Variability in fungi

Variability in fungus
A PRESENTATION ON
PRESENTER
LAXMI PRASAD JOSHI
PLP-09M-2019
DEPARTMENT OF PLANT PATHOLOGY
AGRICULTURE AND FORESTRY UNIVERSITY

General outline
1. What is pathogen variability?
2. Significance of pathogen Variability
3. Stages of variation
4. Mechanism of Variability in fungi
5. Characterization of variability
among plant pathogens.

What is pathogenvariability?
Variability : The property or ability of an organism to change its characteristics
from one generation to another generation is variability.( agrios 2005)
Variant : When progeny of individual show variation in characters from parents
such a progeny
Individuals have different characteristics , not fixed i.e. phenomenon of vatiation
Individual produced by sexual process including sexual spores differ among
themselves and from parents
Low in asexually reproducing ones , some have much higher variability e.g. C.
lindemuthianum .

Physiological Specialization
Within the species of pathogen there exist
certain individuals that are morphologically
similar but differ with respect to their
physiology , biochemical characters and
pathogenicity and differentiated on the basis of
reaction on certain host genera or cultivars .

Significance of pathogenVariability
Genetic variation is the stuff of evolution.
 Phenotypic variation in microorganisms allows rapid adaptation to a
constantly changing environment.
Essential for the survival of pathogen when new host cultivar is introduced .
When a new cultivar is introduced and the existing population of
pathogen show avirulance to the newly introduced cultivar then pathogen have
to produce variability in order to survive. (Sunil Kumar and Shalini
Verma,2019)
 Knowledge of variation in pathogenic characteristics is valuable in planning
effective measures for disease management.

Examples
1. Copper fungicides have been widely used agains blight
of poato( Phytophthora infestans) and Downy mildew of
grape vine (Plasmopara viticola) becoming less effective
now a days (S. G. Georgopoulos and G. Skylakakis ,1986).
2. Metalaxyl, which originally gave excellent control of the
same diseases, was soon rendered ineffective by the
development of resistance by the target fungi (Davidse,
Danial and Vanwesten, 1983).

Stagesof variation
Source : Agrios 2005

1. Genus :- Group of species having common characters.
E.g. Puccinia , Fusarium etc .
2. Species :- Organism with common morphological and phenotypic characters in common .
E.g. Puccinia graminis (cause stem rust of cereals),
Puccinia triticina , Puccinia stiriformis
3. Forma specials (f.sp) :-
Individual of morphologically similar pathogen species but attack particular host or group of
usually related hosts.
Most extensively used taxa in Fusarium and Puccinia .
Puccinia graminis f.sp. tritici , Puccinia graminis f.sp hordei, Puccinia graminis f.sp avenae .
 F.sp assigned based on pathogenicity test on particular host .
Stages of variation…………………

4. Race :-
Sub division of f.sp. (forma specials )
Based on pathogenicity towards particular set of host lines/
varieties/ cultivars .
Some individuals within special form can attack some of the
varieties but not other varieties .
More than 200 races of Puccinia graminis f.sp.tritici (race1 , race15,
race 59, and so on.)
Stages of variation……………….

5. Variant
When progenies show variations in characteristic from
parents it is called as a VARIANT
Individual suddenly attacking or cause sever symptom on a
variety , previously barely infected .
Could be a result of mutation , heteroploidy , parasexuality
etc

6. Biotype
Identical individuals produced asexually by a variant .
Remains genetically pure until mutation occurs
Each race consist of one or several biotypes
e.g. Puccinia graminis f.sp.tritici race 15 (race 15 A , race 15B and so on )
7. Isolates
 Strain that has been isolated from particular area. For eg. Rampur isolate

Cause of variation
1. Environment :- , morphological transitions (MT) are described in most fungi and induced by
environmental signals like starvation, temperature, pH change and mating associated factors.
2. Phenotypic switching (PS). The spontaneous emergence of colony variants. PS occurs in a
small fraction of the population, is random, reversible and represents an epigenetic state.
1st explained in Candida albicans .
Recently found in Candida glabrata, Cryptococcus neoformis
3. Antigenic variation (AV) involves alternating the expression of surface proteins (or
carbohydrates), which happens on cellular level in the entire population but can underlie strong
selection pressure.
Jain et.al. 2008

