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Effect of inoculum source, inoculum pressure and
I
Effect of inoculum source, inoculum pressure and
cultivar on development of black scurf on potatoes in
South Africa
by
SHARIKA BAIJNATH
Submitted in partial fulfillment of the requirements for the degree of MSc (Plant Pathology) in the
Faculty of Natural and Agricultural Sciences
Department of Microbiology and Plant Pathology
University of Pretoria
June 2012
© University of Pretoria
II
DECLARATION
I declare that the dissertation, which I hereby submit for the degree of MSc (Plant Pathology) at
the University of Pretoria, is my own work and has not previously been submitted by me for a
degree at another university. Where secondary material is used, this has been carefully
acknowledged and referenced in accordance with university requirements. I am aware of
university policy and implications regarding plagiarism.
---------------------Signature
-------------------Date
III
ACKNOWLEDGMENTS
I wish to express my sincere thanks and appreciation to the following persons and institution:
 Dr. Jacquie van der Waals, for her continuous support, encouragement, leadership and
understanding which allowed me to complete this study whilst pursuing my career.
 Dr. Mariette Truter, for lending a listening ear to me during my many emotional
moments. Thank you for the constructive criticism, polite editing comments, advice and
comfort.
 Potatoes South Africa for financial support during this study.
 My friends and colleagues, for their help and encouragement. A special thanks to
Estiene Jordaan and Charles Kamau Wairuri for assisting me when I could not be on
campus and for accompanying me over many weekends.
 My parents, for always being there for me with lots of love, support and encouragement.
 To God, for giving me the strength and determination to push through some difficult
periods during this study.
IV
CONTENTS
COVER PAGE
I
DECLARATION
II
ACKNOWLEDGEMENTS
III
LIST OF FIGURES
VII
LIST OF TABLES
IX
CHAPTER 1
1
GENERAL INTRODUCTION
References
3
CHAPTER 2
7
LITERATURE REVIEW
2.1 Casual organism
7
2.2 Taxonomy
7
2.3 Molecular methods of identification
10
2.4 Infection process
11
2.5 Risk factors influencing disease development
13
2.6 Control
15
2.7 References
19
CHAPTER 3
26
EFFECT OF RHIZOCTONIA SOLANI INOCULUM SOURCE AND SOIL TYPE ON
DISEASE DEVELOPMENT IN POTATOES
Abstract
26
3.1 Introduction
26
3.2 Materials and methods
27
3.2.1 Experimental design
27
3.2.2 Inoculation of soil
27
3.2.3 Inoculation of tubers
28
3.2.4 Inoculation of stolons
28
3.2.5 Isolation from stem lesions
29
3.2.6 Disease assessment
29
3.2.7 Statistical analysis
29
V
3.3 Results
30
3.4 Discussion
32
3.5 References
34
CHAPTER 4
38
INFLUENCE OF INOCULUM PRESSURE OF RHIZOCTONIA SOLANI ON DISEASE
DEVELOPMENT UNDER FIELD CONDITIONS IN SOUTH AFRICA
Abstract
38
4.1 Introduction
38
4.2 Materials and methods
39
4.2.1 Experimental design
39
4.2.2 Preparation of inoculum
39
4.2.3 Inoculation of soil
40
4.2.4 Isolation from stem lesions
40
4.2.5 Disease assessment
40
4.2.6 Statistical analysis
41
4.3 Results
42
4.4 Discussion
44
4.5 References
46
CHAPTER 5
49
SUSCEPTIBILITY OF FIVE POTATO CULTIVARS COMMONLY CULTIVATED IN SOUTH
AFRICA, TO RHIZOCTONIA SOLANI
Abstract
49
5.1 Introduction
49
5.2 Materials and methods
51
5.2.1 Pot trials
51
5.2.2 Preparation of inoculum
51
5.2.3 Inoculation of soil
52
5.2.4 Disease assessment
52
5.2.5 Statistical analysis
52
5.3 Results
53
5.4 Discussion
55
5.5 References
56
VI
CONCLUSION
Abstract
61
VII
LIST OF FIGURES
CHAPTER 2
LITERATURE REVIEW
Fig. 1. Mycelium branching of young hyphae
8
Fig. 2. Mycelium branching of older hyphae
8
Fig. 3. Life cycle of R. solani
12
Fig. 4 A & B. Stem canker on potato plant
14
Fig. 5. Sclerotia on tuber
14
CHAPTER 3
EFFECT OF RHIZOCTONIA SOLANI INOCULUM SOURCE AND SOIL TYPE ON
DISEASE DEVELOPMENT IN POTATOES
Fig. 1. nil (tubers with no symptoms)
30
Fig. 2. low (<3% sclerotia on tuber surface)
30
Fig. 3. Moderate (3-25% sclerotia on tuber surface)
30
Fig. 4. High (>25% sclerotia on tuber surface)
30
Fig. 5 A & B. Stem lesions on plants grown in R. solani inoculated soils
31
Fig. 6. Greening of tubers harvested from plants grown in inoculated soils
32
CHAPTER 4
INFLUENCE OF INOCULUM PRESSURE OF RHIZOCTONIA SOLANI ON DISEASE
DEVELOPMENT UNDER FIELD CONDITIONS IN SOUTH AFRICA
Fig. 1. nil (tubers with no symptoms)
41
Fig. 2. low (<3% sclerotia on tuber surface)
41
Fig. 3. Moderate (3-25% sclerotia on tuber surface)
41
Fig. 4. High (>25% sclerotia on tuber surface)
41
o
Fig. 5. Average maximum and minimum temperature ( C) per month
42
Fig. 6 A & B. Total rainfall (mm) per month
43
Fig. 7. Mean disease severity over all four inoculum replicates recorded during the
second trial
43
VIII
CHAPTER 5
SUSCEPTIBILITY OF FIVE POTATO CULTIVARS COMMONLY CULTIVATED IN SOUTH
AFRICA, TO RHIZOCTONIA SOLANI
Fig. 1. nil (tubers with no symptoms)
53
Fig. 2. low (<3% sclerotia on tuber surface)
53
Fig. 3. Moderate (3-25% sclerotia on tuber surface)
53
Fig. 4. High (>25% sclerotia on tuber surface)
53
Fig. 5. Mean disease severity per cultivar in pot trial one
54
Fig. 6. Mean disease severity per cultivar in pot trial two
54
IX
LIST OF TABLES
CHAPTER 3
EFFECT OF RHIZOCTONIA SOLANI INOCULUM SOURCE AND SOIL TYPE ON
DISEASE DEVELOPMENT IN POTATOES
Table. 1. Average disease severity per inoculum treatment and soil type
31
CHAPTER 5
SUSCEPTIBILITY OF FIVE POTATO CULTIVARS COMMONLY CULTIVATED IN SOUTH
AFRICA, TO RHIZOCTONIA SOLANI
Table. 1. Cultivar black scurf susceptibility/ tolerance and growth period
51
CHAPTER ONE
GENERAL INTRODUCTION
The potato is the world’s most important non-grain food crop (Huang et al., 2011) and
South Africa’s third most important food crop after maize and wheat (Potatoes South
Africa, 2010a). In 2009, the gross value of potato production in South Africa accounted
for about 53% of major vegetables, 12% of horticultural products and 3% of total
agricultural production (Potatoes South Africa, 2010b). However, the potato industry in
South Africa encounters numerous challenges such as severe droughts and floods, land
shortages and the economy. The most frequent yield and tuber quality losses to the
industry are attributed to soil and/or tuber borne pathogens (Potatoes South Africa,
2010b).
Rhizoctonia solani is a soil-borne fungus consisting of morphologically similar but
genetically distinct groups, referred to as Anastomosis Groups (AG’s). Research has
identified 13 different AG’ s of which only AG 1, 2.1, 3, 4, 5, 7 and 8 have been
associated with disease in potatoes. AG 7 causes infections on stems, stolons and
tubers but not on roots, while AG 8 infects potato roots (Sneh et al., 1996; Lees et al.,
2002; Woodhall et al., 2007; Okubara et al., 2008). AG 5 is mostly associated with stem;
root canker and black scurf (Balali et al., 1995) whilst AG 4 is only associated with stem
lesions (Sneh et al., 1996; Lees et al., 2002). AG1 has been shown to cause damage to
spouts (Carling & Leiner, 1990). AG 2.1 is associated with stem lesions and superficial
tuber alterations (Campion et al., 2003). Two groups of AG 3 have been identified
namely AG 3PT and AG 3TB, of which only AG 3PT has been reported to be the major
causal agent of black scurf and stem canker (Bounou et al., 1999; Lees et al., 2002,
Yanar et al., 2005). A study by Truter (2005) investigating the different AG’s associated
with potato disease in South Africa showed 99.3% of isolates from tubers infected with
black scurf and 82.1% of isolates from infected stems and stolons, belonged to AG 3.
The development of disease begins when R. solani inoculum is present either as soil- or
tuber-borne sclerotia or hyphae (Scholte, 1992; Gilligan et al., 1996; Atkinson et al.,
2010). Research, however, has yet to determine which inoculum source (seed vs. soil) is
more important for disease development. Stolons have a higher chance of infection by
contact with soil-borne inoculum than tuber-borne inoculum (Frank & Leach, 1980).
1
Some studies show as little as 12-15% tuber-borne inoculum is sufficient to cause
severe stem canker and black scurf symptoms (James & McKenzie, 1972; Atkinson et
al., 2010). Whilst other studies prove even seed tubers not showing black scurf
symptoms could contain Rhizoctonia hyphae, thus resulting in disease development (Du
Plessis, 1999). A study by Tsror & Peretz-Alon (2005) showed that although both
inoculum sources are important for disease development, it is in fact the initial inoculum
load which determines intensity of disease incidence and severity.
R. solani causes disease at all growing stages (sprout development, vegetative growth,
tuber initiation, tuber bulking and tuber maturation) of the potato plant (Banville, 1989;
Simons & Gilligan, 1997; Naz et al., 2008). A study by Ahvenniemi et al. (2007) showed
Rhizoctonia disease develops in four phases. The first and second phases result in
infection of stolon tips prior and after emergence, respectively (Ahvenniemi et al., 2007).
Infected stolons lead to death of sprouts and stems, malformed tubers and the formation
of aerial tubers (James & McKenzie, 1972; Atkinson et al., 2010). The third phase in R.
solani disease development occurs three to four weeks after emergence, resulting in
scabby, sunken areas near the sprout end of young tubers (Ahvenniemi et al., 2007).
The fourth phase is identifiable by the development of black scurf on progeny tubers,
roots, stem bases and stolons (Ahvenniemi et al., 2007). Tubers showing black scurf
symptoms are downgraded on the market and thus responsible for great economic
losses to consumer markets, seed certification programs and the export industry (Platt et
al., 1993; Republic of South Africa, 2002; Hamid et al., 2006).
There are many biotic and abiotic factors that influence the survival and spread of R.
solani in soils such as temperature, soil moisture, soil pH and soil type. Physical soil
characteristics such as pore size, bulk density and tortuosity influence aeration, water
movement, plant root growth, water and nutrient retention, but also determine the ability
of R. solani hyphae to invade available pore spaces and branch out (Otten et al., 2001;
Harris et al., 2003; Ritz & Young, 2004). Further understanding on how physical soil
conditions may be altered by factors such as tillage and irrigation, can assist in
developing an integrated control strategy for R. solani.
Fungicides, applied as either soil and/or seed treatments, are the preferred method of
control (Campion et al., 2003; Boogert & Luttikholt, 2004; Rauf et al., 2007). The efficacy
2
of fungicides, in vivo and in vitro, depends on the morphology, physiology, virulence and
genetic composition of different R. solani sub-groups present in the field (Kataria et al.,
1991). The use of some mycoparasites such as Trichoderma harzianum, T. viridae, T.
hamatum, Gliocladium virens and Verticillium biguttatum have been shown to
significantly reduce R. solani inoculum levels in soils (Wale, 2004; Brooks, 2007; Wilson
et al., 2008). However, both chemical and biological methods are extremely complex,
costly, and time consuming to implement. The use of more tolerant cultivars has been
shown in past studies to be a more convenient, economical and environmentally safe
method to effectively reduce inoculum levels in soils, thereby reducing the risk of
disease development (Otrysko & Banville, 1992, Leach & Webb, 1993).
Most farmers rely on experience and knowledge of field factors to develop their disease
management programs (Wale, 2004). To reduce the associated risk of disease, the
current study focuses on determining the effect of different inoculum sources and
concentrations on disease development and to investigate cultivar susceptibility of a few
commonly planted cultivars in South Africa as a method of reducing soil inoculum levels
for decreasing disease severity and incidence.
1.1 References:
Ahvenniemi, A., Lehtonen, M., Wilson, P. & Valkonen, J. 2007. Disease cycle of seedborne Rhizoctonia-disease, new and old pathogens in changing climate,
abstracts from the proceedings of the EAPR pathology section seminar,
Hattula, Finland, 2-6th July 2007: 26.
