarged seedling with weak roots (root rate 4) and thickened hypocotyl referred to 
as hypertrophy. 

 

 

84 

 

 

Table 3.7 Qualitative Symptoms on Golden Globe Turnip Caused by Streptomyces Spp. Isolates 

GGT 

HL % of control 

Stunting 

Necrosis 

Root Rate 

Note. *For root rating see Table 3.1 

1-1 

36 

11.0 

3 

4.0 

B9.8 

27 

12.0 

5 

4.0 

Isolate 

NV4 

58 

7.0 

0 

1.3 

PBd1 

48 

9.0 

0 

3.0 

V4 

58 

6.0 

0 

2.3 

Quantitative and Qualitative Results of Pathogen Inoculation of Japanese Baby Turnip (JBT) 
Quantitative effects on JBT  

JBT showed the most restriction in HL as a result of the application of NV1, which was 

either non-pathogenic or acted as a growth stimulator in PPT, PTWG and ESGR. Only GGT and 

JBT showed statistically significant shortened hypocotyls from NV1 (Figure 3.8). The most 

detrimental pathogens were NV4, B9.8 and 1-1. Figure 3.9 shows the devastating treatment 

effect caused by 1-1 on JBT. 

l

 

 

o
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C
 
f
o
%
h
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l
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t
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A

A

A

120%

100%

80%

60%

40%

20%

0%

B

B

BC

C

C

Control

TB-CD

V4

PBd1

NV1

NV4

B9.8

1-1

Isolate

 

Figure 3.8 Japanese Baby Turnip Hypocotyl Length  
Note. Different letters indicate significant differences among isolates at .05 significance level.

 

85 

 

Figure 3.9 Effect of 1-1 on JBT Hypocotyl Length 
Note. control (left), 1-1 (right)

 

 

 

86 

 

 

Qualitative Effects on JBT 
 

JBT seedlings also showed more signs of necrosis than other varieties, and two of the 

highest root ratings across all varieties (Table 3.8). In isolate PBd1 there was less stunting 

observed, although the roots had a higher rating. 

Table 3.8. Qualitative Symptoms on JBT Caused by Streptomyces Spp. Isolates 

JBT 

HL as % control 

Necrosis 

Stunting 

Root rate 

1-1 

32 

7 

13 

4 

Isolate  

B9.8 

NV4 

PBd1 

35 

10 

15 

4 

52 

0 

10 

1.7 

58 

1 

4 

2.5 

V4 

89 

0 

0 

1.5 

 
Quantitative and Qualitative Results of Pathogen Inoculation of Early Scarlet Globe Radish 
(ESGR) 
 
Quantitative Effects on ESGR 
 

TB-CD had a definite growth-stimulating effect on radish (Figure 3.11). The other results 

were similar to the treatment effects seen in the turnip varieties, with 1-1 having the biggest 

effect on hypocotyl length.  The pathogens V4 and NV1 were not statistically different from the 

control, and PBd1, NV4, B9.8, and 1-1 were statistically similar. 

 

87 

 

160%

 
l

A

Early Scarlet Globe Radish

 

120%

 

140%

100%

 

o
r
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C
%
 
s
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H

80%

60%

40%

20%

0%

B

B

BC

CD

CD

CD

D

TB-CD

Control

NV1

V4

PBd1

NV4

B9.8

1-1

 
Figure 3.10 Effect of Isolates on ESGR Hypocotyl Length 
Note. Different letters indicate significant differences at .05 significance level. 

 

 

88 

 

 

Figure 3.11 TB-CD Promotes Hypocotyl Growth in ESGR  
Note. Yellow arrows indicate the Petri dish from which the enlarged section was obtained. 

 

 
Qualitative Effects on ESGR 

Table 3.9 Qualitative Symptoms on ESGR Caused by Streptomyces Spp. Isolates 

ESGR 

Necrosis 
Stunting 
Root rate 

1-1 

55% 

3 
n/a 
n/a 

Isolate 

NV4 

54% 

1 
9 
0.7 

B9.8 

47% 

5 
15 
4 

PBd1 

55% 

0 
3 
2.8 

V4 

78% 

0 
1 
1 

 

 

89 

 

Overall Comparison of Host Cultivar Against Streptomyces Isolates 

 

The shortest hypocotyls as a percentage of the control, across all seedling varieties 

including radish (ESGR), were measured in seedlings inoculated with 1-1 (32%) and B9.8 (27%). 