Mechanismof Variabilityin fungi
1. Migration and gene flow
2. Mutation
3. Recombination
4. selection
5. Heterokaryosis
6. Parasexualism
7. Heteropolyploidy
8. Saltation
9. Vegetative incompatibility

1. Migration and gene flow
Migration of one pathogen population from
one place to another.
One of the major and simplest factor of
variability
Its contribution to diversity may be
underestimated.
Leads to development of new species.
Gene flow is the movement of genes
between different gene pools .
Increase variability of gene pool by adding
new allele .

Migration and gene flow……………………………
Higher level of gene flow generally have greater genetic diversity.
Hardy spore or propagules (rust, Powdery mildew, can spread over long
distances, (larger genetic flow ; higher genetic variability )
 Soil borne fungi and nematodes move slowly and limited in certain area
(Lower genetic variability).(Agrios ,2005 p.g.130)
E.g., The introduction of Cryphonectria parasitica to North America,
Phytophthora infestans to Europe, and Puccinia striiformis to Australia. (Sunil
Kumar and Shalini Verma,2019)

Global trade in liveplants, which is nowadays the main
pathway of introduction of alien plant
Once introduced into a new environment,
1. pathogens may adapt their lifestyle to new local hosts
(host jump),
2. evolve by introgressing useful genes for adaptation
through hybridization with resident species or horizontal
gene transfer. (Santini et.al.,2015 )
Migration and gene flow……………………………

2.Mutation
Sudden and heritable change in genetic material of an organism i.e. DNA due to some physical and
chemical shock
Occurs Naturally or induced
Artificially induced by
A. Physical agents (UV rays ,X rays ,Gamma rays) or
B. Chemicals (alkaloids, phenols, epoxides, nitrogen, etc.)
Mutation represent change in sequence of the bases in DNA
a. Substitution of one base for another
b. Addition or deletion of one or many base pairs.
Mutation are spontaneous , Mostly recessive , Tendency for mutation varies in species and strain
Fast and expressed soon in single celled organism .

Mutation………
Also reported in the extra nuclear DNA
(Cytoplasmic DNA ).
Formation of new races in P. stiriiformis
(Gassner and Straib (1993)
Variability has been observed in Phytophthora
infestans , Puccinia graminis and Venturia
inaequalis .
Pathogen develop resistance to fungicide and
antibiotic

3.Heterokaryosis
Co-existance of genetically
different nuclei in cytoplasma .
 Blakeslee in 1904 first showed
that dissimilar nuclei were involved
in the life cycle of a fungus.
Capacity of haploid nuclei to form various associations within
vegetative cells,
The underlying implication of this state is that the fungus may respond
to selection by varying the proportion of dissimilar nuclei in the cells.

Heterokaryosis…………….
Heterokaryotic condition arises by ,
1. Mutation
2. Anastomosis
Fusion mostly intraspecific
eg. development of heterokaryon in basidiomycota
3. Inclusion of dissimilar nuclei in spores after meiosis , in heterothallic fungi.
Meiosis results in the production of genetically different nuclei sharing common
cytoplasma.
E.g. Neurospora tetrasperma, Podospora anserine

Heterokaryosis Examples……………………………
In P. graminis tritici, the dikaryotic mycelium can grow in both
barberry and wheat but the haploid mycelium can grow only in
barberry not on wheat.
Nelson, Wilcoxon, & Christensen(1955) ,inoculated wheat plants with
urediospore mixtures of two different races of Puccinia graminis var.
tritici, and from approximately one million plants. Some urediospores
and mycelial cells of this biotype contained three or four nuclei, instead
of the expected two
Heterokaryosis is involved in the production of fungal variation in
Colchiobolus sativus , Leptosphaeria avenaria and Helminthosporium
graminum ,Puccinia graminis .