Atkinson, D., Thornton, M. K. & Miller, J. S. 2010. Development of Rhizoctonia solani on
stems, stolons and tubers of potatoes I. Effect of inoculum source, American
Journal of Potato Research 87: 374-381.
Balali, G. R., Neate, S. M., Scott, E. S., Whisson, D. L. & Wicks, T. J. 1995. Anastomosis
group and pathogenicity of isolates of Rhizoctonia solani from crops in South
Australia, Plant Pathology 44: 1050-1057.
Banville, G. J. 1989. Yield losses and damage to potato plants caused by Rhizoctonia
solani Kuhn, American Potato Journal 66: 821-834.
Boogert Van den, P. H. J. F. & Luttikholt, A. J. G. 2004. Compatible biological and
chemical control systems for Rhizoctonia solani in potato, European Journal
of Plant Pathology 110: 111-118.
3
Bounou, S., Jabaji-hare, S. H., Hogue, R. & Charest, P. M. 1999. Polymerase chain
reaction-based assay for specific detection of Rhizoctonia solani AG-3
isolates, Mycological Research 103: 1-8.
Brooks, S. A. 2007. Sensitivity to a phytotoxin from Rhizoctonia solani correlates with
sheath blight susceptibility in rice, Phytopathology 97: 1207-1212.
Campion, C., Chatot, C., Perraton, B. & Andrivon, D. 2003. Anastomosis groups,
pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates
collected on potato crops in France, European Journal of Plant Pathology
109: 983-992.
Carling, D. E. & Leiner, R. H. 1990. Effect of temperature on virulence of Rhizoctonia
solani and other Rhizoctonia on potato, Phytopathology 80: 930-934.
Du Plessis, J. C. 1999. Control of black scurf and stem canker on seed potatoes in
South Africa. MSc (Agric) dissertation, University of Pretoria, South Africa.
Frank, A. J. & Leach, S. S. 1980. Comparison of tuberborne and soilborne inoculum in
the Rhizoctonia disease of potato, Phytopathology 70: 51-53.
Gilligan, C. A., Simons, S. A. & Hide, G. A. 1996. Inoculum density and spatial pattern of
Rhizoctonia solani in fields of Solanum tuberosum: effects of cropping
frequency, Plant Pathology 45: 232-244.
Hamid, E. E. H., Himeidan, Y. E. S. & Hassan, S. M. E. 2006. Cultural practices for the
management of Rhizoctonia disease in potato, Agricultural Science 18: 141148.
Harris, K., Young, I. M., Gilligan, C. A., Otten, W. & Ritz, K. 2003. Effect of bulk density
on the spatial organization of the fungus Rhizoctonia solani in soil, FEMS
Microbiology Ecology 44: 45-56.
Huang, S., Buell, C. R. & Visser, R. G. F. 2011. Genome sequence and analysis of the
tuber crop potato. Nature 475: 189-195.
James, W. C. & McKenzie, A. R. 1972. The effect of tuber-borne sclerotia of Rhizoctonia
solani Kuhn on the potato crop, American Potato Journal 49: 296-301.
Kataria, R. H., Hugelshofer, U. & Gisi, U. 1991. Sensitivity of Rhizoctonia species to
different fungicides, Plant Pathology 40: 203-211.
Leach, S. L. & Webb, R. E. 1993. Evaluation of potato cultivars, clones and a true seed
population for resistance to Rhizoctonia solani, American Potato Journal 70:
317-327
4
Lees, A. K., Cullen, D. W., Sullivan, L. & Nicholson, M. J. 2002. Development of
conventional and quantitative real-time PCR assays for the detection and
identification of Rhizoctonia solani AG-3 in potato and soil, Plant Pathology
51: 293-302.
Naz, F., Rauf, C. A., Abbasi, N. A., Irafan-Ul-Haque. & Ahmad, I. 2008. Influence of
inoculum levels of Rhizoctonia solani and susceptibility on new potato
germplasm, Pakistan Journal of Botany 40: 2199-2209.
Okubara, P. A., Schroeder, K. L. & Paulitz, T. C. 2008. Identification and quantification of
Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction,
Phytopathology 98: 837-847.
Otrysko, B. E. & Banville, G. J. 1992. Effect of infection by Rhizoctonia solani on the
quality of tubers for processing, American Potato Journal 69: 645-652.
Otten, W., Hall, D., Harris, K., Ritz, K., Young, I. M. & Gilligan, C.A. 2001. Soil physics,
fungal epidemiology and the spread of Rhizoctonia solani, New Phytologist
151: 459-468.
Platt, H. W., Canale, F. & Gimenez, G. 1993. Effects of tuber-borne inoculum of
Rhizoctonia solani and fungicidal seed potato treatment on plant growth and
Rhizoctonia disease in Canada and Uruguay, American Potato Journal 70:
553-559.
Potatoes South Africa, 2010a, Gross value of different sectors in SA agriculture,
http://www.potatoes.co.za/SiteResources/documents/gross%20value%20of%
20diff%20sectors%20in%20SA%20Agric.- 26 October 2011
Potatoes South Africa, 2010b, The South African potato industry in perspective.
Rauf, C. A., Ashraf, M. & Ahmad, I. 2007. Management of black scurf disease of potato,
Pakistan Journal of Botany 39: 1353-1357.
Republic of South Africa. 2002. Regulation Gazette No. 7495. Government Gazette, 8
November 2002, 449: 1-9.
Ritz, K. & Young, I. M. 2004. Interactions between soil structure and fungi, Mycologist
18: 52-59.
Scholte, K. 1992. Effect of crop rotation on the incidence of soil-borne fungal diseases of
potato, Netherlands Journal of Plant Pathology 98: 93-101.
Simons, S. A. & Gilligan, C. A. 1997. Relationships between stem canker, stolon canker,
black scurf (Rhizoctonia solani) and yield of potatoes (Solanum tuberosum)
under different agronomic conditions, Plant Pathology 46: 651-658.
5
Sneh, B., Jabaji-Hare, S., Neate, S. & Dijst, G. 1996. Rhizoctonia species: Taxonomy,
molecular biology, ecology, pathology and disease control, Kluwer Academic
Publishers, London.
Truter, M. 2005. Etiology and alternative control of potato rhizoctoniasis in South Africa.
MSc (Agric) dissertation, University of Pretoria, South Africa.
Tsror, L. & Peretz-Alon, I. 2005. The influence of the inoculum source of Rhizoctonia
solani on development of black scurf on potato, Journal of Phytopathology
153: 240-244.
Wale, S. J. 2004. Integrating knowledge of soilborne pathogens to minimise disease risk
in potato production, Australasian Plant Pathology 33: 167-172.
Wilson, P. S., Ahvenniemi, P. M., Lehtonen, M. J., Kukkonen, M., Rita, M. & Vaalkonen,
J. P. T. 2008. Biological and chemical control and their combined use to
control different stages of the Rhizoctonia disease complex on potato through
the growing season, Annals of Applied Biology 153: 307-320.
Woodhall, J. H., Lees, A. K., Edwards, S. G. & Jenkinson, P. 2007. Characterization of
Rhizoctonia solani from potatoes in Great Britain, Plant Pathology 56: 286295.
Yanar, Y., Yilmaz, G., Cesmeli, I. & Coskun, S. 2005. Characterization of Rhizoctonia
solani isolates from potatoes in Turkey and screening potato cultivars for
resistance to AG-3 isolates, Phytoparasitica 33: 370-376.
6
CHAPTER TWO
LITERATURE REVIEW
2.1 Causal organism
Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) is a wide-spread,
economically important soil-borne fungus, which is a species-complex consisting of
morphological similar, but genetically distinct species (Beagle-Ristaino & Papavizas,
1985; Carling & Kuninaga, 1990; Lees et al., 2002; Pannecaucque et al., 2008). This
pathogen is the causal agent of fruit and seed decay, damping off, foliar blight and crown
rot of many crops such as soybean, maize, rice, sorghum, sugarcane, cotton and coffee
(Pascual et al., 2000; Stetina et al., 2006; Brooks, 2007; Guleria et al., 2007; Thind &
Aggarwal, 2008). However, on potato crops the symptoms include stem canker, stolon
canker, misshapen tubers, black scurf, cracking, pitting and netting of tuber surfaces
(Carling et al., 1989; Sneh et al., 1996; Stuart et al., 2008).
2.2 Taxonomy
Morphological identification of R. solani is based on a combination of hyphal traits.
These include branching near the distal septum, no clamp connections, pigmentation,
multinucleate and binucleate cells in vegetative hyphae and hyphal interactions
(Parmeter, 1970; Sneh et al., 1996; Lees et al., 2002). Different hyphae have been
shown to vary in shades of brown depending on their age. Young colonies may appear
pale brown or pale yellow at times, whereas the hyphae of older colonies may vary from
brown to dark brown in color (Parmeter, 1970; Hooker, 1981; Guleria et al., 2007). The
brown pigmentation of hyphae cannot be used on its own for the identification of R.
solani, since similar colouration can be present among other fungi (Sneh et al., 1996).
Another characteristic for the identification of R. solani is hyphal branching. In younger
colonies branching is at acute angles (Fig. 1) and near the distal septum of the cells
(Parmeter, 1970; Sneh et al., 1996). In older colonies the hyphae often branch at right
angles (Fig. 2) and at any place along the cell. However since these characteristics are
also common among most fungi, hyphal branching alone cannot be used in accurate
identification (Parmeter, 1970; Sneh et al., 1996).
7
Fig. 1. Mycelium branching of young hyphae
Fig. 2. Mycelium branching of older hyphae
In addition, R. solani hyphae have multiple nuclei, usually four to eight per cell, unlike
typical fungi with only two nuclei per cell. The multinucleate characteristic of R. solani
has become a very reliable method in preliminary identification (Parmeter, 1970; Sneh et
al., 1996; Sharon et al., 2006).
R. solani can produce actively growing mycelium or inactive sclerotia on various
8
substrates (Pascual et al., 2000; Harikrishnan & Yang, 2004; Ritchie et al., 2006;
Brooks, 2007). Genetic variations within R. solani are also reflected in the type of
sclerotia, which are compact masses of cells. The shape, size, shade of brown and
distribution on agar plates vary between the different Anastomosis Groups (AG’s)
(Parmeter, 1970; Sneh et al., 1996; Agrios, 2005; Tsror, 2010). The diversity of this
species complex is also revealed by the presence of several genetically variable AG
sub-groups. So far 13 different AG’s have been identified; AG-1, -2, -3, -4, -5, -6, -7, -8, 9, -10, -11, -12 and -13 (Carling & Kuninaga, 1990; Lees et al., 2002; Harikrishnan &
Yang, 2004; Guleria et al., 2007; Woodhall et al., 2007). Past studies have shown that
different AG’s are associated with different preferred hosts and disease symptoms
(Parmeter, 1970; Sneh et al., 1996). Therefore, a prerequisite for a reliable disease
management strategy for R. solani would be accurate identification of the AG’s present.
Anastomosis reactions of hyphae have been used over the years to place R. solani
isolates into different AG’s (Cubeta & Vilgalys, 1997; Lubeck & Poulsen, 2001; Lees et
al., 2002; Sharon et al., 2006; Yang et al., 2006; Lehtonen et al., 2008). There are four
categories of hyphal reactions that allow grouping and differentiation of isolates, namely,
“C0”, “C1”, “C2” and “C3” (Sneh et al., 1996; Cubeta & Vilgalys, 1997).
“C0” occurs between isolates that are not related and where there is no interaction, while
“C1” occurs between isolates that are distantly related. Though there is contact between
the hyphae, there is no evidence of wall penetration or membrane-membrane contact. In
these reactions often one or both anastomosing cells and adjacent cells die (Cubeta &
Vilgalys, 1997). “C2” occurs between related isolates in which there is a wall connection
and the location of reaction site is visible. The anastomosing and adjacent cells always
die. “C3” hyphal reactions occur between closely related isolates. The walls fuse and
membranes or anastomosis points are frequently not visible. The anastomosing and
adjacent cells may die but generally do not (Cubeta & Vilaglys, 1997).
The reliability of anastomosis grouping of isolates using anastomosis reactions is
however jeopardized due to the occurrence of bridging isolates that can produce hyphal
anastomosis reactions (C1) with isolates from more than one anastomosis group (Sneh
et al., 1996). Moreover, AG’s have been described as being “genetically variable”, thus
they can be considered as different species of R. solani.
9
2.3 Molecular methods of identification
Apart from visual traits employed in R. solani grouping, the diversity of this pathogen is
also reflected at a cellular and molecular level (Liu & Sinclair, 1993; Ceresini et al., 2002;
Justesen et al., 2003; Sharon et al., 2006). Studies employing various molecular
techniques such as Amplified Fragment Length Polymorphism (AFLP), Random
Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphisms
(RFLP) and Polymerase Chain Reaction (PCR) have not only made it possible to identify
R. solani, but also to differentiate among the several AG’s (Liu & Sinclair, 1993; Ceresini
et al., 2002; Justesen et al., 2003; Sharma et al., 2005). These molecular techniques
are normally based on the sequence variation within the internal transcribed spacer
(ITS1 and ITS2) regions, which have been very useful in highlighting R. solani genetic
diversity (Lees et al., 2002; Yang et al., 2006).