Depending on seedling cultivar, all isolates, including NV1, caused at least one significant 

difference in hypocotyl length compared to control (Table 3.10). In the case of isolates TB-CD 

and NV1 however, the statistical difference was often the result of hypergrowth, not stunting. 

Table 3.10 Hypocotyl Lengths as a Percent of Control for Each Variety and Isolate 

Host Variety 

ESGR 

PTWG 

PPT 

GGT 

JBT 

1-1 

44% 

42% 

57% 

36% 

32% 

B9.8 

50% 

39% 

60% 

27% 

35% 

NV1 

99% 

106% 

124% 

70% 

55% 

Isolate 

NV4 

58% 

35% 

72% 

58% 

52% 

PBd1 

62% 

68% 

60% 

48% 

58% 

TB-CD 

143% 

59% 

95% 

64% 

102% 

V4 

87% 

67% 

56% 

58% 

89% 

 
PTWG Compared with PPT 
 

There was a significant difference in HL between Purple Prince turnip (PPT) and the 

other host varieties for most pathogens. PPT seemed the most resistant to pathogens of the 

cultivars tested, including radish. This is seen in the consistently higher HL as % of control in PPT 

compared to other cultivars. PPT performed better than PTWG against each pathogen except 

PBd1 and V4, for which there was no significant difference (Table 3.11). In addition, the lowest 

HL of 56 % (V4) was significantly different from the lowest HL seen in PTWG of 35% (B9.8; Table 

3.10). 

 

90 

 

Table 3.11 Hypocotyl Length as % Control for PTWG vs. PPT 
Isolate 

 

 

PTWG 

PPT 

1-1 

42% 

57% 

B9.8 

39% 

60% 

NV1 

106% 

124% 

LSM p-value 

.0003 
Note. ns indicates lack of significant difference at p <.05. 

.0377 

.0024 

NV4 

58% 

72% 

<.0001 

PBd1 

TB-CD 

68% 

60% 

ns 

59% 

95% 

<.0001 

V4 

67% 

56% 

ns 

PTWG Compared with GGT 
 

Although the data appear to show that GGT performed worse than PTWG against B9.8, 

there was not a significant difference in HL as % of control (Table 3.12). There was a significant 

difference in HL as % of control in isolates NV1, NV4, and PBd1. GGT did not respond to growth 

stimulation effects seen in ESGR or PPT, with no HL above 100% for either NV1 or TB-CD (Table 

3.10).   

Table 3.12 Hypocotyl Length as % Control for PTWG vs. GGT 

NV1 
106% 
70% 

Isolate 
NV4 
35% 
58% 

PBd1 
68% 
48% 

0.0138 

TB-CD 
59% 
64% 
ns 

V4 
67% 
58% 
ns 

p-value 

0.0039 
Note. ns indicates lack of significant difference at p <.05. 

<.0001 

 

PTWG 
GGT 

1-1 
42% 
36% 
ns 

B9.8 
39% 
27% 
ns 

 

91 

 

 

PPT Compared with ESGR 
 

PPT had significantly higher hypocotyl length when compared to ESGR, for all isolates 

except TB-CD and V4, for which there was no significant difference.  

Table 3.13 Hypocotyl Length as % Control for PPT vs. ESGR 

 

ESGR 
PPT 

p-value 

1-1 
44% 
57% 
0.002 

B9.8 
50% 
60% 

0.0017 

NV1 
99% 
124% 
<.0001 

Isolate 

NV4 
58% 
72% 

<.0001 

PBd1 
62% 
60% 
0.01 

TB-CD 
143% 
95% 
Ns 

V4 
87% 
56% 
ns 

Note. ns indicates lack of significant difference at p <.05. 

PPT Compared with GGT 
 

PPT performed better than GGT against all isolates except V4. PPT was susceptible to 

possible growth regulator effects of NV1 whereas GGT was not.  

Table 3.14 Hypocotyl Length as % Control for PPT vs. GGT 

 

 
GGT 
PPT 
p-value 

1-1 

B9.8 

NV1 

36% 
57% 

27% 
60% 

70% 
124% 

0.0049  <.0001 

<.0001 

Isolate 
NV4 

58% 
72% 
0.0102 

PBd1 

TB-CD 

V4 

48% 
60% 

64% 
95% 

58% 
56% 

0.0182  <.0001 

ns 

Note. ns indicates lack of significant difference at p <.05.

