Pathogenicity

In order to confirm pathogenic nature of isolated fungal culture, seedling of cotton variety NHH44 was raised in pots in glass house/screen house. These seeds were sown in sterilized soil:
compost: sand mixture (2:1:1) at the rate 5 seeds/pot (30 cm diameter) on germination 2
seedlings per pot were maintained. These seedlings were inoculated at the stage of 4-6 true
leaves with spore suspension (2x10 6 spores/ml.). Inoculation was carried out by spraying the
suspension with an automizer. For this purified culture was multiplied in conical flask [250ml
containing sterilized PDA broth (100 ml/flask)]. These flasks were kept on mechanical shaker for
72 hrs at slow speed. This growth of fungus was then used for inoculation. Before inoculation,
leaves were injured by rubbing carborandum powder to have small injuries for development of
symptoms. Immediately pots were watered and entire seedlings with pots were covered with
polyethene bags for 48 hrs to maintain humidity. Intermittently pot were watered and the
polythene bags were also taken out for a few minutes to avoid rise in temperature. Observations
were recorded by observing the plants daily and sufficient number of untreated control was
maintained for comparison
Significance:

1. Potential impact of Alternaria macrospora on cotton production in

West Texas
Dr. Jason E. Woodward, Mr. Aaron S. Alexander, Dr. Randal K. Boman, and Dr. Terry A. Wheeler. Texas A&M
University, 1102 East FM 1294, Lubbock, TX 79403

Alternaria macrospora Zimm. is a widespread foliar pathogen of cotton (Gossypium

hirsutum L.) found throughout most production regions of the world. The objectives of this
study were to evaluate the impact of A. macrospora on various aspects of cotton production.
Samples were collected from six locations to compare cotton yield and fiber quality between
healthy and infected areas. Seed and lint yields, and selected fiber properties were significantly
lower, and leaf grades significantly higher from infected areas. As a result the overall crop value
was reduced by approximately $732 ha-1 when infected with A. macrospora. Additional
experiments are currently being conducted to examine the potential for transmission of A.
macrospora on seed, and to evaluate the efficacy of selected seed treatments on growth and
development of A. macrospora. Results from this study will provide cotton producers with
valuable information that can be used to manage this disease more efficiently in the future.
Damage
1.

Relative effects of Alternaria alternata and A. macrospora on cotton crops in Israel

In large commercial fields, the ratio of Alternaria macrospora to A. alternata lesions was 1:0-4
in cv. Pima and 1:19 in cv. Acala. The frequency of A. alternata on Pima and of A.
macrospora on Acala increased in an experimental field with mixed Pima and Acala plots. In

both cultivars disease was inhibited by fungicidal treatment and by removal of flowers. For all
parameters measured (leaf area, number of leaves, flowers and bolls, and yield), the responses to
treatments were greater in Pima than in Acala.
2. Influence of foliar application of nitrogen and potassium on

alternaria diseases in potato, tomato and cotton

ABSTRACT The hypothesis that enrichment of the foliage with nitrogen and potassium may
enhance host resistance toAlternaria and thus reduce disease severity, was examined for potato
and tomato (A.solani) and for cotton (A.macrospora). First, the activity of urea (CO(NH2)2) and
the salts NH4NO3, (NH4)2S04, KNO3, KCl, K2SO4 and KH2P04 againstA. solani andA.
macrospora was determinedin vitro; each of the compounds tested had a weak inhibitory effect
on spore germination of A.solani (ED50 > 1000 g/l) and on mycelial growth of both
A.macrospora andA. solani (ED50 > 10,000 g/l). Next, the effect of foliar application of
selected nutrients onA. solani andA. macrospora was evaluatedin vivo on detached leaves of
tomato and cotton. The diameter of A.solani lesions on leaves sampled from tomato plants
treated with KNO3 was significantly smaller (by 56.5%) than that recorded on leaves sampled
from untreated plants.A. macrospora severity on leaves sampled from cotton plants treated with
urea
was
significantly
lower
than
that
observed
on
untreated
leaves
(70.8% reduction) but KNO3 did not affect disease severity significantly. The following step was
to
examine
the
effects
of
foliar
application
of
urea
and KNO3 onAlternaria development in field experiments, two with potato and one with cotton.
Foliar
application
of
both
nutrients
(8-10
sprays
in total) did not affectAlternaria severity as compared with the untreated control in any of the
experiments.
The
fungicides
chlorothalonil
and
tebuconazole,
on the other hand, significantly suppressed the disease throughout most of the growing season. A
mixture of urea or KNO3 with the fungicides did not further improve the effects of the latter
when
applied
alone.
Based
on
these
results,
it
was
concluded that foliar application of urea or KNO3 does not affect host response toAlternaria.