The intergenic spacer that separates the 28S and 18S rRNA subunits usually contains a
5S rRNA subunit and a 5.8S rRNA subunit. The 5.8S subunit is flanked by two internal
transcribed spacer (ITS1 and ITS2) regions (Cullen et al., 2000; Lehtonen et al., 2008).
Restriction analysis of the ITS1, ITS2 and 5.8S rDNA regions indicates that the AG’s of
R. solani are genetically distinct. Comparison of rDNA-ITS nucleotide sequences has
been shown to provide more genetic information than RFLP, AFLP and RADP-PCR, and
allows for the comparison of results with the GenBank databases around the world and
with other studies related to R. solani (Kuninaga et al., 1997, 2000). R. solani AG-3 has
since been divided into two subgroups; AG-3PT and AG-3TB, however only AG-3PT is
of economic importance on potatoes as it is associated with the tuber blemish, black
scurf (Kuninaga et al., 2000; Woodhall et al., 2007). Moreover, RFLP’s used by
Alabouvette et al. (2003) and Liu & Sinclair (1993) showed variations within each AG
and this necessitates addition of subsets to the different AG’s.
Although RFLP, AFLP and RAPD-PCR are very effective molecular techniques for
differentiating among R. solani AG’s, conventional and real-time PCR are the preferred
molecular techniques as they allow for the rapid detection of a particular R. solani AG
present in plant tissue and in soils (Liu & Sinclair, 1993; Ceresini et al., 2002;; Justesen
et al., 2003). In addition, real time PCR with AG or sub-group specific primers, are highly
sensitive, allowing for the detection of very low concentrations of fungal load (Lees et al.,
2002).
10
2.4 Infection process
Disease development on a potato plant begins with the presence of the pathogen (Fig.
3). R. solani can assume three forms of inoculum; basidiospores, mycelium fragments or
sclerotia. Basidiospores of Thanatephorus cucumeris (A. B. Frank) Donk, the
teleomorph of R. solani, are normally found on soil surfaces, on stems and leaves. They
are only produced under moderate temperatures, high moisture and humidity (Gutierrez
et al., 1997). Basidiospores are air transmitted inoculum mostly causing disease to aerial
parts of the plant (Naito, 1996; Pascual et al., 2000). Mycelium fragments and sclerotia
are present either as soil- or tuber-borne and are commonly associated with disease of
below ground plant parts (Atkinson et al., 2010). However research over the years has
not been able to determine which inoculum source plays a more important role in
disease development (Atkinson et al., 2010; Lees et al., 2010).
Seed inoculum has commonly been associated with disease during the early stages of
plant development, negatively affecting plant emergence (Frank & Leach, 1980). The
close proximity of tuber-borne inoculum to emerging sprouts causes girdling of sprout
and resultant stem cankers. Some studies (Gilligan et al., 1996; Naz et al., 2008) have
shown that soil-borne inoculum can initiate stem and stolon infections as well as black
scurf development. Naz et al (2008) showed a significant increase in black scurf on
progeny tubers and stem canker with an increase in soil-borne inoculum levels. Frank &
Leach (1980) suggested that as stolons move through soils, the probability of contact
with soil inoculum is much greater than that with tuber-borne inoculum. However, some
studies show although both inoculum sources are important for disease development, it
is in fact the initial inoculum load which determines intensity of disease (Tsror & PeretzAlon, 2005).
Optimum temperature and moisture conditions for R. solani can enhance disease
severity and incidence by stimulating the growth and migration of R. solani towards the
plant. When contact with the plant tissue is made, hyphal side branches or T-shaped
hyphal branches give rise to infection structures. These structures, also referred to as
infection cushions, comprise of swollen tips which then form infection pegs. The infection
pegs penetrate the cuticle and epidermis cells and give rise to hyphae within the cells of
11
the plant, the end result being seedling death or stem lesions (Fig. 4) (Keijer, 1996;
Weinhold & Sinclair, 1996).
Fig. 3. Life cycle of Rhizoctonia Solani (Agrios, 2005)
Rhizoctonia solani is capable of causing disease on tubers, stems and stolons during all
five growing stages of the potato plant before harvest (sprout development, vegetative
growth, tuber initiation, tuber bulking and tuber maturation). Symptoms on stem and
stolons consist of dark brown, necrotic lesions (Fig. 4 A & B) (Keijer, 1996; Weinhold &
12
Sinclair, 1996; Simons & Gilligan, 1997; Tsror & Peretz-Alon, 2005). In severely infested
fields these symptoms can result in death of sprouts and stems and girdling of stolons,
resulting in malformed tubers and reduced yields due to poor stand emergence (James
& McKenzie, 1972; Atkinson et al., 2010).
Infected stolons are inhibited from growing to their full length resulting in the
development of tubers close to the soil surface, commonly leading to tuber greening.
Tubers exposed to light change from brown to green in colour due to the production of
amyloplasts from chloroplasts in the potato tuber. Green tubers also form alkaloids
which pose a risk of poisoning (Zhu et al., 1984).
During the last two growth stages of the potato plant, the development of black soil-like
structures on tubers, sclerotia, also referred to as black scurf is common (Woodhall et
al., 2007; Thind & Aggarwal, 2008; Woodhall et al., 2008) (Fig. 5). Sclerotia are made up
of loosely constructed knots of melanised hyphae which are not organized into a rind or
cortex (Sneh et al., 1996; Ritchie et al., 2009). This “loose type” of sclerotia is unique to
R. solani and serves as survival structures. R. solani can survive in the form of sclerotia
in soils or on plant debris for several years (Keijer, 1996; Weinhold & Sinclair, 1996).
Although these structures do not cause any mechanical damage to the tuber, they do
however decrease the tubers’ market value. Therefore, black scurf is considered to be a
disease of great economical importance which results in both quantitative and qualitative
agricultural losses (Lees et al., 2002; Woodhall et al., 2008; Atkinson et al., 2010).
2.5 Risk factors influencing disease development
Although R. solani is a common disease occurring throughout the world, its growth and
disease development is highly influenced by various environmental factors (Simons &
Gilligan, 1997; Ritchie et al., 2006, 2009; Larkin et al., 2010; Tsror, 2010). Past studies
investigating the effects of temperature, pH, water potential and soil type on growth of R.
solani have concluded that all four factors are important for disease development
(Ritchie et al., 2006, 2009).
13
A
B
Fig. 4 A & B. Stem canker on potato plant
Fig. 5. Sclerotia on tuber
14
A study by Richie et al. (2009) showed growth of R. solani (AG3) mycelium to be optimal
at a pH between 5 and 6. Therefore, as a cultural practice, in Scotland, soils are kept at
a very low pH (Ritchie et al., 2009). This reduces the levels of R. solani inoculum in soils,
without having an effect on yield (Ritchie et al., 2009).
Disease incidence and severity are also subject to how temperature affects the hostpathogen relationship. When temperatures are optimum for the host but not the
pathogen, disease development is inhibited (Agrios, 2005). Studies have shown the
optimum temperature range for growth of the potato plant and for Rhizoctonia disease
development are 20-25oC and 10-15oC, respectively (Beukema & Van der Zaag, 1990,
Sneh et al., 1996). Research has shown low temperatures can result in late emergence
of the plant leading to higher disease incidence (Beukema & Van der Zaag, 1990; Rowe
et al., 1993). This poses a huge challenge to the farmer; not only is it impossible to
control temperatures under field conditions, but it is also becoming increasingly difficult
to predict temperatures as well as rainfall due to changing weather patterns around the
world, a consequence of climate change (Norby & Luo, 2004).
2.6 Control
Chemical control of R. solani (black scurf and stem canker) dates back to 1913 (Kataria
& Gisi, 1996). It was and still is one of the most used and relied upon methods of control
for both seed and soil-borne inoculum. Fungicides, either soil or seed treatments or both,
or foliar applications used to control R. solani belong to many different chemical groups
viz. aromatic benzimidazoles, B-methaoxyacrylates, carboxamides, dicarboximides,
hydrocarbons, morpholine, phenylpyrroles, phenylurea, triazoles and validamycin
(Kataria & Gisi, 1996; Nel et al., 2003, Tomlin, 2006).
Although a wide range of fungicides are available for the control of R. solani, the
different Rhizoctonia species and the different AG’s vary in levels of sensitivity to the
active ingredients (a.i.). Research by Kataria et al. (1991), with 14 active ingredients
(benodonil, benomyl, carboxin, cyproconazole, fenarimol, fenpropimorph, fusilazole,
imazalil, iprodione, prochloraz, pencycuron, propiconazole, triadimenol and tolclofos
methyl) and five Rhizoctonia species (R. cerealis, R. sacakii, R. solani, R. zeae and R.
oryzae) showed varying levels of control by the different chemical groups on the five
Rhizoctonia species. Although the different levels of control could be attributed to the
15
varying toxicity levels of the a.i., it is in fact the distinct morphology, physiology,
virulence, genetic-constitution and different teleomorphic forms between the various
Rhizoctonia species that determine the effectiveness of the chemical employed (Kataria
et al., 1991).
In South Africa, chemical groups registered with the regulating body (Department of
Agriculture: Act 36 of 1947) for the control of black scurf and stem canker on potato,
either as seed treatments, furrow applications or both, include dichlorophen, fludioxonil,
imazalil/iprodione, pencycuron, quintozene, thiabendazole, tolclofos–methyl and thiram
(Nel et al., 2003). Research by Truter (2005) on chemical inactivation of R. solani in
South Africa, showed that of the twenty disinfectants tested OA5 DP was the most
effective in killing sclerotia of R. solani and prevented progeny tubers from infection,
however acute phytotoxicity towards the tubers was noted. Furthermore tolclofos-methyl
was the only fungicide which gave total control of potato rhizoctoniasis (Truter, 2005).
Although registered fungicides have been proven to effectively control R. solani, some
contain active ingredients that are highly toxic to humans and animals and result in
adverse environmental consequences (Eddleston et al., 2002; Tomlin, 2006). Therefore
recent studies have focused on investigating integrated control management systems
combining biological agents (Rhizobacteria, Trichoderma spp., Gliododium spp.,
Actinomycetes spp., non-pathogenic Rhizoctonia spp.), with the appropriate chemical
control and cultural techniques (Strashnow et al., 1985; Wale, 2004; Brooks, 2007;
Wilson et al., 2008).
Research by Wilson et al. (2008) on the combined use of biological and chemical control
is one such study that highlights the importance of determining the compatibility of the
different components within an integrated control strategy. In addition, this study also
proved the antagonist T. harzianum to be compatible with the seed dressing Flutolanil,
reducing the incidence of black scurf on progeny tubers by 11% to 31%. Truter (2005)
reported the most inhibition of mycelial growth in vitro was achieved with Kresoximmethyl followed by volatiles from roots and shoots of B. napus, B. oleracca, var. capitata,
R. sativus, S. alba and T. minuta. In soils the biocontrol formulation Trykocide (T.
harzianum) eradicated the pathogen.
16
Furthermore cultural techniques such as crop rotation are recommended to the potato
grower as the planting of non-host crops reduces soil inoculum levels (Gilligan et al.,
1996; Larkin et al., 2010). A study by Larkin et al. (2010) used three cropping systems;
the Standard {SQ-2 years rotation of barley (Hordeum vulgare L.) with red clover
(Trifolium pretense L.) followed by potato}, Soil–Conserving system {SC-3 year rotation
with barley and forage grass timothy (Phleum pratense L.) followed by potato in the third
year}, Soil-Improvement system {SI- 3 year rotation with barley/timothy- timothy- potato}
and Disease–Suppressive system {DS-oriental and white mustard seed (Brassica juncea
L. and Sinapis alba L.) followed by rapeseed (Brassica napus L.) in the first year. In the
second year, sorgum- sudan- grass hybrid was used and winter rye (Secale cereale L.)
with potato in the third year}, to investigate the effects of crop rotation on black scurf and
stem canker. The results showed all rotation systems reduced stem canker by 10- 50 %.
SQ, SC and DS systems reduced black scurf by 18- 58%. Black scurf was also reduced
under non-irrigated conditions in the SI system.
Other recommended cultural techniques include use of disease free propagation
material, use of pathogen-free soil, soil management, irrigation, timing of harvest and
haulm destruction (Beukema & Van der Zaag, 1990; Rowe et al., 1993; Wale, 2004;
Hamid et al., 2006; Tsror, 2010). Black scurf development on progeny tubers has been
shown to increase after haulm destruction (Beukema & Van der Zaag, 1990; Rowe et al.,
1993; Tsror, 2010). Sclerotial development on progeny tubers is at a maximum 3-4
weeks after vine-killing (Gudmestad et al., 1979; Kempenaar & Struik, 2007). This is
mainly due to potato tubers exuding volatiles (Beukema & Van der Zaag, 1990; Rowe et
al., 1993; Tsror, 2010). Therefore, the time between haulm destruction and harvest
should be as short as possible to decrease risk of disease development.