Significance of Heterokaryosis
Substitute for heterozygosity and variability
Heterokaryosis and pathogenicity- e.g. in rusts
and smuts
Origin of new races
Initial steps in Parasexual cycle .

4 Parasexualism
First discovered by Pontrcorvo and Roper (1956) in Aspergillus nidulans.
Parasexuality is defined as a cycle in which plasmogamy , karyogamy and meiosis (haplodization ) takes
place in sequence but not at specified time or at specified points in the life cycle of an organism .
 Also called Somatic Recombination
Occasional fusion of two nuclei and formation of diploid nucleus results in parasexuality or mitotic
recombination.
Generlly occurs in those fungi in which true sexual cycle doesn’t takes place . Generally in Class
Deuteromycotina also In Ascomycetes and Basidiomycetes.
Parasexuality has been reported in many rusts , including P.graminis tirtici, P. coronata and in some
smuts Ustilago hordei and U. maydis etc.

1. Formation of heterokaryotic
mycelium
2. Karyogamy: Fusion between two
nuclei
a) Fusion between like nuclei
b) Fusion between unlike nuclei
3. Multiplication of diploid nuclei
4. Occasional mitotic crossing over
5. Sorting out of diploid nuclei.
6. Occasional haploidization of
diploid nuclei
7. Sorting of new haploid strains
Parasexual cycle steps ………..
Fig : Parasexual cycle Aspergillus niger

Steps of parasexual cycle
1. Formation of heterokaryotic mycelium
Anastomosis of somatic hyphae .
Introduction of foreign nuclei into a mycelium .
Mutation in one or more nuclei of
homokaryotic mycelium .
2. Fusion between two nuclei
a. Fusion between like nuclei
b. Fusion between unlike nuclei
If A and B are the unlike nuclei present in the
heterokaryotic mycelium ,then 5 types of nuclei can be formed,
A ,B,AA,BB,AB

3. Multiplication of diploid nuclei
All 5 types of nuclei multiply at about the same rate but
number of haploid nuclei present in much greater than diploid .
4. Occasional mitotic crossing over
During multiplication of diploid nucleus mitotic crossing
over may take place .
Results in the formation of new gene combination .
Give the fungus some of the advantage of sexual reproduction .
Source https://www.biologydiscussion.com/wp-
content/uploads/2016/11/clip_image002_thumb-120.jpg

5. Sorting out of Diploid nuclei
In those fungi which produce uninucleate
conidia ,sorting out of the diploid nucleus
Occurs by their incorporation into conidia
which germinate to produce diploid mycelia .
6. Occasional haploidization of diploid nuclei
 Occasionaly , some hyphae of diploid mycelium found to
form haploid conidia which form haploid mycelia on germination.
It indicates that haploidization occurs in some diploid nuclei .

After Parasexual cycle ,
The mycelium may contain following type of nuclei
A. Haploid nuclei like those of both parent.
B. Haploid nuclei with various new genetic recombinations .
C. Several types of diploid homozygous nuclei .
D. Several types of diploid heterozygous nuclei .

Comparison Between sexual and
parasexual cycle
SEXUAL CYCLE
SEXUAL CYCLE PARASEXUAL CYCLE
1.Nuclear fusion is in specialized structures 1. Rare nuclear fusion in vegetative structures
2. Zygote usually persist one nuclear generation only 2. Zygote persist through many mitosis .
3. Recombination by meiosis crossing over in all
chromosome number random assortment of members of
each chromosome pair
3. Recombination by rare accidents of mitosis
a. Mitotic crossing over , at each events usually
confined to one exchange on Singal; chromosome
arm .
b. Haplodization independent of crossing over , random
assortment of members of each chromosome pair.
4. Products of meiosis readily recognized and isolated 4. Recombinant occur among vegetative cells ,recognize
only by use of suitable genetic markers

Parasexualism importance
Importance in industrial process (belong to Deuteromycotina)
Some of impotramt antibiotic producing fungi like Penecillium
chrysogenum and Acremonium chrysogenum lack sexual reproduction .
Discovery of parasexual cycle provide suitable techniques to obtain
higher antibiotic strain .
Parasexuality can also be applied in the analysis of genetic and
physiological processes of perfect and imperfect fungi.
Employed in the genetic control of Pathogenicity and host range in
Several speies of Fusarium.
.