Way to control the fungus

Tuesday, September 11, 2007 - 5:00 PM

Development of sensitive molecular diagnostic tools for detection of
economically important fungal pathogens of cotton
Dr. P. K. Chakrabarty, Mr. R.L. Chavhan, Ms. S.V. Sable, Mr. A.V. Narwade, Dr. D. Monga, and Dr. B.M. Khadi.
Central Institute for Cotton Research, Post Bag No 2., Shankar Nagar, P.O., Nagpur -440010, India
1

PCR protocols for detection and differentiation of strains of Rhizoctonia solani, R. bataticola, Ramularia
areola and Alternaria macrospora, four economically important fungal pathogens of cotton were developed. Based on
nucleotide sequence of the internal transcribed spacer regions of ribosomal RNA genes of these pathogens four sets
of primers were developed. Primers pRSol and pRBat were specific to strains of R. solani and R. bataticola and
supported amplifications of rDNA fragments of 255 and 400 bp, respectively. Primer pRare indiscriminately detected
four strains of R. areola isolated from each of the only four cultivated species of cotton by supporting amplification of
an universal amplicon of 372 bp. Strains of A. macrospora could be identified by amplification of a DNA fragment of
542 bp using primer pAmac and differentiated from other species of Alternaria by PCR-RFLP of the rDNA product
with BanII,HaeIII and MseI restriction endonucleases.
Cultivated cotton (Gossypium arboreum, G. herbaceum, G. hirsutum and G. barbadense) in India suffers from large
number of diseases that affect both above and underground parts of the plant causing considerable losses in quality
and yield (Hillock, 1992; Srinivasan, 1994; Chakrabarty and Mayee, 2004). Besides bacterial and viral diseases,
fungal diseases provide a real challenge to successful cultivation of cotton. Fungal foliar diseases such as Grey
mildew, which is caused by Ramularia areola, was predominantly a pathogen of diploid cotton (G.
arboreum and G. herbaceum) which now infects tetraploid cotton (G. hirsutum and G. barbadense) as well (Mukewar
et al. 1994). The disease causes extensive defoliation and has become a menace in central and South India. Leaf
spot and blight caused by Alternaria macrospora is another destructive foliar disease of cotton that affects production
of cotton in different regions (Srinivasan, 1994). Root rot and wilt, caused by the soil-borne fungal plant
pathogens, Rhizoctonia spp. and Fusarium oxysporum f.sp.vasinfectum, are two major diseases that exist in different
cotton growing regions of the country (Srinivasan 1994 ; Monga and Raj, 2003).
Identification of the cause and prevalence of a disease is very essential for adequate and timely plant disease
management, which in turns depends on accurate diagnosis and early detection of the pathogen. Often it may be
desirable to examine the soil for prevalence of any potential pathogen even before the crop is sown. Early detection
enables one to make decisions regarding cultivar choice and chemical control that can be used most effectively to
prevent development of a potential plant disease epidemic (Ward et al. 2004). Diagnosis of the causal agent is also
important for studies on epidemiology (NOTE: Some diseases and/or declines have been studied without knowing the
biological cause such as Para wilt of cotton (Raj et al. 1991), yield loss relationships and designing new strategies for
disease management. Traditional or classical methods of disease diagnosis and pathogen identification could be
relatively slow, often requiring skilled taxonomists to reliably identify the pathogens at the genus or species level.
Delays are damaging when quick diagnosis is needed so that appropriate disease control measures may be taken to
prevent plant injury especially when high value cash crops like cotton and other important plant species are at stake.
JOURNAL OF COTTON SCIENCE, Volume XXX, Issue XXX, 2007
Advances in Biotechnology have intensified efforts in recent years to develop novel methods for detection and
identification of plant pathogens. Nucleic acid has increasingly been used in recent years to develop diagnostic assay
for plant pathogens (Ward et al. 2004). Molecular approaches mainly the polymerase chain reaction have been used
widely as the tool for detection of fungal pathogens (Martin et al. 2000, Schaad and Frederick, 2002). Rapid PCR
assay based on amplification of sequence of internal transcribed spacer (ITS) region of rDNA or pathogenicity genes
have been developed and used for detection of several plant pathogens (Henson and French 1993). Molecular
techniques, if not alone, can be used in conjunction with classical methods where the latter approaches can at least
narrow pathogen diagnosis to genus level. Once genus is narrowed by morphology, symptomatology, host-specificity,
etc., then PCR can be used to differentiate species.
We developed PCR based diagnostic methods to detect strains of R. solani, R. bataticola, Ramularia areola and A.
macrospora
MATERIALS AND METHODS
Fungal strains and maintenance. The sources of fungal species for which diagnostic tools were developed are
given in Table 1. Fungal strains, except that of R. areola, were grown and maintained on potato dextrose agar (PDA).
For long term storage, they were stored in mineral oil at 4oC in 15 ml screw-capped Corning glass tubes. For DNA
isolation the fungal strains except that of R. areola were grown in potato dextrose broth (PDB). PDB (100 ml) was
inoculated with a 5 mm diameter plug of culture agar cut from the edge of 5 days old culture of each isolate grown on
a Petri dish. The inoculated broth was incubated at 28 2oC for 7 days.