Soil characteristics such as pore size, aeration, ability to retain nutrients and water also
influence the development and severity of disease. Otten et al, (2001), Harris et al.
(2003) and Ritz & Young (2004) have shown growth and spread of R. solani is restricted
in high density soils with tortuous and discontinuous air-filled pore space. Earlier work
regarding soil physics in relation to R. solani suggest the manipulation of the physical
properties of soils by tillage and irrigation can offer some control to the spread and
growth of R. solani (Otten et al., 2001; Harris et al., 2003). Tillage practices such as
moldboard plowing, chisel plowing and disking have been shown to successfully reduce
17
disease incidence and severity (Leach & Webb, 1993). Schroeder & Paulitz (2008)
suggested tillage of infested soils assists in breaking or disrupting the mycelia network of
R. solani thereby reducing the pathogen’s vigour. Some tillage methods such as the
moldboard plowing technique also removes R. solani from the upper 10cm of soil and
has the ability to bury sclerotia to depths preventing germination and infection (Leach &
Webb, 1993).
Soils with high bulk densities have been shown to reduce the spread of R. solani. The
more compact the soil, the higher its bulk density with limited pore space. The narrow
pore spaces in compact soils retain more water than pore spaces in loose sandy soils
(Otten et al., 1999, 2001; Harris et al., 2003 and Ritz & Young, 2004). As a result, water
blocked pores restrict hyphal growth, reducing the spread of R. solani. Whilst some
studies show R. solani growth is restricted by high moisture levels (>1500 bars) due to
poor aeration (Ploetz & Mitchell, 1985), other studies show moisture levels have no
influence on R. solani growth (Junior et al., 2007; Olanya et al., 2010). The varying
results may be a result of moisture levels affecting the various AG’s differently. This
further highlights the importance of accurate identification of AG’s present in soils before
cultivation.
Although biological, chemical and cultural techniques reduce the incidence and severity
of black scurf on progeny tubers, the implementation of these methods is extremely
complex, costly and time consuming. Research has shown the severity and incidence of
black scurf on potato tubers can also be reduced by the type of potato cultivar planted
(Otrysko & Banville, 1992; Leach & Webb, 1993). Although the use of tolerant cultivars is
the most practical, economical and convenient method of controlling R. solani, this
method is not used as no resistant cultivars are available for commercial use (Harris,
1978; Otrysko & Banville, 1992; Leach & Webb, 1993)
Studies conducted by Bains et al. (2002) and Yanar et al. (2005) on AG3 tolerant
cultivars showed that none of the tested cultivars were resistant. However, Bains et al.
(2002) found that late-maturing cultivars showed low levels of disease as compared to
early and mid-season cultivars. Yanar et al. (2005) also demonstrated that of the 22
tested cultivars, five showed low susceptibility levels. Although there may be no resistant
18
cultivars currently available for the farmer, cultivars which are less susceptible can be
incorporated into integrated disease management systems.
In South Africa some of the commonly planted cultivars include; Astrid, Aviva, BP1,
Buffelspoort, Calibra, Caren, Columbus, Darius, Devlin, Eryn, Esco, Evan, Fabula,
Fianna, Hermes, Herta, Hoevelder, Lady Rosetta, Liseta, Mondial, Mnandi, Pentland
Dell, Ronn, Ropedi, Sandvelder, Shepody, Up-To-Date and Vanderplank (Potatoes
South Africa, 2010). A study by Du Plessis (1999) investigating susceptibility levels of 24
cultivars showed Buffelspoort had a higher tolerance to R. solani compared to the other
cultivars tested. However, future studies should focus on determining exactly why some
cultivars show more tolerance than others. Perhaps more consideration should also be
given to unraveling the genetic composition of various cultivars to identify potential
resistance genes.
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Stetina, K. C., Stetina, S. R. & Russin, J. S. 2006. Comparison of severity assessment
methods for predicting yield loss to Rhizoctonia foliar blight in soybean, Plant
Disease 90: 39-43.
Strashnow, Y., Elad, Y., Sivan, A. & Chet, I. 1985. Integrated control of Rhizoctonia
solani by methyl bromide and Trichoderma harzianum, Plant Pathology 34:
146-151.
Stuart, J. W., Harold, W. P., Ligel, D. C. 2008. Page 75 In: diseases, pests and disorders
of potatoes: a colour handbook, Academic Press, United States of America.
Tomlin, C. D. S. 2006. The Pesticide Manual, British Crop Production Council, United
Kingdom.
24
Thind, T. S. & Aggarwal, R. 2008. Characterization and pathogenic relationships of
Rhizoctonia solani isolates in a potato-rice system and their sensitivity to
fungicides, Phytopathology 156: 615-621.
Truter, M. 2005. Etiology and alternative control of potato rhizoctoniasis in South Africa.
MSc (Agric) dissertation, University of Pretoria, South Africa.
Tsror, L. & Peretz-Alon, I. 2005. The influence of the inoculum source of Rhizoctonia
solani on development of black scurf on potato, Journal of Phytopathology
153: 240-244.
Tsror, L. 2010. Biology, epidemiology and management of Rhizoctonia solani on potato,
Journal of Phytopathology 158: 649-658.
Wale, S. J. 2004. Integrating knowledge of soilborne pathogens to minimise disease risk
in potato production, Australasian Plant Pathology 33: 167-172.
Weinhold, A. R. & Sinclair, J. B. 1996. Rhizoctonia solani: Penetration, colonisation, and
host response. Pages 163-174 In: Sneh, B., Jabaji-Hare, S., Neate S. & Dijst,
G. (eds). Rhizoctonia species: taxonomy, molecular biology, ecology,
pathology and disease control, Kluwer Academic, Dordrecht.
Wilson, P. S., Ahvenniemi, P. M., Lehtonen, M. J., Kukkonen, M., Rita, M. & Vaalkonen,
J. P. T. 2008. Biological and chemical control and their combined use to
control different stages of the Rhizoctonia disease complex on potato through
the growing season, Annals of Applied Biology 153: 307-320.
Woodhall, J. H., Lees, A. K., Edwards, S. G. & Jenkinson, P. 2007. Characterization of
Rhizoctonia solani from potatoes in Great Britain, Plant Pathology 56: 286295.
Woodhall, J. H., Lees, A. K., Edwards, S. G. & Jenkinson, P. 2008. Infection of potato by
Rhizoctonia solani: effect of anastomosis group, Plant Pathology 57: 897905.
Yanar, Y., Yilmaz, G., Cesmeli, I. & Coskun, S. 2005. Characterization of Rhizoctonia
solani isolates from potatoes in Turkey and screening potato cultivars for
resistance to AG-3 isolates, Phytoparasitica 33: 370-376.
Yang, G. H., Naito, S. & Dong, W. H. 2006. Identification and pathogenicity of
Rhizoctonia spp. causing wirestem of Betula nigra in China, Phytopathology
54: 80-83.
Zhu, Y. S., Merkle-Lehman, D. L. & Kung, S. D. 1984. Light-induced transformation of
amyloplasts into chloroplasts in potato tubers, Plant Physiology 75: 142-145.
25
CHAPTER THREE
EFFECT OF RHIZOCTONIA SOLANI INOCULUM SOURCE AND SOIL TYPE ON
DISEASE DEVELOPMENT IN POTATOES
ABSTRACT
Rhizoctonia solani inoculum can be present either as soil- or tuber-borne sclerotia or
hyphae. Although both inoculum sources play a role in disease development, it is not
clear which of the two is more important. Physical soil characteristics such as pore size,
bulk density and tortuosity determine the ability of R. solani hyphae to grow and spread
in soils. Two pot trials were conducted to determine the effect of tuber- and soil-borne
inoculum, and stolon inoculations on R. solani disease development in sandy and clay
loam soils. Of the two soil types, tubers harvested from inoculated sandy soils developed
significantly more disease than those harvested from clay loam soils. Of the three
inoculum sources, stolon inoculation and tuber-borne inoculum resulted in significantly
more disease on progeny tubers than those from R. solani spiked soils. Although soil
inoculum resulted in the lowest incidence and severity of black scurf symptoms, other
symptoms, such as tuber greening and stem canker were more prominent in this
treatment.
3.1 INTRODUCTION
Black scurf and stem canker on potato crops is caused by Rhizoctonia solani Kuhn
(teleomorph Thanatephorus cucumeris (A. B. Frank) Donk). This pathogen causes
disease on various parts of the potato plant (Ritchie et al., 2006; Al-Mughrabi, 2008;
Lehtonen et al., 2009; Ritchie et al., 2009). Stem and stolon infections are characterized
by dark brown necrotic lesions which may result in death of sprouts and stems, girdling
of stolons, malformed tubers and the formation of aerial tubers (Zhu et al., 1984; Tsror &
Peretz-Alon, 2005; Naz et al., 2008; Atkinson et al., 2010). Infected tubers develop black
soil-like sclerotia on their surfaces, a cosmetic symptom referred to as black scurf
(Parmeter, 1970; Sneh et al., 1996; Lahlali & Hijri, 2010).
Disease development begins when R. solani inoculum is present either as soil- or tuberborne sclerotia or hyphae (Sneh et al., 1996; Agrios, 2005). Although both inoculum
sources play a role in disease development, it is not clear which of the two is more
important. However, research has shown that when both are present disease (black
26
scurf, stem canker and stolon canker) incidence and severity is intensified (Frank &
Leach, 1980; Platt et al., 1993; Gilligan et al., 1996; Simons & Gilligan, 1997). Other
studies have however suggested each inoculum source plays a role in disease
development at different stages of the plant growth (Frank & Leach, 1980; Tsror &
Peretz-Alon, 2005; Atkinson et al., 2010).
Disease development is also influenced by other factors such as temperature, pH, water
potential and soil properties. Physical soil characteristics such as pore size, bulk density
and tortuosity influence aeration, water movement, plant root growth, water and nutrient
retention, but also determine the ability of R. solani hyphae to invade available pore
spaces and branch out (Otten et al., 2001; Harris et al., 2003; Ritz & Young, 2004).
Limited or restricted pore space has been shown to inhibit the growth of R. solani in soils
(Otten et al., 1999; Harris et al., 2003; Ritz & Young, 2004). Therefore, further
understanding on how physical soil conditions may be altered by factors such as tillage
and irrigation, can assist in developing an integrated control strategy for R. solani.
The aim of this study was to determine the effect of inoculum source on development of
R. solani on stems, stolons and tubers of potato plants cultivated in sandy and clay loam
soils.
3.2 MATERIALS AND METHODS
3.2.1 Experimental design:
Pot trials were conducted at the University of Pretoria under greenhouse conditions. The
temperature was maintained at 15oC at night and 20oC during the day. Disease-free
mini-tubers (cv. BP1) were used for the trials. The trials were set out in a factorial
design, consisting of three treatments (soil inoculum, seed inoculum and stolon
inoculation) and two soil types (sandy and clay loam). Before planting and inoculation
soil samples of each soil type were taken for soil analysis. Uninoculated soil, tubers and
stolons were used as control for each soil type. Each inoculum treatment (including
controls) was replicated five times for each soil type. The experiment was repeated.
3.2.2 Inoculation of soil:
An R. solani (PPRI 9527) isolate from the Potato Pathology Program culture collection
which had previously been identified as AG3 using conventional PCR, in a separate
27
study, was obtained. The isolate was plated on fresh PDA plates and allowed to grow for
five days at 25oC. Wheat seeds were soaked overnight in sterile distilled water with
250µg/ml chloramphenicol and drained. The moist wheat seeds were autoclaved at
120oC for 1 hour. Each bag of seeds weighing 200g was inoculated with five (5mm x
5mm) colonized agar blocks cut under aseptic conditions. The wheat seeds were shaken
every three days during the incubation period of 14 days at 25oC. Soil autoclaved at
120oC for 1 hour, weighing 3.4kg was potted in 4kg-capacity pots. Each pot was
inoculated with 40g of R. solani colonized wheat seeds (inoculum). The inoculum was
thoroughly mixed into the soil before planting one mini-tuber (cv. BP1) in each pot at a
depth of 100mm. Soil moisture was maintained by irrigating with 200ml distilled water
three times a week. After plant emergence 200ml of Culterra plant food (Multi-Kelp) was
applied at a rate of 5ml in 1.5L water once a week. Uninoculated soil was used as
control for each soil type.