In rust fungi as P. graminis tritici, mitotic recombination may
represent a most important method of generating new races especially
in countries such as India where sexual stage of the fungus is rare
due to scarcity of the alternate host, the barberry.
For other fungi with no known sexual stage such as P.striiformis,
mitotic recombination is the only means of genetic assortment.

4. Selection
Directional process by which fittest variants in environment increase their frequency in
population (positive selection), while less fit variants decrease their frequency (negative selection)
Quantification of pathogen fitness :
a. Measurement of reproductive rate ,
b. Rate of multiplication ,
c. Efficiency of infection , and
d. Amount of disease caused (aggressiveness )
Results in decrease in diversity within population but increase in diversity between population .

5. Recombination
Most changes in the characteristics of pathogens are the result of recombination
occurs during sexual processes.
 When two haploid nuclei (1N) containing different genentic material unite to
form diploid (2N) nucleus called a Zygote, when undergo meiotic division
produce new haploid .
Occurs during meiotic division of zygote as a result of crossing over in which
part of chromatid of one chromosome of a pair are expressed with that of the
other.
 Recombination can also occur during mitotic division of cell in the course of
growth of the individual and is important in fungi Puccinia graminis.

Recombination……..
 The majority of fungi being haploid, have only a very brief diploid phase, very often
undergoing meiosis very soon after karyogamy.
Some Oomycetes are predominantly diploid
Many Basidiomycetes are functionally diploid by virtue of their extended dikaryon phase.
In case of homothallic fungi which are essentially self compatible and in a single thallus.
In these fungi, the extent of out-crossing will be close to zero and there will be little
opportunity for recombination between genetically different individuals.
 On the other hand, there are heterothallic fungi with physiologic distant mycelia upon
which are produced compatible male and female gametes. Here the extent of outcrossing
might reach 100%.
A good example is the black stem rust fungus,

6. Saltation (sectporing)
Appearance of morphologically different sectors in fungal
colonies .
Frequently seen In colonies of Fusarium and
Helminthosporium
May be induced by chemical and dilute antiseptic and at
time by antagonism of one fungus over other (Gayed, 1963)
May be influenced by compaction and thickness of
culture media .

H.sativum (barley). Large number
of sectors formed on a shallow
layer of Brown's starch agar (
M.Mitra. 1931)
Sectors have appeared from time to time in
the cultures. They occur on almost all
media and more particularly on prune-juice
agar and Brown's starch agar.

8. Vegetative incompatibility
Inability of fungal hypha belonging to same species to
fuse when come in contact .
Heterokaryotes obtained by somatic fusion are unstable ,
In some fungi, Appears to be defence mechanism that
protects individual from harmful nuclei, mitochondria,
plasmid and viruses that can reach them trough anastomosis .

9. Heteroploidy
 Heteroploidy is the existance of cells tissues or whole organisms with numbers of
chromosomes per nucleus that differ from the normal 1N or 2N complements for the particular
organism .Agrios ,2005
Heteroploids may be haploids, diploid, triploid or tetraploids i.e. have one or more extra
chromosomes from normal euploid number e.g.N+1
The contribution of aneuploids to variability also is potentially much greater because of the
variety of aneuploids that are possible. (Tolmsoff ,1983)
Shown to affect the growth rate ,spore size, and rate of spore production, hyphal colour
,enzyme activities and pathogenicity .
For e.g. Verticillium albo-atrum, cause of wilt in cotton , lose ability to infect cotton plant even
when derived from highely virulent haploids

Characterizationof variability
amongplant
 Assessment of variability provides a basis of breeding cultivar with durable resistance and
designing strategies for long term management of major diseases.
All the disease management strategies based on host resistant require the knowledge of
variability in pathogens.
The choice of method for characterization of pathogen isolates should be based upon
simplicity, reproducibility and cost effectiveness.( Kumar et.al.,2019)
Methods of characterization of genetic variability(Kumar et.al., 2019):
I. Traditional methods
II. Molecular or biotechnological methods

I. Traditional methods
Study the variability in pathogens are based on the use of
A. Differential host
Set of plant varieties used to define strains of plant pathogens
Based upon susceptibility or resistance reaction.
B. Cultural characteristics
Colour of colony
Hyphae colour
 Conidia production etc.