Isolation of genomic DNA. The mycelial mat was filtered through Whatman No 1 filter paper and dried at room
temperature. The genomic DNA was extracted from fresh mycelium by a modified DNA extraction protocol
(Chakrabarty 2004). Approximately 0.5 g of dry mycelial mat was transferred to a clean sterile mortar. Added 1.5 g of
White quartz sand (HiMedia, India), 2.5 ml extraction buffer (100 mM Tris, pH 8.0, 20 mM EDTA, 0.5 M NaCl, 1%
SDS, 0.5M glucose) and 1.25 ml buffer saturated phenol/chloroform/isoamyl alcohol (25:24:1) at pH 8. The mixture
was ground thoroughly with a pestle and the homogeneous slurry was transferred into several microfuge tubes using
a wide-bore tip and centrifuged at 13,000 rpm for 5 min at room temperature. The aqueous phase from each tube
was transformed to 1.5 ml microfuge tubes to a volume of 750l and re-extracted with equal volume of
chloroform/isoamyl alcohol (24:1). The contents of the tube were mixed by inverting several times followed by
centrifugation at 13000 rpm for 5 min. The aqueous phase was again transferred to a new tube and the DNA was
precipitated with 0.1 volume 3M sodium acetate (pH 5.2) and 1 volume isopropanol at room temperature for 10 min.
The DNA was pelleted by centrifugation at 13,000 rpm for 10 min at 40oC, rinsed with 70% ethanol, and resuspended
in 200 l of TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer containing 20 g/ml RNAse. Using this method, genomic
DNA was extracted from strains of A. macrospora, R. solani, and R. bataticola. Spores from the surface of the lesions
of mildew infected leaves were scraped with a sterile tooth-pick moistened with sterile distilled water. The spore mass
were boiled for 5 min and used as the template in PCR reaction.
ITS-PCR and cloning of rDNA sequences. PCR amplification of rDNA sequences for all fungal species was
conducted in 50 l reaction volumes using conserved ITS1 and ITS4 primers (White et al. 1990). Each reaction
consisted of 2 l of 50 ng/l DNA template, 5 l of 10X PCR buffer, 0.5 l of 25mM dNTPs, 1.5 l of 15 mM MgCl 2,
0.3 l of 1.25U Taq DNA polymerase, 1 l each of 10 M primers ITS1(5' TCC GTA GGT GAA CCT GCG G 3 ') and
ITS 4 (5 TCC TCC GCT TAT TGA TAT GC 3 ') and 38.7l sterile distilled water. The PCR protocol was standardised
to amplify rDNA sequences from a strain each of R. solani, R. bataticola, A. macrospora and four strains of R.
areolainfecting four cultivated species of cotton:, G. arboreum, G. herbaceum, G. hirsutum and G. barbadense. The
standardised protocol had cycling parameters of initial denaturation at 94oC for 4 min followed by 33 cycles of
denaturation at 94o C for 1 min, annealing at 55oC for 1 min and extension at 72oC for 1.5 min. A final extension at
72 oC for 5 min was done at the end of amplification. Negative controls were used to test for false priming and
amplification.
A 10-l PCR amplification product for each of the fungal species was visualized in a 1%
agarose gel and viewed under UV light following staining with ethidium bromide.
Cloning of rDNA fragments. Gel purified fragments of ~ 650 bp comprising partial sequences of 18S and 28S rRNA
genes, and complete sequences of ITS1, 5.8S and ITS2 of each fungal strain were cloned in pGEMT (Promega,
Madison, WI, USA), following manufacturers protocol, unless stated otherwise. The ligation reaction was incubated
overnight at 40oC. The ligation mix was transformed in Escherichia coli (XL-1 Blue) by heat shock method. The tube
containing the competent cells (200 l) was removed from 70oC and allowed to thaw on ice. Ligation reaction
mixture (2 l) was added to the tube of competent cells following incubation on ice for 5 min. The cells were subjected
to heat shock at 42oC for 30 second and transferred on ice for 2 min. Heat-shocked cells were dispensed in 250 l LB
in micro centrifuge tube. The transformation mix was incubated at 37oC in an orbital shaker at 220 rpm for 45 min to
allow expression of the plasmid. The entire transformation mixture was then plated on LB agar containing Ampicillin
(70ug/ml), Xgal (80 g/ml) and IPTG (50 M). The plate was incubated overnight at 37o C. The recombinant clones
were identified by blue white colony selection. The white putative recombinant colonies were streaked on LB agar
supplemented with Ampicillin (70 g/ml). The plasmid isolation from the putative transformants was done by the rapid
miniprep protocol (Chakrabarty, unpublished). The recombinant clones were confirmed by digesting plasmid DNA
with AatII and PstI.
Sequencing of ITS amplicons and multiple alignment of sequence data. The cloned ribosomal RNA genes and
the ITS regions of each fungal strains were sequenced using T7 and SP6 vector based primers at M/S Bangalore
Genie Pvt. Ltd. Bangalore (India). The rRNA sequences of each fungal pathogens, comprising of partial sequences of
18S rRNA and 28S rRNA; and complete sequences of ITS 1, 5.8S rRNA and ITS 2 were submitted in GenBank. The
DNA sequences of each accession were aligned among themselves as well as with other published sequences
available in GenBank using BlastN and http://www.justbio.com.
Development of species-specific primer and PCR detection protocol. Following multiple alignments of the rDNA
sequences, regions of dissimilarity in ITS 1 and ITS 2 sequences were determined and used to design primers
specific to three fungal species: R. solani, R. bataticola and R. areola and an Alternaria genus-specific primer for A.
macrospora. To test specificity of primers in detecting strains of respective cotton pathogen only, the genomic DNA of
each pathogen was subjected to PCR amplification with each set of primer. For A. macrospora, sequence variability