 

92 

 

 

PPT Compared with JBT 
 

PPT performed better than JBT with a significant difference against all isolates except 

TB-CD and V4, for which there was no difference in HL. 

Table 3.15 Hypocotyl Length as % Control for PPT vs. JBT 

 
JBT 
PPT 
p-value 

1-1 

B9.8 

NV1 

Isolate 
NV4 

PBd1 

TB-CD 

32% 
57% 

35% 
60% 

55% 
124% 

0.0008  <.0001 

<.0001 

52% 
72% 
0.0002 

58% 
60% 

102% 
95% 

0.0437  ns 

V4 
89% 
56% 
ns 

Note. ns indicates lack of significant difference at p <.05. 

 

93 

 

Effect of Phytotoxins 
Thaxtomin Effects on Hypocotyl Length 

 

The effect of thaxtomin application to seedlings was dependent upon thaxtomin 

concentration. At very low concentrations of 0.18-1.83 nmoles thaxtomin, two of the turnip 

cultivars, GGT and PTWG, and the radish, ESGR, grew hypocotyls of equal or greater length than 

controls. At moderate toxin concentrations of 9.1 nmoles (50 ng) thaxtomin ESGR, PPT, and 

PTWG hypocotyls were stunted to less than half of the control hypocotyls. A noted decrease in 

seedling health was seen at all concentrations greater than 50 ng (9 nmoles) of toxin. At 100 ng 

thaxtomin the seedlings lack substantial roots, thickened hypocotyls were observed, and some 

necrosis of root and hypocotyl was common. Above 100 ng, most seedlings lacked root 

structures and tissues were blackened, clubbed, and disintegrated. At higher toxin 

concentrations of 18.30–182.60 nmoles there was universal stunting of hypocotyls along with 

deformation, blackening, and disintegration of hypocotyls, roots, and cotyledons.

 

94 

 

 

l

o
r
t
n
o
C
 
f
o
%
 
s
a
 
L
H

 

140%

120%

100%

80%

60%

40%

20%

0%

1 ng

10 ng

50 ng

100 ng

500 ng

1000 ng

Thaxtomin Concentration (ng)

PTWG

GGT

JBT

ESGR

PPT

 

Figure 3.12 Effect of Different Concentrations of Thaxtomin on Hypocotyl Length in Seedlings of 
Five Varieties 
 
PTWG 

PTWG response to thaxtomin application was representative of the response seen in 

most other varieties. In addition to hypocotyl length, qualitative effects were rated and are 

listed in Table 3.16. Seen in Figure 3.13, thaxtomin has caused hypertrophy on PTWG seedlings, 

roots that have failed to grow past the toxin strip, chlorosis, and some browning of hypocotyls. 

Table 3.16 Qualitative Effects of Thaxtomin Application to PTWG 

ngrams 
nmoles 
necrosis 
stunting 
hypertrophy 
curling 
chlorosis 
root rate 

50 ng 
9.13 

PTWG 
100 ng 
18.26 

500 ng 
91.32 

1000 ng 
182.65 

0 
13 
11 
1 
1 
3 

0 
15 
13 
4 
4 
3 

10 
15 
15 
6 
1 
4 

10 
15 
15 
3 
4 
4 

 

95 

 

 

Figure 3.13 PTWG in 100 ng (18.3 nmoles) Thaxtomin  
Note. This is a close-up of one of three Petris dishes in the replication.  
 

 

Most of the plant varieties were affected by thaxtomin at low concentrations, although 

the difference between control and low thaxtomin concentrations often resulted in longer 

hypocotyl lengths. Both ESGR and PTWG grew longer hypocotyls in 0.18 nmoles than in 

controls, and PTWG and GGT grew longer hypocotyls in 1.83 nmoles than in controls. Only JBT 

and GGT had HL that were statistically the same as the control at 9.10 nmoles (Table 3.17). 