C. Morphological characteristics
Spore size
Nature of conidiogenous cells
Micro and microconidia etc.
D. Study of virulence reactions using different disease rating scale
on set of host differentials,
E. Biochemical tests
Ability of pathogen to utilize disaccharides e.g. sucrose, maltose,
lactose etc.
Pigments produced by pathogen in different media.

Disadvantage if traditional methods
1.Distinguish pathogens on the basis of their physiological characters
i.e. pathogenicity and growth behavior
2.Highly influenced by the host age, inoculum quality and
environmental conditions
3. Time consuming and laborious
4. Differential hosts are not available in most of the host- pathogen
system, thus variability cannot be assessed

II. Molecular or biotechnological methods
most precise tools for differentiation between species, and identification of new strain/
isolates collected from infected samples.
The molecular methods vary with respect to discriminatory power, reproducibility, ease of
use and interpretation (Lasker, 2002
Comparison at the DNA sequences level
provides accurate classification of fungal species
Molecular markers monitor the variations in DNA sequences within and between the species
and provide accurate identification.

In recent years, different marker system have been developed
A. Restriction Fragment Length Polymorphisms (RFLP), :
B. Random Amplified Polymorphic DNA (RAPD),
C. Sequence Tagged Sites (STS),
D. Amplified Fragment Length Polymorphisms (AFLP),
E. Simple Sequence Repeats (SSR) or microsatellites
F. Single Nucleotide Polymorphism (SNPs)

Referencs
Agrios, G.N. (2005) Plant Pathology. 5th Edition, Elsevier Academic Press, Amsterdam.
S. G. Georgopoulos and G. Skylakakis ,1986 . Genetic variability in the fungi and the problem of fungicide resistance . Crop protection 5 (5), 299-305
.
Davidse, L. C., Danial, D. L. and vanwesten, C. J. (1983). Resistance to metalaxyl in Phytophothora infestans in the Netherlands. Netherlands Journal
of Plant Pathology 89, l-20.
P. Narayanasamy, Microbial Plant Pathogens-Detection and Disease Diagnosis: Fungal Pathogens, Vol. 1, DOI 10.1007/978-90-481-9735-4_4,
Nelson ,R.R., Wilcoxon,R.D., and Christensen, J. J., Heterokaryosis as a basis for variation in Puccini graminis var.tritici,Phytopathology 45,639-
43(1955)
Gassner, G. and Straib, W. 1993. (Schmidt) Erikss. and Henn.Molecular Genetics and Genomics 63:154–180.
Sunil Kumar and Shalini Verma. 2019. Variability in Plant Pathogens and Tools for its Characterization. Int.J.Curr.Microbiol.App.Sci. 8(02): 2887-
2902. doi: https://doi.org/10.20546/ijcmas.2019.802.338
Sharma, T.R., Prachi, and Singh, B.M. 1999. Application of polymerase chain reaction in phytopathogenic microbes. Indian Journal of Microbiology
39: 7991.
http://agris.fao.org/agris-search/search.do?recordID=US201300681731
Mitra, M.1931.Saltation in the genus Helminthosporium .Transaction of the British Mycological Society.16(2-3):115-127.
https://doi.org/10.1016/S0007-1536(31)80025-7
Jain, N., Hasan, F., & Fries, B. C. (2008). Phenotypic Switching in Fungi. Current fungal infection reports, 2(3), 180–188.
https://doi.org/10.1007/s12281-008-0026-y

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