with respect to other Alternaria species infecting other economically important plants was not good enough for
designing species-specific primers. Therefore, restriction fragment length polymorphism analyses of amplified rDNA
fragments with different restriction enzymes were used to differentiate A. macrospora from other Alternaria species.
PCR amplified ITS regions of A. macrospora and seven other Alternaria species were digested with restriction
enzymes viz., BalI, BalII, BanII, ClaI, HaeIII,HindIII, HphI, MboI, MseI, NlaIV, SacI, TfiI, SalI, Sau3aI, SmaI, XhoI
and XmaI. Restriction digestion reaction was carried out in 15 l volumes and consisted of 0.5 l restriction
endonuclease (5U/l), 1.5l restriction buffer (10X), 11l sterile distilled water and 3l of PCR product. The digestion
was carried out at 37oC for 2 h. The digested PCR product was resolved on 2 percent agarose gel, stained with
ethidium bromide (0.5 g/ml) and visualized under UV to analyze nucleotide polymorphism in amplified fragment.
RESULTS
DNA based PCR diagnostic protocols were developed to identify four fungal pathogens of cotton, including R. solani,
R. bataticola, A. macrospora and R. areola (Table 1). PCR Amplification of cotton fungal species with conserved
primers ITS1 and ITS4 yielded an ~600 base pair rDNA product which were cloned in plasmid pGEMT (Fig. 1 a & b).
Analysis of rDNA fragments from fungal strains revealed presence of partial sequences of 18S and 28S rRNA genes
and complete sequences of ITS 1 and ITS 2 along with 5.8S rRNA gene. The sequences of the entire ITS 1/5.8S/ITS
2 regions together with short termini from large and small subunit genes, were obtained for each of the four
pathogens. The sequences were deposited in GenBank and accession numbers obtained for each of them (Table 1).
There was significant variation in the sequences of the ITS regions, especially within ITS1 and ITS 2, although
several highly conserved regions were present in both regions. Regions of significant sequence variability in R.
solani, R. bataticola, R. areola and A. macrospora, were good enough to design species-specific oligonucleotide
primers for strains of first three species. Four different sets of primers capable of differentially detecting these four
pathogens were designed. The pathogens, the primers and the sizes of the diagnostic amplicons are given in Table 2.
Primers pRsol, pRbat, pAmac and pRare could specifically detect strains of R. solani, R. bataticola, A.
macrospora and R. areola by amplification of rDNA fragments of 255, 400, 542 and 372 bp, respectively (Fig. 2).
Primers pRsol and pRbat can specifically amplify strains of R. solani and R. bataticola, respectively. pRsol
successfully detected strains of R. solani tested but did not detect R. bataticolastrains infecting cotton (Fig. 3a). On
the other hand pRbat could amplify a DNA fragment of 400 bp from strains of R. bataticola but not from the strains
of R. solani collected from different cotton growing zones of the country (Fig. 3b).
Primer pRare indiscriminately detected four strains of R. areola, each isolated from G. hirsutum, G. barbadense, G.
arboreum and G. herbaceum by universal amplification of a DNA fragment of 372 bp. pAmac amplified a rDNA
fragment of 542 bp from strains of A. macrospora. Each set of primer supported amplification of the strains of
respective target pathogen but failed to detect members of other three pathogens tested (Fig. 4a-d).
The primer pAmac however, was not specific to A. macrospora of cotton but supported amplification of the rDNA
fragment from several species of Alternaria, such as A. alternata strains from sorghum and sunflower, A. longipes, A.
porri, A. dianthicola, A. citri and A. brassicae (Kadam 2005). Lack of adequate variability in nucleotide sequences in
the ITS region of different species of Alternaria did not allow designing species-specific primers for A. macrospora.
Strains of A. macrospora could however, be identified and differentiated by possession of two unique restriction
endonuclease sites such as BanII and MseI in the rDNA repeat unit. These two enzymes sites are not present in any
other Alternaria species studied. There was a single BanII site in the ITS1 region of A. macrospora which cleaved the
linear PCR amplified rDNA repeat into two fragments of 448 and 127bp size (Fig. 5). The rDNA region also
possessed two MseI sites one each in ITS2 region and 28S rRNA gene that generated three fragments of 418, 136
and 21bp size. Also unlike in all Alternaria spp. which possessed single HaeIII site, A. macrospora had two HaeIII
sites one each in ITS1 and ITS2 regions and generated three DNA fragments of 368, 140 and 67 bp. Besides,
comparison of the rDNA sequences amplified using conserved ITS1 and ITS4 primers in A. macrospora against other
Alternaria species, revealed that the former has the highest number of nucleotides (575 bp) in the ITS region.
DISCUSSION
Four sets of pathogen-specific primers developed as a part of this study enabled successful diagnosis of cottonspecific strains of R. solani, R. bataticola and R. areola, while PCR-RFLP method could differentiate strains of A.
macrospora from several other species of this pathogen. Detection of polymorphism using PCR-RFLP analysis of the
ribosomal DNA- ITS region has been successfully used for identification of several species of fungi (Martin et al.
2000). This simple technique requires only minute amounts of DNA and two specific conserved primers flanking the
ITS region of rDNA genes. This is one of the groups of genes most frequently targeted for phylogenetic studies and
codes for rRNA. The main reasons for the popularity of rDNA are that it is a multicopy, non-protein-coding gene,