Table 3.17 Hypocotyl Length as Percent of Control in Thaxtomin Treatments for All Varieties 

ng 

nmoles 
ESGR 
PPT 

PTWG 
GGT 
JBT 

Thaxtomin Concentration 

1  
0.18 
122% 
99% 
102% 
96% 
76% 

10  
1.83 

99% 
81% 
113% 
103% 
54% 

50  
9.10 

100  
18.30 

500  
91.30 

1000  
182.60 

48% 
23% 
39% 
96% 
87% 

44% 
8% 
28% 
43% 
50% 

26% 
3% 
13% 
13% 
40% 

24% 
7% 
10% 
16% 
44% 

Note. Dark background indicates hypocotyl length statistically different from control at .05 significance 
 

One isolate, GGT, showed some resistance to changes in hypocotyl length at thaxtomin 

concentrations up to 50 ng. This result was statistically significant compared to the hypocotyl 

lengths of all the other cultivars, except JBT, in 50 ng thaxtomin. This possible resistance to the 

toxins was broadly seen at thaxtomin concentrations from 0.18–9.10 nmoles (1 ng–50 ng; Table 

3.18). 

 

96 

 

 

Table 3.18 Pairwise Comparison of Different Varieties to GGT 
 

Treatment Level 
100 
18.30 

500 
91.30 

1000 
182.60 

ng 

nmoles 
ESGR 
PPT 

PTWG 

1 

0.18 
ns 

0.0013 

ns 

10 
1.83 
0.0034 

ns 
ns 

JBT 

0.0007 

<.0001 

50 
9.10 
0.0027 
0.0006 
0.0003 
0.017 

ns 

0.0006 

ns 
ns 

ns 
ns 
ns 
ns 

ns 
ns 
ns 
ns 

Note. ns indicates lack of significant difference at p <.05. 

 

 

97 

 

There was also a visible difference in qualitative symptoms between GGT vs. PPT, ESGR, 

and PTWG at a thaxtomin concentration of 9.10 nmoles (50 ng; Figure 3.14). 

 

 

Figure 3.14 Comparison of GGT, PPT, PTWG, and ESGR in 9.10 nmoles Thaxtomin 

The difference between GGT and the other seedling cultivars is striking. Whereas the 

roots in each replicate of GGT have grown far past the toxin strip, only ESGR has grown roots in 

one replicate that extend past the toxin strip. PPT has signs of necrosis that is lacking in GGT, 

and PPT, PTWG and ESGR all have severely stunted hypocotyls that GGT does not.  

 

98 

 

 

Coronatine 
 

Coronatine treatments also shortened hypocotyls, but the effect was not as acute, and 

the seedlings were able to tolerate higher molar concentrations than thaxtomin. For example, 

18.26 nmoles (100 ng) of thaxtomin reduced hypocotyl length of all tested varieties to 50% or 

less of control, whereas 25.05 nmoles of coronatine (100 ng) shortened just three of the 

varieties below 50%. At 91.32 nmoles (500 ng) of thaxtomin the seedlings were universally 

compromised with hypocotyls between 3-40% of control. At 125.23 nmoles (500 ng) 

coronatine, some HL were still between 29-48% control. 

Seedlings treated with coronatine also had fewer visible signs of distress, with no 

incidence of necrosis or hypertrophy. The most common symptoms were stunted hypocotyls 

and roots, and chlorotic cotyledons. Figure 3.15 lists the results of hypocotyl length as % of 

control for all cultivars. 

Figure 3.15 Hypocotyl Length as % of Control for all Varieties Treated with Coronatine 
 

 

 

99 

 

The hypocotyl lengths of PTWG were significantly different at all concentrations except 

10 ng. Although there was a trend of coronatine causing hypergrowth in both JBT and PTWG at 

low concentrations, the results were not statistically significant. PPT showed the most 

susceptibility to coronatine, with stunted hypocotyl length values that were statistically 

 

significant at all concentrations.  

Figure 3.16 shows a comparison of select PTWG seedlings following treatment with 

thaxtomin or coronatine. The two seedlings photographed were selected as representative of 

the most and least affected in each treatment replicate. There is some variation between the 

most and least affected seedlings at 50 ng concentrations for both thaxtomin and coronatine, 

but at 100 ng thaxtomin all seedlings were dramatically stunted, most were clubbed and 

exhibited a severe loss of root structure. The coronatine application of 100 ng did not cause 

such a consistent reduction in hypocotyl length as thaxtomin, but the seedlings were still 

affected, particularly in the lack of robust root formation. Although the roots of the seedlings 

treated with 100 ng coronatine are longer than in the 100 ng thaxtomin, they lacked the lateral 

roots seen in healthy seedlings.