3.2.3 Inoculation of tubers:
An R. solani (PPRI 9527) isolate from the Potato Pathology Program culture collection
was obtained. The isolate was plated on fresh PDA plates and allowed to grow for five
days at 25oC. Soil autoclaved at 120oC for 1 hour, weighing 3.4kg was potted in 4kgcapacity pots. Each mini-tuber (cv. BP1) was coated with a sludge (mud) prepared by
mixing five (5mm x 5mm) colonized agar blocks with 500ml of distilled water and 700g
soil (Simons & Gilligan, 1997). One coated mini-tuber was planted in each pot at a depth
of 100mm. Soil moisture was maintained by irrigating with 200ml distilled water three
times a week. After plant emergence 200ml of culterra plant food (Multi-Kelp) was
applied at a rate of 5ml in 1.5L water once a week. Tubers planted without the inoculum
coating were used as control for each soil type.
3.2.4 Inoculation of stolons:
An isolate of R. solani AG3 (PPRI 9527), from the Potato Pathology Program culture
collection was obtained. The isolate was plated on fresh PDA plates and allowed to grow
for five days at 25oC. Soil autoclaved at 120oC for 1 hour, weighing 3.4kg was potted in
4kg capacity pots. Each pot received one mini-tuber planted at a depth of 100mm. Two
weeks after planting stolons below the soil surface were injected with 0.1ml hyphal
suspension using a sterile hypodermic syringe. The hyphal suspension was prepared by
blending five (5mm x 5mm) colonized agar blocks with 100ml of distilled water. Soil
28
moisture was maintained by irrigating with 200ml water three times a week. After plant
emergence 200ml of Culterra plant food (Multi-Kelp) was applied at a rate of 5ml in 1.5L
water once a week. Stolons not injected with the hyphal suspension were used as
control for each soil type.
3.2.5 Isolation from stem lesions:
After five weeks, plants grown in R. solani-spiked soils showing symptoms of stem
canker lesions were used for fungal isolation to confirm the causal agent. Pieces (5mm
x 5mm) were cut from the stem lesions and plated on fresh PDA plates and allowed to
grow for five days at 25oC. The cultures were microscopically examined to identify R.
solani.
3.2.6 Disease assessment:
At harvest, potato tubers were placed into four disease severity categories: 0-nil (tubers
with no symptoms) (Fig. 1), 1-low (<3% sclerotia on tuber surface) (Fig. 2), 2-moderate
(3-25%) (Fig. 3) and 3-high (>25%) (Fig. 4). The disease severity index (s.i.) was
calculated using the following formula (Tsror & Peretz-Alon, 2005):
s.i.= (0 x n) + (1 x l) + (2 x m) + (3 x h)
Total number of tubers
At harvest, stems and stolons were evaluated using a scale of 0-5, where 0 = healthy
tissue, 1 = several brown to black lesions, 2 = up to 15% of the tissue is covered with
lesions, 3 = up to 30% of the tissue is covered with lesions, 4 = up to 60% of the tissue is
covered with lesions and 5 = >60% of the tissue is covered with lesions (Tsror & PeretzAlon, 2005).
3.2.7 Statistical analysis:
Data were analyzed statistically using GenStat® (Payne et al., 2011). Analysis of
variance was used to test for differences between variables and means were separated
by means using of Fisher’s protected F-test least significant difference.
29
Fig. 1. nil (tubers with no symptoms)
Fig. 2. low (<3% sclerotia on tuber
surface)
Fig. 3. Moderate (3-25% sclerotia on
Fig. 4. High (>25% sclerotia on
tuber surface)
tuber surface)
3.3 RESULTS
Two soil types were used in this study. The laboratory analysis showed the first was a
clay loam with 68% coarse sand, 10% silt and 20% clay, with a pH of 7.3. The second
was a sandy soil with 84.6% coarse sand, 5% silt and 10% clay, with a pH of 6.3.
Of the two soil types, tubers harvested from inoculated sandy soils developed
significantly more disease than those harvested from clay loam soils (P≤0.003) (table.
1). Of the three inoculum sources stolon inoculation and seed inoculum caused
significantly more disease on progeny tubers than those from R. solani spiked soils
(P≤0.001) (table. 1).
No disease development was observed on plants and progeny tuber from the control
pots in this trial, the mean disease severity for all the control pots was 0.0. Therefore, the
statistical calculation does not include the controls.
30
Table. 1. Mean disease severity per inoculum treatment and soil type. .
Soil type
Mean Disease severity*
Soil inoculum
Stolon inoculation
Soil type
Seed inoculum
means
(P ≤0.003)
Clay loam
2.1 ab
3.0 b
2.5 ab
2.6 a
Sandy soil
2.6 ab
3.4 c
3.2 c
3.1 b
Inoculum
2.4 b
3.2 a
2.9 a
means
(P≤0.001)
* Values followed by the same letter do not differ significantly according to Fisher’s F-test
least significant difference.
Plants grown in inoculated soils developed stem canker (Fig. 5 A & B) and green tubers
(Fig. 6). R. solani was successfully isolated from the stem cankers. A Fusarium species
was also isolated from the infected stem tissue.
A
B
Fig. 5 A & B. Stem lesions on plants grown in R. solani inoculated soils
31
Fig. 6. Greening of tubers harvested from plants grown in inoculated soils.
3.4 DISCUSSION
Disease development on a potato plant begins with the presence of the pathogen as
inoculum. R. solani can assume three forms of inoculum basidiospores, mycelium
fragments or sclerotia. Basidiospores are air transmitted inoculum mostly causing
disease to aerial parts of the plant (Naito, 1996; Pascual et al., 2000). Mycelium
fragments and sclerotia are present either as soil- or tuber-borne inoculum and are
commonly associated with disease of below ground parts (Atkinson et al., 2010).
There is controversy surrounding the role and importance of each inoculum source (soilborne vs. tuber-borne) in disease development. Some studies show that as little as 1215% tuber-borne inoculum is sufficient to cause severe stem canker and black scurf
symptoms (James & McKenzie, 1972; Atkinson et al., 2010). Other studies show
significant increases in black scurf and stem canker with an increase in soil-borne
inoculum (Gilligan et al., 1996; Naz et al., 2008). A comparison of disease symptoms
from all three inoculum sources in the current study showed only plants grown in R.
solani inoculated soils developed stem canker. In addition, both stolon and seed
inoculum produced progeny tubers with significantly more severe black scurf symptoms
than soil inoculation. These results may suggest that soil-borne inoculum plays a more
important role in the development of stem canker, whilst tuber-borne inoculum and
stolon infections are more important for the development of black scurf.
32
Although soil-borne inoculum resulted in the least black scurf symptoms, other
symptoms such as tuber greening and stem canker were more prominent. Research has
shown as stolons grow through soils there is a higher chance of infection by contact with
soil-borne inoculum than with tuber-borne inoculum (Frank & Leach, 1980). Infected
stolon tips result in stolon pruning, preventing them from growing to their full length
(Struik et al., 1990). Shallow growth results in tuber development close to the soil
surface and exposure to sunlight. Tubers exposed to sunlight develop poisonous
alkaloids and chloroplasts from amyloplasts which turn tubers green in colour (Zhu et al.,
1984).
One of the symptoms of R. solani disease is stem canker, described as dark brown
necrotic lesions on underground stems (Keijer, 1996; Weinhold & Sinclair, 1996; Simons
& Gilligan, 1997; Tsror & Peretz- Alon, 2005). R. solani and a Fusarium specie was
isolated from the stem lesions observed in the current study. It is, however, not clear
which of the two pathogens was responsible for this symptom or if a relationship exists
between the two pathogens resulting in the development of the stem cankers. The stem
lesions observed in the current study were not characteristic to stem canker, possibly
due to the presence of the Fusarium species. A repeat of this experiment should include
Koch’s postulates to determine if the Fusarium species isolated in this study played a
role in the development of the stem cankers.
Recent research has shown how soil structure and texture can influence soil-borne fungi
such as R. solani (Otten et al., 2001; Harris et al., 2003; Ritz & Young, 2004). Physical
soil characteristics determine the ability of soils to retain water and essential nutrients. It
also determines the connectivity and tortuosity of the air filled pore spaces within soils
(Otten et al., 2001; Harris et al., 2003; Ritz & Young, 2004). Otten et al. (2001) and
Harris et al. (2003) proved that loose sandy soils promote the spread and growth of R.
solani, whilst compact soils inhibit hyphal spread due to the limited available pore space
resulting in small, dense colonies. Results from the current study confirm those of Otten
et al. (2001) and Harris et al. (2003). Black scurf symptoms were significantly more
severe on tubers harvested from loose sandy soils compared to clay loam when data of
the different inoculation methods were combined. These results suggest cultivating in
clay loamy soils is of advantage as it reduces the disease development by limiting the
33
growth and spread of R. solani. However other studies have also shown certain soil
conditions influence the pathogenicity of R. solani (James & McKenzie, 1972).
Another soil condition shown to influence R. solani mycelial growth and sclerotial
development is pH. Research by Ritchie et al. (2009) has shown the optimum pH for
mycelial and sclerotia growth is 5-6. The sandy and clay loam soil used in the current
study had pH readings of 6.3 and 7.3, respectively. The near optimum pH conditions of
the sandy soil used in this study may have contributed to more severe black scurf
symptoms observed on harvested tubers. Future studies however should focus more on
investigating the role soil pH plays on disease development and to confirm results of this
study.
In summary, all three inoculum sources resulted in black scurf development, however
tuber-borne and stolon inoculum proved to be of primary importance. Soil inoculum was
found to be more important for stolon infection and stem canker. Furthermore, the soil
structure and texture of sandy soils used in this study promoted more disease
development than clay loam soils. Future work should confirm the findings of this study
and focus on further investigating the possibility of clay loam soils and soil pH limiting
disease development by reducing the pathogenicity of R. solani.
3.5 REFERENCES
Agrios, G. N. 2005. Plant Pathology, Academic Press INC., London.
Al-Mughrabi, K. I. 2008. Salicylic acid resistance in potatoes against Rhizoctonia solani,
the cause of black scurf and stem canker, International Journal of Biological
Chemistry 2 :14-25.
Atkinson, D., Thornton, M. K. & Miller, J. S. 2010. Development of Rhizoctonia solani on
stems, stolons and tubers of potatoes I. Effect of inoculum source, American
Journal of Potato Research 87: 374-381.
Frank, A. J. & Leach, S. S. 1980. Comparison of tuberborne and soilborne inoculum in
the Rhizoctonia disease of potato, Phytopathology 70:51-53.
Gilligan, C. A., Simons, S. A. & Hide, G. A. 1996. Inoculum density and spatial pattern of
Rhizoctonia solani in fields of Solanum tuberosum: effects of cropping
frequency, Plant Pathology 45: 232-244.
34
Harris, K., Young, I. M., Gilligan, C. A., Otten, W. & Ritz, K. 2003. Effect of bulk density
on the spatial organization of the fungus Rhizoctonia solani in soil, FEMS
Microbiology Ecology 44: 45-56.
James, W. C. & McKenzie, A. R. 1972. The effect of tuber-borne sclerotia of Rhizoctonia
solani Kuhn on the potato crop, American Potato Journal 49: 296-301.
Keijer, J. 1996. The initial steps of the infection process in Rhizoctonia solani. Pages
149-162 In: Sneh, B., Jabaji-Hare, S., Neate S. & Dijst, G. (eds). Rhizoctonia
species: taxonomy, molecular biology, ecology, pathology and disease
control, Kluwer Academic, Dordrecht.
Lahlali, R, & Hijri, M. 2010. Screening, identification and evaluation of potential
biocontrol fungal endophtes against Rhizoctonia solani AG3 on potato plants,
FEMS Microbiology Letters 311: 152-159.
Lehtonen, M. J., Wilson, P. S., Ahvenniemi, P. & Valkonen, J. P. T. 2009. Formation of
canker lesions on stems and black scurf on tubers in experimentally
inoculated potato plants by isolates of AG2-1, AG3 and AG5 of Rhizoctonia
solani: a pilot study and literature review, Agricultural and Food Science 18:
223-233.
Naito, S. 1996. Basidiospore dispersal and survival. Pages 197-207 In: Sneh, B., JabajiHare, S., Neate S. & Dijst, G. (eds). Rhizoctonia species: taxonomy,
molecular
biology,
ecology,
pathology
and
disease
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Naz, F., Rauf, C. A., Abbasi, N. A., Irafan-Ul-Haque. & Ahmad, I. 2008. Influence of
inoculum levels of Rhizoctonia solani and susceptibility on new potato
germplasm, Pakistan Journal of Botany 40: 2199-2209.
Otten, W., Gilligan, C. A., Watts, C. W., Dexter, A. R. & Hall, D. 1999. Continuity of airfilled pores and invasion thresholds for a soil-borne fungal plant pathogen,
Rhizoctonia solani, Soil Biology and Biochemistry 31: 1803-1810.
Otten, W., Hall, D., Harris, K., Ritz, K., Young, I. M. & Gilligan, C.A. 2001. Soil physics,
fungal epidemiology and the spread of Rhizoctonia solani, New Phytologist
151: 459-468.