whose repeated copies in tandems are homogenized by concerted evolution and is therefore treated as a single
locus gene. Furthermore, the ribosomes are present in all organisms and ribosomal RNA genes are the most
commonly used target for fungal and bacterial diagnostics (Ward et. al 2004). The amplified products of ITS region of
11 fungal species from different crops (Kadam 2005), including strains of R. solani, R. bataticola, A.
macrospora and R. areola reported in the present study, ranged between 569-575 bp, coinciding with the sizes
obtained from similar fungal pathogens from other strains of the same species. The multiple alignments of the rDNA
sequences using sequences available in GenBank and sequences from this study revealed significant variability in
ITS1 and ITS2 regions directly allowing us to design species-specific primers. Considerably greater sequence
variations is found in the internal transcribed spacer (ITS) regions between the rRNA genes within a rRNA repeat unit
(Henson and French 1993). Nazar et. al. (1991) found adequate sequence differences in the ITS regions of the cotton
wilt fungi, Verticillium dahliae and V. alboatrum, to design primers that specifically amplify the DNA of each species.
Primers based on differences in ITS 1 sequences of Leptosphaeria maculans allowed specific amplification of weak
or virulent isolates of this fungal pathogen (Xue et. al. 1992). Specific primers were also designed and developed
based on the ribosomal genes to detect and differentiate several species of the genus Phytophthora, an economically
important fungal pathogen of crop plants Ristaino et. al. 1998; Appiah et. al. 2004). For fungal species such
as Alternaria alternata, A. longipes, A. dianthicola, A. citri, A. brassicae, A. macrospora and A. porri, where significant
variability in the nucleotide sequence of rDNA did not exist, inter and intra-specific variation was evaluated by
analysis of the ITS region of rDNA using restriction fragment length polymorphism. Cleavage of amplified fragments
with specific restriction enzymes revealed extensive polymorphism that allowed further differentiation of
these Alternaria species. A. macrospora possessed certain unique restriction sites like BanII and MSeI. The presence
of these unique restriction sites are consistent with observed nucleotide sequence variability in the rDNA sequences
of these Alternariaspecies that included addition or deletion of several conserved nucleotides. Such substitutions are
responsible for obliteration of some conserved restriction sites or creation of some unique sites. The inter-specific
variation among several species of Phytophthora infecting cocoa could be clearly distinguished by restriction analysis
of the PCR amplified rDNA regions with unique restriction enzymes (Appiah et al 2004). The PCR-RFLP analysis of
rDNA-ITS region has also been successfully used for identification and differentiation of several species of
ectomycorrhizal fungi (Amicucci et al.1996, Eliane et al. 2002). Previously, Chakrabarty et al (2005) developed PCR
based diagnostic protocols for detection of Xanthomonas axonopodis pv. malvacearum and cotton leaf curl virus, two
major pathogens of cotton, by developing primers based on their pathogenicity genes. A PCR protocol for detection
of Alternaria radicina on carrot seed was developed by Pryor and Gilbertson, (2001). The primers were designed
based upon the sequence of a cloned RAPD fragment of the pathogen.
The results obtained during the present investigation showed that the internal transcribed spacer regions of the
ribosomal RNA gene sequences can be used to design species-specific diagnostic tools. Furthermore, the ITSrestriction fragment length polymorphism analysis has potential to serve as markers for differentiation of closely
related species or the strains belonging to same species.
REFERENCES
Amicucci, A.; I. Rossi, L. Potenza, A. Zambonelli, D. Agostini, F. Palma, and V. Stocchi.
1996. Identification of ectomycorrhizae from tuber species by RFLP analysis of the ITS region. Biotechnol. lett. 18:
821-826.
Table 1: The fungal strains, sources and GenBank accessions of their ITS sequence.

