FEMS Microbiology Letters 115 (1994) 83-86
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
83
FEMSLE 05751
Variations in culture morphology and pathogenicity
among protoplast-regenerated strains
of Rhizoctonia solani
H u a A. Yang a, Jumei Z h o u
a Krishnapillai Sivasithamparam .,a and Philip A. O'Brien b
a Soil Science and Plant Nutrition, School of Agriculture, The Unicersity of Western Australia, Nedlands, W.A. 6009, Australia, and
b Biotechnology Program, School of Biological and Environmental Science, Murdoch Unit,ersity, W.A. 6150, Australia
(Received 4 October 1993; revision received and accepted 20 October 1993)
Abstract: Protoplast-regenerated cultures derived from mycelia of cereal-infecting field isolates of Rhizoctonia solani exhibited
major variations in cultural morphology and in pathogenicity. Each field isolate yielded three or four distinct morphological types
of protoplast cultures. The presence of the new morphological phenotypes was attributed to the selection of homokaryons arising
from protoplasts with single nuclei. Highly pathogenic field isolates produced protoplast cultures with higher virulence than those
from weakly virulent pathogenic isolates, and homokaryotic strains were generally less pathogenic than the parental field isolate.
Key words: Rhizoctonia solani; Heterokaryosis; Protoplast; Pathogenicity
Introduction
Rhizoctonia solani Kiihn, an important soilborne plant pathogen distributed worldwide [1], is
characterized by multinucleate hyphal cells. Field
isolates are heterokaryotic [2]. Heterokaryosis
contributes much of the variability in this species
[3]. As R. solani does not produce asexual spores,
studies on heterokaryosis has relied on basidiospore cultures [2,3]. Two problems are encountered in dealing with homokaryotic cultures arising from basidiospores. One is that the sporula-
* Corresponding author.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 3 ) E 0 4 4 3 - G
tion is very difficult to induce, as none of the
available methods can guarantee success. The
other problem is that a basidiospore culture does
not represent the genetic constitution of the component nuclei in vegetative hyphae, as basidiospores are the products of potential intensive
genetic recombinations during meiosis. Therefore, it is difficult to assess the genetic differences
between the sibling nuclear types in vegetative
mycelia by analyzing basidiospore cultures. Recently, protoplasts were used to generate
homokaryotic strains from heterokaryotic field
isolates [4,5]. However, the extent of variation in
cultural morphology and pathogenicity among
protoplast-regenerated cultures in R. solani has
not been reported.
84
Materials and Methods
Field isolates
The test field isolates of R. solani, belonging
to anastomosis group-8 (AG-8), were obtained
from infected wheat roots sampled from barepatch disease in a wheat field site at Newdegate,
Western Australia.
Protoplast-regenerated strains
Each field isolate of R. solani was cultured in
a Petri plate containing 15 ml of Difco potato
dextrose broth (PDB). After 48 h incubation at
25°C, the mycelia were transferred into protoplast formation solution which contained 10 mg
ml i Novozym 234 (Sigma L - 2 2 6 5 ) a n d 1 M
sodium chloride. The reaction mixture was filtrated with double layer Miracloth (Calbiochem,
475855), and centrifuged at 800 rpm for 5 rain.
Protoplasts in the supernatant fraction were sus-
pended in PDB amended with 2% Bacto agar
and 1.0 M mannitol. Single regenerated protoplasts were picked up under a microscope and
transferred onto potato-dextrose agar (PDA).
Transferred cultures received the alphabet 'P' in
addition to their number to denote their cultural
origin. The homokaryotic and heterokaryotic nature of each protoplast-regenerated culture was
identified using a system based on tuft formation
[3]. Detailed procedures used for protoplast
preparation, regeneration and homokaryon testing were as described previously [4].