Parmeter, J. R. 1970. Rhizoctonia solani, biology and pathology, University of California
Press Berkeley, Los Angeles.
35
Pascual, C. B., Toda, T., Raymondo, A. D. & Hyakumachi, M. 2000. Characterization by
conventional techniques and PCR of Rhizoctonia solani isolates causing
banded leaf sheath blight in maize, Plant Pathology 49: 108-118.
Platt, H. W., Canale, F. & Gimenez, G. 1993. Effects of tuber-borne inoculum of
Rhizoctonia solani and fungicidal seed potato treatment on plant growth and
Rhizoctonia disease in Canada and Uruguay, American Potato Journal 70:
553-559.
Payne, R.W., Murray, D.A., Harding, S.A., Baird, D.B. & Soutar, D.M. 2011. GenStat®
for Windows™ 14th Edition, Introduction. VSN International, UK.
Ritchie, F., Bain, R. A. & McQuilken, M. P. 2009. Effects of nutrient status, temperature
and pH on mycelial growth, sclerotial production and germination of
Rhizoctonia solani from potato, Journal of Plant Pathology 91: 589-596.
Ritchie, F., McQuilken, M. P. & Bain, R. A. 2006. Effects of water potential on mycelial
growth, sclerotial production, and germination of Rhizoctonia solani from
potato, Mycological Research 110: 725-733.
Ritz, K. & Young, I. M. 2004. Interactions between soil structure and fungi, Mycologist
18: 52-59.
Simons, S. A. & Gilligan, C. A. 1997. Relationships between stem canker, stolon canker,
black scurf (Rhizoctonia solani) and yield of potatoes (Solanum tuberosum)
under different agronomic conditions, Plant Pathology 46: 651-658.
Sneh, B., Jabaji-hare, S., Neate, S. & Dijst, G. 1996. Rhizoctonia species: taxonomy,
molecular, biology, ecology, pathology and disease control, Kluwer Academic
Publishers, London.
Struik, P. C., Haverkort, A. J., Vreugdenhil, D., Bus, C. B. & Dankert, R. 1990.
Manipulation of tuber-size distribution of a potato crop, Potato Research 33:
417-432.
Tsror, L. & Peretz-Alon, I. 2005. The influence of the inoculum source of Rhizoctonia
solani on development of black scurf on potato, Journal of Phytopathology
153: 240-244.
Weinhold, A. R. & Sinclair, J. B. 1996. Rhizoctonia solani: Penetration, colonisation, and
host response. Pages 163-174 In: Sneh, B., Jabaji-Hare, S., Neate S. & Dijst,
G. (eds). Rhizoctonia species: taxonomy, molecular biology, ecology,
pathology and disease control, Kluwer Academic, Dordrecht.
36
Zhu, Y. S., Merkle-Lehman, D. L. & Kung, S. D. 1984. Light-induced transformation of
amyloplasts into chloroplasts in potato tubers, Plant Physiology 75: 142-145.
37
CHAPTER FOUR
INFLUENCE OF INOCULUM PRESSURE OF RHIZOCTONIA SOLANI ON DISEASE
DEVELOPMENT UNDER FIELD CONDITIONS IN SOUTH AFRICA
ABSTRACT
Successive cultivation of potato crops increases Rhizoctonia solani soil inoculum load
resulting in an escalation in disease incidence and severity. Two field trials were
conducted over two growing seasons, using three different inoculum levels and an
uninoculated control treatment. Four mini plots (1.5m wide x 5m long) were used,
consisting of four rows each. The first trial was planted during summer months
(November 2009-February 2010) and the second trial was planted during winter months
(April 2010-July 2010), where temperatures ranged between 16-29oC and 5-24oC,
respectively. The cultivation of potatoes in the same soil over two growing seasons
resulted in an increase in diseased (black scurf) tubers. Furthermore, results from the
second trial showed black scurf was most severe on tubers from soils inoculated with the
highest concentration of inoculum. There were significant disease severity differences,
with initial soil inoculum levels being directly proportional to final disease severity.
4.1 INTRODUCTION
The potato is the world’s most important non-grain food crop (Huang et al., 2011). Due
to its high nutritional value and ability to produce high yields under diverse environmental
conditions, the potato plays an important role in the fight against hunger and poverty in
many developing countries. The frequent cultivation of potatoes has led to an increased
population of soil-borne pathogens resulting in higher incidence and severity of disease.
One such soil-borne pathogen is R. solani AG3.
This soil- and tuber-borne pathogen is capable of causing disease on tubers, stems and
stolons at all five growing stages of the potato plant (sprout development, vegetative
growth, tuber initiation, tuber bulking and tuber maturation) (Banville, 1989; Simons &
Gilligan, 1997; Naz et al., 2008). Symptoms on stems and stolons consist of dark brown,
necrotic lesions (Simons & Gilligan, 1997; Tsror & Peretz-Alon, 2005). In severely
infested fields these symptoms can result in death of sprouts and stems and girdling of
stolons, resulting in malformed tubers and reduced yields due to poor stand emergence
(James & McKenzie, 1972; Atkinson et al., 2010). During the last two growth stages
38
(tuber bulking and tuber maturation) of the potato plant, the development of black soillike structures on tubers referred to as black scurf is common (Woodhall et al., 2007;
Thind & Aggarwal, 2008; Woodhall et al., 2008).
The development of disease begins when R. solani inoculum is present either as soil- or
tuber-borne sclerotia or hyphae (Scholte, 1992; Gilligan et al., 1996; Atkinson et al.,
2010). Although tuber-borne inoculum has been shown in some studies to be the most
important source (James & McKenzie, 1972; Gudmestad et al., 1979; Atkinson et al.,
2010), it is soil-borne inoculum which proves the most difficult to control due to the wide
host range of R. solani and its ability to survive in soils for several years, even after the
cultivation of non-host crops (Gilligan et al., 1996; Sneh et al., 1996; Naz et al., 2008).
Studies have shown the successive cultivation of potato crops resulted in an increase in
soil inoculum load, intensifying disease (stem canker, stolon canker and black scurf)
incidence and severity (Scholte, 1992; Leach & Webb, 1993; Jager & Velvis, 1995, Mohr
et al., 2011The objective of this study is to investigate the effect of soil inoculum levels
on the development, incidence and severity of stem canker, stolon canker and black
scurf, over two growing seasons under field conditions.
4.2 MATERIALS AND METHODS
4.2.1 Experimental design:
Field trials were conducted over two growing seasons in the Gauteng province in South
Africa. During each trial, temperature and rainfall data was recorded daily at the weather
station situated on the farm. The first trial was planted during summer months
(November 2009-February 2010) and the second trial was planted in the same soils
during winter months (April 2010-July 2010), where temperatures ranged between 1629oC and 5-24oC, respectively. Twenty disease free mini-tubers (cv. Mondial) were
planted by hand in pre-marked rows at a depth of 100mm and 30cm apart.
Approximately 105 days after planting, plant vines were physically cut off. Two weeks
after haulm destruction tubers were harvested by hand.
4.2.2 Preparation of inoculum:
An R. solani (PPRI 9527) isolate from the Potato Pathology Program culture collection
which had previously been identified as AG3 using conventional PCR, in a separate
39
study, was obtained. The isolate was plated onto fresh Potato Dextrose Agar (PDA)
plates and allowed to grow for five days at 25oC. Wheat seeds were soaked overnight in
sterile distilled water with 250µg/ml chloramphenicol and drained. The moist wheat
seeds were autoclaved at 120oC for 1 hour. Each bag of seeds weighing 150g, 250g and
500g, respectively, received five (5mm x 5mm) colonized agar blocks cut up under
aseptic conditions. The wheat seeds were shaken every three days during the
incubation period of 14 days at 25oC.
4.2.3 Inoculation of soil:
The trials were planted in loamy-clay soil, in four mini plots, 1.5m (wide) x 5m (long),
consisting of four rows each. Each mini plot consisted of three different inoculum levels
(150g, 250g & 500g inoculum per row) and an uninoculated control treatment. R. solani
inoculated wheat seeds were evenly distributed in each row after mini-tubers were
placed in the furrows. Irrigation pipes were placed alongside each row in the plots.
Irrigation was applied daily and adjusted to maintain soil moisture to field capacity.
4.2.4 Isolation from stem lesions:
Plants with stem lesions were used for fungal isolation to confirm the causal agent.
Pieces (5mm x 5mm) were cut from the stem lesions and plated on fresh PDA plates
and allowed to grow for five days at 25oC. The cultures were microscopically examined
to identify R. solani.
4.2.5 Disease assessment:
Disease assessment of all tubers and plants was conducted for each inoculum treatment
and their respective replicates, separately. Potato tubers harvested from each plant were
placed into four disease severity categories: 0-healthy (tubers with no symptoms) (Fig.
1); 1-low (<3% sclerotia on tuber surface) (Fig. 2); 2-moderate (3-25%) (Fig. 3) and 3high (>25%) (Fig. 4). The disease severity index (s.i.) was calculated using the following
formula (Tsror & Peretz-Alon, 2005):
s.i.= (0 x n) + (1 x l) + (2 x m) + (3 x h)
Total number of tubers
40
Fig. 1. nil (tubers with no symptoms)
Fig. 2. low (<3% sclerotia on tuber
surface)
Fig. 3. Moderate (3- 25% sclerotia on
Fig. 4. High (>25% sclerotia on
tuber surface)
tuber surface)
At harvest, underground stems and stolons were evaluated using a scale of 0-5, where 0
= healthy tissue, 1 = several brown to black lesions, 2 = up to 15% of the tissue is
covered with lesions, 3 = up to 30% of the tissue is covered with lesions, 4 = up to 60%
of the tissue is covered with lesions and 5 = >60% of the tissue is covered with lesions
(Tsror & Peretz-Alon, 2005).
4.2.6 Statistical analysis:
Data were analyzed statistically using GenStat® (Payne et al., 2011). Analysis of
variance was used to test for differences between variables and means were separated
using Fisher’s protected F-test least significant difference (P≤0.001).
41
4.3 RESULTS
Maximum temperatures averaged at 29oC and 24oC for the first and second trials,
respectively, while temperatures dropped to a minimum of 14oC and 5oC, respectively
(Fig. 5). Rainfall data collected showed the summer months received a total of 157.5mm
more seasonal rain than the winter months (Fig. 6).
Avg. Temperature (oC)
35
30
25
20
15
10
Max 5
Min
0
Month Fig. 5. Average minimum and maximum temperature (oC) per month
The first field trial planted during November 2009 resulted in only 4 diseased tubers out
of a total of 429 harvested tubers, results are therefore not shown. At harvest of the
second field trial, 216 tubers showed black scurf symptoms. Although disease severity
increased with increasing initial inoculum, there were no significant differences between
the 0 and 150 inoculum levels and the 250 and 500 inoculum levels, respectively (Fig.
7).
Stem lesions not typical to Rhizoctonia disease were observed on above ground parts of
plant stems. Isolation from these lesions did not yield R. solani but resulted in the
positive identification of a Fusarium species.
42
Total Rainfall (mm)
140
120
100
80
60
40
20
0
A
Rainfall (mm)
November December January '10 February '10
'09
'09
Total Rainfall (mm)
Summer months
200
180
160
140
120
100
80
60
40
20
0
B
Rainfall (mm) April '10
May '10
June '10
July '10
Winter months Mean of disease
severity
Fig. 6 A & B. Total rainfall (mm) per month
3
2.5
2
1.5
1
0.5
0
b
b
a
a
0
150
250
500
Amount of inoculum (g of inoculated seed/row)
Fig. 7. Mean disease severity index over all four inoculum replicates recorded during the
second trial. Disease severity categories: 0-healthy (tubers with no symptoms), 1-low
(<3% sclerotia on tuber surface), 2-moderate (3-25%) and 3-high (>25%) (Tsror &
Peretz-Alon, 2005). Bars followed by the same letter do not differ significantly according
to Fisher’s F-test least significant difference (P≤0.001).
43
4.4 DISCUSSION
Infection of potato by R. solani AG3 results in black scurf, stem and stolon canker
(Woodhall et al., 2007; Thind & Aggarwal, 2008; Woodhall et al., 2008). The incidence
and severity of these symptoms is determined by many biotic and abiotic factors as well
as initial inoculum load present in soils (Scholte, 1992; Gilligan et al., 1996). Studies
investigating the role inoculum plays in disease development have shown crop rotation
to be an effective method in reducing inoculum concentrations in soils. Although rotation
with non-host crops such as barley, sorghum and certain grasses has been shown to
reduce soil inoculum, the correct rotation intervals are also necessary for this method to
be effective. Gilligan et al. (1996) showed that when potato crops are cultivated
consecutively a 70-80% increase in diseased tubers and plants can be expected. The
cultivation of potatoes in the same soil over two growing seasons in the current study
may have led to the 56% increase in diseased tubers (black scurf) observed in the
second trial. Furthermore, results from the second trial showed black scurf was most
severe on tubers from soil infested with the highest initial inoculum concentrations.