Sr.
No

Pathogen species/ strain

Sexual/ asexual form

Source

GenBank
Accession

Rhizoctonia solani Kuhn.

Thanatephorus cucumeris(Frank)
Donk

Infected roots of cotton

DQ339103

Rhizoctonia bataticola (Taub.), Butler

Macrophomina phaseolina (Tassi)

Gold.

Infected roots of cotton

DQ339102

Alternaria macrospora Zimm.

Infected leaves of cotton

DQ156342

Ramularia areola Atk. (hirsutum)

Mycosphaerella areola (Ehrlich and

Wolf.)

Mildewed leaves of G.
hirsutum

DQ459076

R. areola Atk. (barbadense)

Mycosphaerella areola (Ehrlich and

Wolf.)

Mildewed leaves of G.
barbadense

DQ631897

R. areola Atk. (arboreum)

Mycosphaerella areola (Ehrlich and

Wolf.)

Mildewed leaves of G.
arboreum

DQ459081

R. areola Atk. (herbaceum)

Mycosphaerella areola (Ehrlich and

Wolf.)

Mildewed leaves of G.
herbaceum

DQ459082

Table 2. The fungal pathogens, diagnostic primers and sizes of the amplified products.

Sr.
No

Pathogen species

Primers

amplicon (bp)

Rhizoctonia solani

pRsol

255

Rhizctonia bataticola

pRbat

400

Ramularia areola

pRare

372

pAmac

542

4
Alternaria macrospora

Fungicide treatment and varietal effects onAlternaria leaf spot

of Pima cotton
Mary W. Olsen, Plant Pathology Department
Lee Clark, Safford Agricultural Center
Hal Moser, Maricopa Agricultural Center
Abstract

The effect of foliar treatments for prevention of Alternaria leaf spot was evaluated in
the field on six varieties of Pima cotton. Disease was significantly reduced by
protective sprays of mancozeb and micronized sulfur but not by foliar applications of
urea in trials at the University of Arizona Safford Agricultural Center in Safford, AZ..
Treatments had no significant effects on yields. Significantly fewer lesions developed
on Pima variety UA 4 than on the other varieties. Disease pressure was relatively
light, and even though scheduled preventive sprays with mancozeb were effective,
fungicide applications probably would not increase yields under the environmental
conditions of this experiment.

Introduction

Alternaria leaf spot of cotton, caused by the fungus Alternaria macrospora, causes
lesions on leaves, bracts, and bolls of cotton. Disease is common in Arizona only
under very humid conditions, and is usually associated with the onset of rains in the
summer months at higher elevations. In the Safford Valley and other cotton growing
areas of Graham, Greenlee, Cochise, and Pima Counties, disease can be severe on
Pima cotton, causing defoliation if rain and high humidity are persistent in July,
August and September. Variations in susceptibility among cotton cultivars have been
reported (Cotty, 1987). Pima variety S5 was more susceptible than S6, and both Pima
varieties were more susceptible than DP90 or other upland varieties. There is no data
available comparing susceptibility of newer Pima varieties. Although older leaves are
believed to be more susceptible to infection, disease development is not related to
plant age (Shtienberg, 1993). Late season infections usually are not considered a
problem since yields are probably not affected.
There are currently no fungicides registered for use on cotton for control
of Alternarialeaf spot in Arizona. In other cotton growing regions of the world where
disease is a problem and causes yield losses, fungicides such as maneb, mancozeb,
difenoconazole and tebuconazole are used as protectant sprays (Shtienberg, 1991,
1992). Fungicides may be applied as often as every ten days to two weeks and
initiated before flowering. Because of the restricted occurrence ofAlternaria leaf spot
in the United States, especially in the Southwest, it is unlikely that new fungicide
labels will be forthcoming for disease control. Therefore, it is important to determine
the efficacy of foliar treatments that have current labels on cotton for the control
of Alternaria leaf spot.
The objectives of this study were to (1) determine if preventive sprays of candidate
foliar treatments would reduce disease incidence significantly; (2) demonstrate
varietal susceptibility of Pima cotton to Alternaria leaf spot; (3) determine the effect
of disease on yield; and (4) generate data for effective foliar treatments that would
lead to a label for use on cotton for control of Alternaria leaf spot of cotton.
Materials and Methods