Et,'aluation of" cultural morphology
The parent isolates and their resultant protoplast-regenerated strains were grown on P D A for
5 days. A plug of 5 mm diameter from the edge of
the colony was transferred onto a Petri plate
containing 20 ml Difco PDA, and incubated at
25°C. Hyphal growth was determined after 72 h
Fig. 1. Field isolate 33052 and its three resultant protoplast-regenerated strains 33052P2, 33052P14 and 33052P20 of Rhizoctonia
solani (AG-8) grown on potato-dextrose agar at 25°C for 6 days. Note the distinct cultural morphology.
85
incubation. Two perpendicular diameters were
measured for each colony, and hyphal growth
rate, expressed as radial colony increase per 24 h,
was calculated on the basis of three replicates.
Colony pigmentation, abundance of aerial hyphae, sclerotia and moniloid cells were observed
after incubation for four weeks [6].
Pathogenicity test
Pot trials for pathogenicity tests were based on
the method of McDonald and Rovira [7]. Four g
of millet seeds was soaked in a 10-ml McCartney
glass bottle containing 4 ml deionized water
overnight. The bottle was autoclaved three times
for 20 rain at 121°C on three separate days. Each
bottle was inoculated with two PDA plugs of an
isolate. The bottle was capped with a cotton plug
and incubated at 25°C for two weeks. Colonized
millet seeds were spread over a tissue and airdried in a laminar flow cabinet for 2 h. Seven
millet seeds, colonized by R. solani, were placed
in a pot containing pasteurized sand. After two
weeks incubation at 18°C/15°C (day/night), five
pregerminated wheat seeds (cv. Spear) were
planted in each pot. Each isolate had six replicate
pots. The plants were harvested four weeks after
sowing. Disease severity was estimated by measuring fresh root weight.
Results and Discussion
Variation in culture morphology
Thirteen viable protoplast-regenerated cultures were obtained from field isolate 33052. Four
distinct morphological types were identified: type
1 (representative strain: 33052P2) had the same
pigmentation as the mother field isolate, but was
characterized by reduced hyphal growth rate and
denser aerial hyphae; type 2 (representative
strain: 33052P14) had very slow hyphal growth
rate, deep brown pigmentation, and a moderate
amount of aerial hyphae; type 3 (representative
strain: 33052P20) was characterized by the pale
pigmentation and dense aerial hyphae; and the
fourth morphological type was morphologically
similar to their mother field isolate, as they all
had fast hyphal growth rate, medium brown pig-
Table 1
Cultural morphology of representative protoplast-regenerated
cultures and their parent field isolates of Rhizoctonia solani
Test
isolates
Radial hyphal
growth rate
( r a m / 2 4 h) a
Pigmentation b
Abundance of
aerial hyphae c
11034
11034P2
11034P3
11034P7
11102
11102P3
11102P8
11102P12
11082
11082P1
11082P19
23074
23074P21
23074P36
12092
12092P1
12092P3
7.30
6.28
4.00
5.39
7.28
4.25
5.41
4.69
7.20
4.06
5.28
7.28
4.45
5.39
7.88
4.00
7.76
MB
MB
DB
Pale
MB
DB
Pale
Pale
MB
DB
Pale
MB
DB
Pale
MB
DB
DB
1
1
0
3
1
0
3
3
1
0
2
1
0
3
1
2
1
Means of increase of radial growth on PDA at 25°C determined 72 h after inoculation.
b Colonies grown on PDA at 25°C for 4 weeks. MB = midbrown; DB = deep brown.
Abundance of aerial hyphae of colonies grown on PDA at
25°C for 4 weeks 0 = nil; 1 = very sparse; 2 = medium; 3 =
dense.
a
mentation, and sparse aerial hyphae (Fig. 1). All
of the protoplast cultures produced sclerotia and
moniloid ceils.
Variations in cultural morphology among protoplast-regenerated cultures have also been observed for other heterokaryotic isolates tested.