 

100 

 

 

 

Figure 3.16 PTWG in Various Toxin Treatments  
Note. a. 50 ng thaxtomin b. 100 ng thaxtomin c. 50 ng coronatine d. 100 ng coronatine  
 

Table 3.19 lists in more detail the effects of coronatine on PTWG at various 

concentrations. From 25.05 nmoles and higher concentrations the effects on root formation 

correlates with a loss of growth similar to inoculation with pathogenic Streptomyces spp. 

isolates and higher levels of thaxtomin. 

In seedlings treated with coronatine, stunting and lack of lateral root formation 

correlates more with chlorotic tendencies than with the necrosis and hypertrophy seen in 

thaxtomin treatments (Table 3.19).  

 

101 

 

Table 3.19 Qualitative Effects of Coronatine Application to PTWG 

 

ngrams 
nmoles 
necrosis 
stunting 
hypertrophy 
curling 
chlorosis 
root rate 

 

50 ng 
12.52 

0.0 
4.0 
0.0 
0.0 
0.0 
1.7 

Coronatine 

100 ng 
25.05 

0.0 
14.0 
0.0 
2.0 
2.0 
3.0 

500 ng 
125.23 

0.0 
14.0 
0.0 
1.0 
3.0 
3.0 

1000 ng 
250.47 

0.0 
15.0 
1.0 
4.0 
8.0 
3.7 

Effect of Both Toxins Combined 
 

The application of both toxins to the seedlings resulted in similar effects to that seen in 

the separate applications of each toxin, but symptoms appeared at lower concentrations. The 

seedlings performed poorly at lower toxin concentrations than was seen with the application of 

one toxin at a time. The effect on PTWG from increasing concentrations of both thaxtomin and 

coronatine could be seen in the progression from a hypocotyl that reached about 4 cm with full, 

green cotyledons to a seedling with 0.5 cm hypocotyl length and chlorotic cotyledons. These 

seedlings’ roots were missing. The experiment was not repeated with 1000 ng of each toxin 

since 500 ng of each was consistently toxic to the plants. 

 

102 

 

Figure 3.17 Effect of Both Toxins Combined on PTWG  
Note. A. Control B. 50 ng each C. 100 ng each D. 500 ng each

 

 

 

103 

 

 

Histological Examination of Toxin Effects 
 

To examine the effects of toxin application on a cellular level, treated tissues were 

examined by light microscopy at 10X magnification. PTWG seedlings grown in 50 ng thaxtomin, 

50 ng coronatine, and 50 ng of both toxins were sampled at 72 and 144 hours post inoculation 

(hpi).  

Normal plant growth of rectangular, elongated cells is seen in Figure 3.18 A. The effects 

of thaxtomin in increasing the width of the cell are apparent in Figure 3.18 B., as well as green 

spots. In the control the cells are disk shaped and in the presence of thaxtomin they have 

become nearly square. In 50 ng CT, the normally rectangular epidermal cells have become 

practically round (Figure 3.18 C.), and this trend continues with 50 ng of each toxin but now the 

cells are showing a tendency to be more tall than wide (Figure 3.18 D.), a reversal of growth 

seen in the control. Rather than disk-like and more rectangular, the growth has become oval, 

almost square. This hypertrophy seen on a cellular level was observable in the stunted, 

hypertrophied appearance documented in both the pathogen inoculation and in the application 

of toxins. 

Measurements of representative cell lengths indicate a trend towards shortened 

epidermal cells with the addition of toxins, with the most reduction of epidermal cell length 

seen in 50 ng of each toxin (Table 3.20). The ratio of length to width went from 4:1 in control at 

72 hpi, to 1:1 in 50 ng of each toxin at 72 hpi. 

 

104 

 

 

Table 3.20 Measurement of Representative Cell Lengths at 10X Magnification 

 

Average 

µm 

Length  
Length  
Width  
Width  

 

 

Time (hpi) 

72 hpi 
144 hpi 
72 hpi 
144 hpi 

 0 
45 
58 
12 
13 

Toxin ng 

50 thaxtomin  

50 coronatine 

50 Each 

31 
64 
17 
25 

28 
33 
17 
20 

8 
22 
13 
13 

Figure 3.18 Comparison of Treated Tissue at 72-hpi in Thaxtomin, Coronatine, and Combined 
Toxins at 10X Magnification 
Note. A. control, B. 50 ng thaxtomin, C. 50 ng coronatine, D. 50 ng both toxins combined  

 

 

105 

 

 

 