There were significant differences between disease severity and soil inoculum levels,
with inoculum levels being directly proportional to final disease severity.
The two most important abiotic factors influencing disease development are temperature
and moisture (Sneh et al., 1996; Agrios, 2005). Typical climatic conditions common to
the Gauteng province in South Africa are hot, wet summers and cold, dry winters.
Research has shown the optimum temperature range for R. solani disease development
to be between 0-15oC (Beukema & Van Der Zaag, 1990, Sneh et al., 1996). Some
studies show high moisture conditions promote the development of black scurf (Rowe et
al., 1993; Gilligan et al., 1996; Simons & Gilligan, 1997), whilst others show high
moisture levels produce harvests with less extensive sclerotia (Frank & leach, 1980) The
variation in results between the two field trials in the current study could be explained by
the different weather patterns experienced during the two growing seasons.
The first trial was planted during the warmer months and received a total of 415.5mm of
rainfall for the duration of the trial. The second field trial, which was planted during the
cooler months and received a total of 258mm of rainfall, resulted in more black scurf
diseased tubers than in the first trial. Rainfall during the first trial fell throughout the
growing season, during the second trial rainfall was mostly received during the early
44
parts of the growing season. However the role the rainfall patterns played in the increase
of disease severity cannot be conclusive, as the mini plots used in the current study
were also irrigated daily Although some studies have shown R. solani growth is
restricted by high moisture levels (>1500 bars) (Ploetz & Mitchell, 1985), other studies
show moisture levels have no influence on R. solani growth (Junior et al., 2007; Olanya
et al., 2010). Unfortunately, in this study tensiometer readings to determine soil water
content were not taken. A repeat of this experiment should include measurement of
moisture and aeration of soils to investigate their role in disease development.
The development of these symptoms is influenced by temperature, soil moisture and
inoculum levels (Simons & Gilligan, 1997). Stem or stolon canker symptoms were not
observed during either trial, however stem lesions on above ground parts of stems were
observed. These lesions did not fit the typical characteristic stem canker symptoms
caused by R. solani, and isolation of R. solani from these lesions was unsuccessful. The
absence of stem or stolon canker symptoms may suggest that the environmental
conditions did not favour disease development. In addition, the absence of canker
symptoms could also be attributed to the lack of seed inoculum, which has been
associated with stolon and stem infections at early stages of the growing period (Frank &
Leach, 1980).
In recent years, studies have investigated the important role soil characteristics play in
soil inoculum levels and disease development (Otten et al., 2001; Harris et al., 2003;
Ritz & Young, 2004). Soil structure and texture affects pore size, aeration, water
movement, plant root growth and retention ability of nutrients and water, thereby
influencing the growth and spread of soil-borne pathogens. Research has shown hyphal
growth and spread is restricted in high density soils, with high water retention capacity,
as pore space is limited and pores are often filled with water (Otten et al., 2001; Harris et
al., 2003; Ritz & Young, 2004). The narrow and limited pore space available in high
density soils, such as loamy-clay used in the current study may have inhibited disease
development by preventing complete R. solani colonization of the soil (Otten et al., 1999;
Otten et al., 2001; Harris et al., 2003; Ritz & Young, 2004). Disease incidence and
severity may have been higher if this experiment was conducted using loose, sandy
soils. Further research investigating the role of soil type (sandy soil vs. dense soil) is
discussed in chapter three.
45
Although the relationship between inoculum levels and disease severity has been
established, our attempts at quantifying the amount of inoculum present in soils after
inoculation using real time PCR in a separate study, proved unsuccessful. Knowledge of
inoculum levels in soils prior to planting is essential for better disease management.
Therefore, future studies should focus on using diagnostic tools such as nested PCR,
microarray or real time PCR to detect and quantify R. solani in soils. Further research
into the role of interactions between R. solani and the environment and other microorganisms on disease development, is necessary for better disease management
decisions.
4.5 REFERENCES
Agrios, G. N 2005. Plant Pathology, Academic Press INC., London.
Atkinson, D., Thornton, M. K. & Miller, J. S. 2010. Development of Rhizoctonia solani on
stems, stolons and tubers of potatoes I. Effect of inoculum source, American
Journal of Potato Research 87: 374-381.
Banville, G. J. 1989. Yield losses and damage to potato plants caused by Rhizoctonia
solani Kuhn, American Potato Journal 66: 821-834.
Beukema, H. P. & Van der Zaag. 1990. Introduction to potato production, Agriculture
University Wageningen.
Frank, A. J. & Leach, S. S. 1980. Comparison of tuberborne and soilborne inoculum in
the Rhizoctonia disease of potato, Phytopathology 70: 51-53.
Gilligan, C. A., Simons, S. A. & Hide, G. A. 1996. Inoculum density and spatial pattern of
Rhizoctonia solani in fields of Solanum tuberosum: effects of cropping
frequency, Plant Pathology 45: 232-244.
Gudmestad, N. C., Zink , R. T. & Huguelet, J. E. 1979. The effect of harvest date and
tuber-borne sclerotia on the severity of Rhizoctonia disease of potato,
American Potato Journal 56: 35-41.
Harris, K., Young, I. M., Gilligan, C. A., Otten, W. & Ritz, K. 2003. Effect of bulk density
on the spatial organization of the fungus Rhizoctonia solani in soil, FEMS
Microbiology Ecology 44: 45-56.
Huang, S., Buell, C. R. & Visser, R. G. F. 2011. Genome sequence and analysis of the
tuber crop potato. Nature, International weekly journal of science,
http://www.nature.com/nature/journal/v475/n7355/full/nature10158.html-
26
October 2011
46
Jager, G. & Velvis, H. 1995. Dynamics of Rhizoctonia solani (black scurf) in successive
potato crops, European Journal of Plant Pathology 101: 467-478.
James, W. C. & McKenzie, A. R. 1972. The effect of tuber-borne sclerotia of Rhizoctonia
solani Kuhn on the potato crop, American Potato Journal 49: 296-301.
Junior, J. T., Rotter, C. & Hau, B. 2007. Effects of soil moisture and sowing depth on the
development of bean plants grown in sterile soil infested by Rhizoctonia
solani and Trichoderma harzianum, European Journal of Plant Pathology
119: 193-202.
Leach, S. L. & Webb, R. E. 1993. Evaluation of potato cultivars, clones and a true seed
population for resistance to Rhizoctonia solani, American Potato Journal 70:
317-327.
Mohr, M. R., Volkmar, K., Derksen, A. D., Irvine, R. B., Khakbazan, M., McLaren, L. D.,
Monreal, M. A., Moulin, P. A. & Tomasieqwiez, J. D. 2011. Effect of rotation
on crop yield and quantity in an irrigated potato system, American Journal of
Potato Research 88: 346-356.
Naz, F., Rauf, C. A., Abbasi, N. A., Irafan-Ul-Haque. & Ahmad, I. 2008. Influence of
inoculum levels of Rhizoctonia solani and susceptibility on new potato
germplasm, Pakistan Journal of Botany 40: 2199-2209.
Olanya, M. O., Porter, A. G., Lambert, H. D., Lakin, P. R. & Starr, C. G. 2010. The
effects of supplemental irrigation and soil management on potato tuber
diseases, Plant Pathology Journal 9: 65-72.
Otten, W., Gilligan, C. A., Watts, C. W., Dexter, A. R. & Hall, D. 1999. Continuity of airfilled pores and invasion thresholds for a soil-borne fungal plant pathogen,
Rhizoctonia solani, Soil Biology and Biochemistry 31: 1803-1810.
Otten, W., Hall, D., Harris, K., Ritz, K., Young, I. M. & Gilligan, C.A. 2001. Soil physics,
fungal epidemiology and the spread of Rhizoctonia solani, New Phytologist
151: 459-468.
Payne, R.W., Murray, D.A., Harding, S.A., Baird, D.B. & Soutar, D.M. 2011. GenStat®
for Windows™ 14th Edition, Introduction. VSN International, UK.
Ploetz, R. C. & Mitchell, D. J. 1985. Influence of water potential on the survival and
saprophytic activity of Rhizoctonia solani AG4 in natural soil, Canadian
Journal of Botany 63: 2364-2368.
Ritz, K. & Young, I. M. 2004. Interactions between soil structure and fungi, Mycologist
18: 52-59.
47
Rowe, R. C., Curwen, D., Ferro, D. N., Loria, R. & Secor, G. A.
1993. Potato
Management, The American Phytopathological Society, United States of
America.
Scholte, K. 1992. Effect of crop rotation on the incidence of soil-borne fungal diseases of
potato, Netherlands Journal of Plant Pathology 98: 93-101.
Simons, S. A. & Gilligan, C. A. 1997. Relationships between stem canker, stolon canker,
black scurf (Rhizoctonia solani) and yield of potatoes (Solanum tuberosum)
under different agronomic conditions, Plant Pathology 46: 651-658.
Sneh, B., Jabaji-Hare, S., Neate, S. & Dijst, G. 1996. Rhizoctonia species: taxonomy,
molecular, biology, ecology, pathology and disease control, Kluwer Academic
Publishers, London.
Thind, T. S. & Aggarwal, R. 2008. Characterization and pathogenic relationships of
Rhizoctonia solani isolates in a potato-rice system and their sensitivity to
fungicides, Phytopathology 156: 615-621.
Tsror, L. & Peretz-Alon, I. 2005. The influence of the inoculum source of Rhizoctonia
solani on development of black scurf on potato, Journal of Phytopathology
153: 240-244.
Woodhall, J. H., Lees, A. K., Edwards, S. G. & Jenkinson, P. 2007. Characterization of
Rhizoctonia solani from potatoes in Great Britain, Plant Pathology 56: 286295.
Woodhall, J. H., Lees, A. K., Edwards, S. G. & Jenkinson, P. 2008. Infection of potato by
Rhizoctonia solani: effect of anastomosis group, Plant Pathology 57: 897905.
48
CHAPTER FIVE
SUSCEPTIBILITY OF FIVE POTATO CULTIVARS COMMONLY CULTIVATED IN
SOUTH AFRICA, TO RHIZOCTONIA SOLANI
ABSTRACT
In addition to chemical and biological control, the use of tolerant cultivars can be an
effective method for reducing Rhizoctonia solani inoculum levels in soils, thereby
decreasing disease severity and incidence. Two pot trials were conducted to determine
susceptibility of five cultivars, BP1, Fianna, Mondial, Up-To-Date (UTD) and Valor, to R.
solani. The first pot trial was grown at 0-10oC at night and 15–20oC during the day;
during the second trial temperatures were 21-23oC at night and 25-26oC during the day.
Results showed none of the cultivars to be resistant to R. solani, however BP1 was less
susceptible to R. solani at temperatures between 21-26oC. Furthermore, in comparison
to the other cultivars UTD had the lowest severity index values for both trials suggesting
it may be less susceptible to black scurf at a wider range of temperatures (0-26oC). A
comprehensive analysis of the results from both trials showed more severe disease
symptoms on all cultivars were observed under cooler temperatures.
5.1 INTRODUCTION
Rhizoctonia solani Kuhn (teleomorph Thanatephorus cucumeris (A.B. Frank) Donk) is a
soil-borne fungus causing various disease symptoms on many different host crops
(Brooks, 2007; Guleria et al., 2007; Thind & Aggarwal, 2008). Symptoms associated with
the potato crop include black scurf, stolon canker and stem canker (Woodhall et al.,
2007; Thind & Aggarwal, 2008; Woodhall et al., 2008). There are 13 different
Anastomosis Groups (AG’s) within this species complex (Lees et al., 2002; Harikrishnan
& Young, 2004; Guleria et al., 2007; Woodhall et al., 2007). Each AG has been shown to
differ in disease symptoms, host range and control methods (Sneh et al., 1996; Keijer et
al., 1997; Tewoldemedhin et al., 2006). However, research has commonly attributed
black scurf on potato crops to AG 3 (Parmeter, 1970; Sneh et al., 1996; Lees et al.,
2002; Virgen-Calleros et al., 2000; Back et al., 2006; Lehtonen et al., 2009; Lahlali &
Hijri, 2010).
Black scurf is often referred to as a “cosmetic” disease, identifiable as black soil-like
structures (sclerotia), on tuber surfaces. Potato tubers showing black scurf symptoms
49
are downgraded in consumer markets resulting in immense economic losses (Platt et al.,
1993; Hamid et al., 2006; Al-Mughrabi, 2008). Seed markets are also susceptible to
such losses as the certification of potato seed tubers in South Africa is governed by the
Plant Improvement Act (ACT No. 57 of 1976) which permits the following (Republic of
South Africa, 2002):
 0% infection in G0
 0.5-20% infection in G1-3
 1-20% infection in G4-6
 1-20% infection in G7-8
Reducing inoculum levels in soils and on seed tubers is therefore essential. Fungicides,
applied as either soil and/or seed treatments, are the preferred method of control
(Campion et al., 2003; Boogert Van der & Luttikholt, 2004; Rauf et al., 2007). However,
efficacy of these fungicides is dependent on toxicity levels of the active ingredient (a.i.)
and on the morphology, physiology, virulence and genetic constitution of different
Rhizoctonia species (Kataria et al., 1991). Furthermore, the use of fungicides over time
could result in development of resistance, residue build-up on harvested products and
adverse effects on the environment, animal and human health (Eddleston et al., 2002;
Tomlin, 2006).