This study was conducted at the University of Arizona Safford Agricultural Center.
Six varieties of Pima cotton were planted according to standard practices in 4 row
plots 15 m long with 3 replications in a randomized block design. Varieties were OA

361, UA 4, OA 312, OA 325, S6 and S7. Varieties S6 and S7 are currently available
for commercial use and are planted in the Safford Valley; varieties OA 361, OA 312
and OA 325 are short season varieties developed by Olvey and Associates and are
well suited to the Safford Valley; UA 4 is a short season variety developed by the
University of Arizona Pima Breeding group.
Foliar treatments for disease control - mancozeb, sulfur, and urea - were selected on
the basis of their potential availability for use and their cost. Mancozeb already has a
registration on cotton for prevention of cotton rust and is registered for use on other
crops for control ofAlternaria diseases; micronized sulfur was applied because of its
combined fungicidal and insecticidal potential and current registration on cotton for
mite control; and urea, a known greening agent, was evaluated since disease has been
shown to be related to leaf age (Shtienberg, 1993). Mancozeb was applied as 1.75
lb/ac Penncozeb 75df, sulfur as 7.5 lb/ac Microthiol Special, and urea as 5 lb/ac foliar
urea by ground spray application on August 15, September 1, and September 15,
1997. These three application dates at two week intervals were chosen since more
than two or three applications are considered economically unfeasible at current
cotton prices and the six week interval was considered reasonable for disease control
based on other studies (Shtienberg, 1992 ). Control plots were not treated. Treatments
were applied along the varietal plots in a split-plot design.
Disease was assessed on September 19, 1997 by counting the number of lesions on
leaves at the fifth node down from the terminal node. Ten leaves were sampled from
each plot and taken to the laboratory where Alternaria lesions were counted. Lint
yields were determined at harvest by mechanical harvesting of plots and assuming
35% lint. Data were analyzed using the General Linear Models Procedure and
Duncan's Multiple Range Test of SAS.
Results and Discussion

As shown in Table 1, the number of lesions per leaf was reduced significantly by the
application of mancozeb and sulfur compared to the urea treatment or untreated
control. Mancozeb was the most effective treatment and reduced lesions by more than
50%. However, the average number of lesions per leaf, even in the untreated controls,
was low. Environmental conditions were not favorable for disease in 1997, and
disease pressure was relatively light compared to years with higher humidity and more
rain in July and August. Treatments had no significant effect on yields (Table 1).

There was a varietal effect on the number of lesions per leaf (Table 2). Variety OA 325
had significantly higher numbers of lesions per leaf than S-6 or UA 4, with UA 4
having significantly fewer lesions per leaf than all other varieties. OA 312 had a
significantly higher yield than UA 4, but was not different from the other varieties.
Results indicate that Alternaria leaf spot will not reduce yields of Pima cotton under
the environmental conditions of these trials, and foliar applications would not be
warranted. However, treatments of Pima cotton should be repeated in years of higher
disease pressure and at different application dates in order to compare higher lesion
numbers on yields. Trials carried out when weather conditions are more conducive to
disease development would give growers the information needed to decide if and
when to make foliar treatments.
Literature cited

1. Cotty, P. J. 1987. Evaluation of cotton cultivar susceptibility

to Alternaria leaf spot. Plant Disese 71:61082-1084.
2. Shtienberg, D. and J. Dreishpoun. 1991. Suppression
of Alternaria leaf spot in Pima cotton by systemic fungicides.
Crop Protection 10: 381-385.
3. Shtienberg, D. 1992. Development and evaluation of guidelines
for the initiation of chemical control of Alternaria leaf spot in
Pima cotton in Israel. Plant Disease 76: 1164-1168.
4. Shtienberg, D., Y. Kremer and A. Dinoor. 1993. Influence of
physiological age of Pima cotton on the need for fungicide
treatment to suppress Alternaria leaf spot. Phytopathology 83:
1235-1239.
3. This is a part of publication AZ1006: "Cotton: A College of Agriculture Report," 1998,
College of Agriculture, The University of Arizona, Tucson, Arizona, 85721. Any
products, services, or organizations that are mentioned, shown, or indirectly implied
in this publication do not imply endorsement by The University of Arizona. The
University is an Equal Opportunity/Affirmative Action Employer.
This document located at http://ag.arizona.edu/pubs/crops/az1006/az100610a.html
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Why homeopathic medicines are use for Alterneria