There were 2 to 3 new morphological phenotypes
(which were different from the parental isolate)
of protoplast cultures from each field isolate (Table 1). The cultural morphology of each protoplast strain was stable on successive subculturing
during the 18-month period of experiment. Tuft
formation tests suggested that each new morphological type of protoplast culture resulted from a
field isolate carrying a distinct heterokaryon incompatibility factor [4]. It has been reported that
26% of protoplasts in the supernatant fraction
subjected to low speed centrifugation contained
one nucleus per cell [8], and mononucleate proto-
86
Acknowledgement
1600
H.A. Yang thanks the Australian Government
for the award of an Overseas Postgraduate Research Scholarship.
8oo
References
0
Fig. 2. Pathogenicity of protoplast-regenerated strains and
their parent field isolate of Rh~octonia so~ni. Vertical lines
represent standard deviations.
plast regenerated cultures are homokaryotic [3].
Therefore, the variation in culture morphology
among protoplast-regenerated strains from a field
isolate is attributed to the selection of homokaryotic single nuclear types during protoplast formation.
Variation in p a t h o g e n i c i t y
T h e p a t h o g e n i c i t y level of t h e r e p r e s e n t a t i v e
cultural m o r p h o l o g y types o f p r o t o p l a s t strains is
p r e s e n t e d in Fig. 2. As all th e t e s t e d field isolates
were more pathogenic than their respective
h o m o k a r y o t i c p r o t o p l a s t cultures, th e g e n e t i c factors c o n t r o l l i n g p a t h o g e n i c i t y in field isolates of
R. solani a p p e a r to be d o m i n a n t . F i e l d isolates
with a high level o f p a t h o g e n i c i t y y i e l d e d p r o t o plast c u l t u r e s with h i g h e r v i r u l e n c e t h a n th o s e
arising f r o m weakly v i r u l e n t isolates, which suggests that p a t h o g e n i c i t y o f a field isolate is th e
c o n s e q u e n c e o f additive effects f r o m t h e c o m p o n e n t n u c l e a r types.
1 Domsch, K.H., Gams, W. and Anderson, T.H. (1980)
Compendium of Soil Fungi. Academic Press, London.
2 Flentje, N.T., Stretton, H.M. and McKenzie, A.R. (1970)
Mechanisms of variation in Rhizoctonia solani. In: Rhizoctonia solani: Biology and Pathology (Parmeter, J.R., Ed.),
pp. 52-65. University of California Press, Berkeley.
3 Adams, G.C. (1988) Thanatephorus cucumeris (Rhizoctonia
solani), a species complex of wide host range. In: Advances in Plant Pathology. Vol. 6: Genetics of Plant
Pathogenic Fungi. (Sidhu, G.S., Ed.), pp. 535-552. Academic Press, New York.
4 Yang, H.A., Tommerup, I.C., Sivasithamparam, K. and
O'Brien, P.A. (1992) Heterokaryon formation with
homokaryons derived from protoplasts of Rhizoctonia
solani anastomosis group eight. Exp. Mycol. 16, 268-278.
5 Phillips, A.J.L. (1993) The use of protoplasts for the
preparation of homokaryons from heterokaryotic isolates
of Rh&octonia solani. Mycol. Res. 97, 456-460.
6 Roberts, F.A. and Sivasithamparam, K. (1986) Identity
and pathogenicity of Rhizoctonia spp. associated with bare
patch disease of cereals at a field site in Western Australia. Neth. J. Plant Pathol. 92, 185-195.
7 McDonald, H,J. and Rovira, A.D. (1985) The development
of an inoculation technique for Rhizoctonia solani and its
application to screening cereal cultivars for resistance. In:
Ecology and Management of Soil-borne Plant Pathogens.
(Parker, C.A., Rovira, A.D., Moore, K.J. and Wong,
P.T.W., Eds.), pp. 174-176. Am. Phytopathol. Soc., St.
Paul, Minnesota.
8 Hashiba, T. and Yamada, M. (1984) Intraspecific protoplast fusion between auxotrophic mutants of Rhizoctonia
solani. Phytopathology. 74, 398-401.