Recent studies have investigated the use of biological agents for reducing disease
incidence and severity. Some mycoparasites such as Trichoderma harzianum, T.
viridae, T. hamatum, Gliocladium virens and Verticillium biguttatum have been shown to
significantly reduce R. solani inoculum levels in soils (Wale, 2004; Brooks, 2007; Wilson
et al., 2008). However, geographical variations in temperature, soil type, soil moisture
and cultural practices can limit the use of biological control agents.
In addition to chemical and biological control, the use of tolerant cultivars is the most
economical yet effective method for reducing inoculum levels in soils (Otrysko &
Banville, 1992, Leach &Webb, 1993). Research by Du Plessis (1999) showed varying
disease susceptibility levels of 16 different cultivars grown in South Africa. However
there are approximately 79 different cultivars in South Africa, of which the most
prominent on the consumer market is Mondial (65%), BP1 (14%) and Up-To-Date (UTD)
50
(5%) (Potatoes South Africa, 2010). Further research on susceptibility levels of these
cultivars to R. solani is therefore required. This study focuses on determining
susceptibility levels of five commonly planted cultivars; BP1, Mondial, Valor, Fianna and
UTD, to R. solani.
5.2 MATERIALS AND METHODS
5.2.1 Pot trials:
Pot trials were conducted at the University of Pretoria under greenhouse conditions.
Disease free mini-tubers (cv. BP1, Fianna, Mondial, UTD and Valor) were used for the
trials (table. 1). The pot trials were set out in a randomized complete block design and
consisted of five cultivars planted in inoculated soil and uninoculated soil (control). The
first pot trial started in June 2009 and was maintained at temperatures of 0-10oC at night
and 15–20oC during the day. The second trial started in October 2010 and was
maintained at temperatures of 21-23oC at night and 25-26oC during the day. Each
treatment was replicated five times. Approximately 105 days after planting, plant vines
were cut off manually. Tubers were harvested two weeks after haulm destruction.
Table. 1. Cultivar black scurf susceptibility/ tolerance and growth period (Visser, 2011)
Cultivar
Black scurf susceptibility/ tolerance
Growth period
BP1
Highly susceptible
Medium (90-110 days)
Fianna
Moderately susceptible
Medium (90-110 days)
Mondial
Highly tolerant
Medium (90-110 days)
UTD
Susceptible
Medium to long (90-120 days)
Valor
Moderately susceptible
Medium (90-110 days)
5.2.2 Preparation of inoculum:
An R. solani (PPRI 9527) isolate from the Potato Pathology Program culture collection
which had previously been identified as AG3 using conventional PCR, in a separate
study, was obtained. The isolate was plated on fresh Potato Dextrose Agar (PDA) plates
and allowed to grow for five days at 25oC. Wheat seeds were soaked overnight in sterile
distilled water with 250µg/ml chloramphenicol and drained. The moist wheat seeds were
autoclaved at 120oC for 1 hour. Each bag of seeds weighing 200g was mixed with five
(5mmx5mm) agar blocks of actively growing mycelium cut up under aseptic conditions.
51
The wheat seeds were shaken every three days during the incubation period of 14 days
at 25oC (Sneh et al., 1986).
5.2.3 Inoculation of soil:
Soil autoclaved at 120oC for 1 hour, weighing 3.4kg was potted in 4kg-capacity pots.
Each pot was inoculated with 40g of R. solani colonized wheat seeds. The inoculum was
thoroughly mixed into the soil before planting one seed tuber in each pot at a depth of
100mm. Soil moisture was maintained with 200ml distilled water three times a week.
Plants were fertigated with 200ml of Superfeed every three weeks, applied at a rate of
1g in 1L water.
5.2.4 Disease assessment:
At harvest, potato tubers harvested from each plant were placed into four disease
severity categories: 0-nil (tubers with no symptoms) (Fig. 1), 1-low (<3% sclerotia on
tuber surface) (Fig. 2), 2-moderate (3-25%) (Fig. 3) and 3-high (>25%) (Fig. 4). The
disease severity index (s.i.) was calculated using the following formula (Tsror & PeretzAlon, 2005):
s.i.= (0 x n) + (1 x l) + (2 x m) + (3 x h)
Total number of tubers
At harvest, underground stems and stolons were evaluated using a scale of 0-5, where 0
= healthy tissue, 1 = several brown to black lesions, 2 = up to 15% of the tissue is
covered with lesions, 3 = up to 30% of the tissue is covered with lesions, 4 = up to 60%
of the tissue is covered with lesions and 5 = >60% of the tissue is covered with lesions
(Tsror & Peretz-Alon, 2005).
5.2.5 Statistical analysis:
Data were analyzed statistically using GenStat® (Payne et al., 2011). Analysis of
variance was used to test for differences between variables and means were separated
using Fisher’s protected F-test least significant difference.
52
Fig. 1. nil (tubers with no symptoms)
Fig. 2. low (<3% sclerotia on tuber
surface)
Fig. 3. Moderate (3-25% sclerotia on
Fig. 4. High (>25% sclerotia on
tuber surface)
tuber surface)
5.3 RESULTS
Results from the first pot trial planted during June 2009 showed varying levels of black
scurf in all five cultivars. Cultivar Fianna and BP1 developed the highest level of disease
symptoms, compared to the other three cultivars which showed high to moderate levels
of disease symptoms. UTD showed the least amount of disease, although not
significantly less than Mondial (Fig. 5). However in the second pot trial planted in
October 2010 all cultivars had low (<3%) to moderate (3-25%) disease symptoms with
no significant differences between the cultivars (Fig. 6). No disease development was
observed on plants and progeny tuber from the control pots in this trial, the mean
disease severity for all the control pots was 0.0.
53
3.5
Mean of disease severity
3
d
d
2.5
c
2
1.5
1
b
b
0.5
0
UTD
Mondial Valor
BP1
Fianna
Potato cultivar
Fig. 5. Mean disease severity per cultivar in pot trial one. Disease severity categories: 0nil (tubers with no symptoms), 1-low (<3% sclerotia on tuber surface), 2-moderate (325%) and 3-high (>25%) (Tsror & Peretz-Alon, 2005). Bars followed by the same letter
do not differ significantly according to Fisher’s F-test least significant difference
(P≤0.001).
Mean of disease severity
2.5
a
2
1.5
a
a
a
UTD
Fianna
a
1
0.5
0
BP1
Valor
Mondial Potato cultivar
Fig. 6. Mean disease severity per cultivar in pot trial two. Disease severity categories: 0nil (tubers with no symptoms), 1-low (<3% sclerotia on tuber surface), 2-moderate (325%) and 3-high (>25%) (Tsror & Peretz-Alon, 2005). Bars followed by the same letter
do not differ significantly according to Fisher’s F-test least significant difference
(P≤0.001).
54
5.4 DISCUSSION
Disease incidence and severity increases when favorable temperatures exist for the
pathogen and not for the host. When temperatures are optimum for the host but not for
the pathogen, disease development is inhibited (Agrios, 2005). Studies have shown that
the optimum temperature range for the healthy growth of a potato plant is 20-25oC, while
cooler temperatures 10-15oC results in weaker plant growth, allowing Rhizoctonia to
cause more disease (Beukema & Van Der Zaag, 1990, Sneh et al., 1996). Results from
the current study supports this as more severe disease symptoms were observed under
cooler temperatures (0-20oC). These results also support those of Du Plessis (1999)
showing higher disease incidence and severity of black scurf at temperatures between
12-15oC than at temperatures ranging between 21-28oC.
Tuber development and maturity in early cultivars occurs earlier in the growing season
than that of late cultivars (Beukema & Van Der Zaag, 1990; Rowe et al., 1993). Bains et
al. (2002) found that late maturing cultivars developed low levels of black scurf as
compared to early and mid-season cultivars. He suggested the reason for these results
were the differences in the time of tuber maturity between the cultivars. However the
exact mechanism of how the time of tuber maturity affects disease development is
unknown. Although none of the five cultivars used in the current study were resistant to
R. solani, results showed varying levels of black scurf symptoms between the cultivars.
Statistical analysis of the results showed that Mondial, a mid-season cultivar,had fewer
disease symptoms when compared to the other mid-season cultivars (BP1, Fianna, and
Valor). These results therefore do not agree with the findings of Bains et al. (2002).
Furthermore these results are not in keeping with that of Visser (2011) who reported
Mondial to be highly tolerant to black scurf (table. 1). Future research should focus more
on unraveling the genetic composition of the potato tuber to identify potential resistance
genes in different cultivars.
The killing of vines (haulm destruction) to induce early tuber maturation is a common
practice in the potato production industry. Studies have shown the time between haulm
destruction and harvest influences the development of black scurf on progeny tubers
(Beukema & Van Der Zaag, 1990; Rowe et al., 1993; Kempenaar & Struik, 2007). A
study by Gudmestad et al. (1979) showed that if tubers are harvested within 3-4 weeks
after vine killing, disease symptoms are minimal. In the current study, tubers were
55
harvested two week after vine killing; however progeny tubers showed medium to high
levels of black scurf symptoms. These results may suggest if initial inoculum levels are
high under favorable biotic and abiotic conditions; the influence of haulm destruction on
disease development may be negligible. Banville (1989) during his research on cultivar
susceptibility also made mention of cultivars showing greater susceptibility levels being a
result of the amount of inoculum and environmental conditions.
The role of inoculum source (seed vs soil) on disease development has been the subject
of many studies (Frank & Leach, 1980; Platt et al., 1993; Gilligan et al., 1996; Tsror &
Peretz-Alon, 2005; Atkinson et al., 2010; Lees et al., 2010). Research by Tsror & PeretzAlon (2005) showed that when seed and soil inoculum is present black scurf incidence
and severity increases. Furthermore, the presence of both inoculum sources under cold
and moist conditions also increases the incidence and severity of stem canker and
stolon canker (Frank & Leach, 1980; Platt et al., 1993; Rowe et al., 1993; Gilligan et al.,
1996; Simons & Gilligan, 1997; Al-Mughrabi, 2008; Ritchie et al., 2009; Atkinson et al.,
2010). In this study inoculated wheat seeds were the only source of inoculum which
could explain why no stem or stolon canker was observed on any of the potato plants. It
could also be that the temperature range and moisture levels in this study were not
favorable for the development of stem and stolon cankers.
In summary, none of the cultivars used in this study were resistant to R. solani and no
stem or stolon canker symptoms were observed. However, higher incidence of disease
symptoms were observed on progeny tubers cultivated under cooler temperatures.
Future research should focus on the possibility of cv. UTD being less susceptible to
black scurf at temperatures ranging from 0-26oC; the importance of timing of lifting after
haulm destruction in disease development and unraveling the genetic composition of
various cultivars which could potentially provide more insight into cultivar resistance to
R. solani.
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ABSTRACT
Rhizoctonia solani inoculum can be present either as soil- or tuber-borne sclerotia or
hyphae. Although both inoculum sources play a role in disease development, it is not
clear which of the two is more important. Successive cultivation of potato crops
increases R. solani soil inoculum load resulting in an escalation in disease incidence and
severity. The use of tolerant cultivars, however, can effectively reduce inoculum levels
thereby decreasing disease intensity. Four pot trials were conducted; the objective of the
first two pot trials was to determine the effect of tuber and soil-borne inoculum and stolon
inoculations on disease development in sandy and clay loam soils. The second two pot
trials were aimed at determining susceptibility levels of five cultivars. Two field trials were
planted over two growing seasons in the same soils, using three inoculum levels.
Results from the pot trials showed that tubers harvested from inoculated sandy soils
developed significantly more disease than those harvested from clay loam soils. Of the
three inoculum sources, stolon inoculation and seed-borne inoculum resulted in
significantly more disease on progeny tubers than those from R. solani spiked soils.
Although none of the cultivars proved to be tolerant to R. solani, BP1 was less
susceptible to R. solani at temperatures between 21-26oC. More severe disease
symptoms were observed under cooler temperatures on all cultivars. Results from the
field trial showed the cultivation of potatoes in the same soil over two growing seasons
resulted in an increase in diseased (black scurf) tubers. Furthermore, black scurf was
most severe on tubers from soils infested with the highest concentration of inoculum.
There were significant disease severity differences, with initial soil inoculum levels being
directly proportional to final disease severity. Future studies in South Africa should focus
on investigating the genetic composition of various cultivars; the effect of soil type and
pH on the pathogenicity of R. solani and the use of molecular diagnostic tools to detect
and quantify R. solani in soils.
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