Figure 3.19 Comparison of Treated Tissue at 144-hpi in Thaxtomin, Coronatine, and Combined 
Toxins at 10X Magnification 
Note. A. control, B. 50 ng thaxtomin, C. 50 ng coronatine, D. 50 ng both toxins combined  
 

 

106 

 

 

 
Symptoms Caused by Streptomyces Isolates 
 

DISCUSSION 

There was a high degree of variation in symptoms caused by different Streptomyces spp. 

isolates on the same host, and differences between hosts in response to the same 

Streptomyces spp. isolate. The same strain of Streptomyces could produce different symptoms 

on different hosts. The most consistently pathogenic isolates—B9.8, 1-1, NV4, and PBd1—

caused a statistically significant hypocotyl length reduction in every host tested, although the 

degree of reduction varied.  

Every isolate, including the “non-pathogenic” NV1 caused statistically significant 

hypocotyl length reduction in at least one host. Growth stimulation by NV1 and TB-CD was 

observed on some hosts. This could be due to the production of the plant hormone indole-3-

acetic acid (IAA) by the bacteria. IAA has been shown to be produced in some Streptomyces 

that act as plant growth-promoting rhizobacteria (PGPR) (Salwan et al., 2020). It is possible that 

NV1 and TB-CD have characteristics of PGPR, and this trend may warrant further study, 

especially in alternate hosts such as potato, turnip, and radish.  

There was a surprising degree of variation in the type of symptom produced by 

Streptomyces spp. in different hosts, although the Streptomyces scabies isolate 1-1 consistently 

caused disease that included necrosis. 

It would be interesting to repeat the histological examination following inoculation with 

Streptomyces isolates to see how closely plant defense responses mimic what is seen with 

 

107 

 

phytotoxins. Additional replications that would allow for statistically accurate comparisons of 

 

epidermal cell’s lengths and widths would also be informative.  

Indications of Resistance in Turnip Varieties 

Of all the hosts tested, PPT showed the most evidence of resistance to the pathogen 

and further study of this turnip for Streptomyces resistance may be indicated. It would be 

interesting to see how this variety performs in growth chamber or field tests when inoculated 

with various strains of Streptomyces spp. It was surprising that PPT was not resistant to the 

toxins after showing resistance to the isolates. It is possible that the plant is producing defense 

compounds to fight the bacteria rather than de-toxifying phytotoxins or does not induce the 

production of the toxins. 

The isolates GGT and JBT showed a strong trend to be the most susceptible to the 

pathogens, although surprisingly, GGT may have some resistance to thaxtomin. GGT was also 

different from the other turnip varieties in showing susceptibility to inoculation with NV1. 

 JBT appeared to show resistance in all three replicates to thaxtomin at 50 ng (9.13 

nmoles) but at no other concentration. This result probably needs to be retested since it seems 

likely to be an experimental error that caused the hypocotyls to evade the toxin. It is possible 

that they escaped encountering the toxin rather than resisting it. 

Effect of Phytotoxins  
 

Thaxtomin did not induce a significant growth difference to most hosts until the 50 ng 

(9.13 nmoles) concentration. At and above this level, the health of seedlings dropped off 

sharply. Higher concentrations of coronatine seemed to be tolerated and when qualitative 

ratings were considered, 100 ng coronatine affected roots more than hypocotyls and further 

 

108 

 

study may reveal that seedlings could be “rescued” if removed from the toxin. Rescue of 

seedlings inoculated with the same of amount of thaxtomin seems unlikely due to the severe 

 

tissue symptoms seen.  

The application of both toxins caused tissue death more quickly than with either toxin 

alone. Since it is likely the pathogen produces more than one toxin in nature, it seems likely 

that less of each would be needed to cause disease.  

It would be interesting to repeat the histological examination with additional 

replications to allow statistically accurate comparisons of epidermal cell’s lengths and widths.  

Further Studies 
 

Testing the effects of the necrogenic protein nec1 by applying the toxin to tissue 

seedlings inoculated with various strains of the pathogen would be interesting as this is the first 

toxin produced during infection. It would be informative to see if this necrosis-inducing protein 

would lead to the release of plant cell wall sugars like cellobiose, thereby causing thaxtomin 

production to increase.  

The next step in analyzing host specificity may be to inoculate potato seedlings with 

Streptomyces strains that were not capable of causing symptoms of disease on tissue discs to 

see if the host specificity indicated by the assays is seen in seedlings.  

 

109 

 

 

 

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110 

 

 

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