Alternaria leaf spot of cotton

Submitted by naipagropediaraichur on Thu, 17/05/2012 - 12:33 Posted in

Alternaria leaf spot of Cotton

Alternaria leaf spot is one of major foliar disease. This disease occurs in almost all the cotton
growing countries of the world. Hybrids are more susceptible to this disease. Disease infect
on leaves resulting in suppression of plant growth and reduction of yield. High severity of the
infection causes strong defoliation of cotton, sharp decrease of yield and crude fiber quality.
Etiology

Causal organisum : Alternaria macrospora

Conidia are light-brown, 22-27 x 9-11 . They affect cotton cotyledons in
seedlings, also bolls and their fiber. Mycelium of A. macrospora is dark-brown.
Conidiophores are light brown, single or in groups. Conidia are red-brown, 90-180 x
15-22 in size.
Disease cycle
The undecomposed crop residues and infected seeds provide the primary
source of inoculum, giving rise to infected cotyledons, which support the early
stages of an epidemic. Primary infection of lower canopy leaves can be initiated
from conidia splashed up from infected crop residues or blown into the crop from
other foci of infection. Alternaria spp., also attacks the bolls and grow on exposed
lint if bolls open in wet weather, giving rise to contaminated seed. The disease cycle
is completed when infected leaves fall to the ground.
Symptoms

Small, pale to brown, round or irregular spots

Leaves become dry and fall off.
Cause cankers on the stem.
Infection spreads to the bolls and finally falls off.

Brownish spots on leaves

Symptoms on boll
infected field with Alternaria leaf spot

Severely

Epidemiology

Favourable condition for pathogy was high humidity, intermittent rains and
moderate temperature of 25-28OC. The pathogen survives in the dead leaves as
dormant mycelium. The pathogen primarily spreads through irrigation water. The
secondary spread is mainly by air-borne conidia.

Diseases
Alternaria gossypii (Jacz.) Nisik., K. Kimura and
Miyaw.; Alternaria macrospora Zimm. - Alternaria
Leaf Spot of Cotton.

Object map

Systematic position.

Kingdom Fungi, phylum Ascomycota, class Ascomycetes, order Pleosporales, family Pleosporaceae, genus
Alternaria.

Synonyms.
Macrosporium gossypii Jacz.; Alternaria longipedicellata Snowden.

Biological group.
Saprotroph.

Morphology and biology.

The causative agents of Alternaria Leaf Spot of Cotton (Macrosporium Leaf Spot) are fungi Alternaria gossypii
and A. macrospora developing in their life cycle in only anamorphous stages. Mycelium of A. gossypii is dark
brown. Conidiophores are brown, single or in groups. Conidia are light-brown, 22-27 x 9-11 mkm in. They
affect cotton cotyledons in seedlings, also bolls and their fiber. Mycelium of A. macrospora is dark-brown.
Conidiophores are light brown, single or in groups. Conidia are red-brown, 90-180 x 15-22 mkm in size. They
affect leaves, bracts in seedlings and adult plants, bolls. The causative agents cause necroses on cotyledons,
leaves, and bolls of cotton in form of dark-green and then brown, rounded or various shaped spots with
clearly expressed zonality. At high humidity light pink or dark conidial sporulation appears on necrotic spots.
Affected boll fiber is brownish-red. Systematic position of the causative agents of cotton alternariosis and
macrosporiosis is sometimes considered indistinct; they may represent one or two different species
(Gorlenko, 1968). Sources of the infection are affected cotton crop residues, seeds, and also weeds.
Additional vectors of the infection are aphids parasitizing cotton plants.

Distribution.
The Alternaria Leaf Spot is spread on cotton everywhere in the Central Asian and Caucasian countries of the
former USSR.

Ecology.
The causative agents affect cotton more intensively at high air humidity and at daily average temperature
about 25C. Strong severity of the disease is observed on old leaves with slow metabolic process.

Economic significance.
At the moderate affection of cotton by the causative agents, assimilative processes are broken in leaves
resulting in suppression of plant growth and reduction of yield. High severity of the infection causes strong
defoliation of cotton, sharp decrease of yield and crude fiber quality. Gossypium barbadense cotton varieties
are more strongly affected than varieties of Gossypium hirsutum. Control measures are crop rotations
including alternation with cereals and unaffected crops, application of the biological preparation of
Trichoderma, duly weed control in crops, the use of chemical control against the disease or aphids, if
necessary.

Reference citations:
Gorlenko M.V. 1968. Agricultural phytopathology. Moscow: Visshaya Shkola, 434 p. (In Russian).
CABI Bioscience Databases. 2004. http://www.SpeciesFungorum.org .

Peresypkin V.F. 1974. Agricultural phytopathology. Moscow: Kolos, 560 p. (In Russian).
Peresypkin V.F. 1987. Atlas of diseases of field cultures. Kiev: Urozhai, 144 p. (In Russian).
Pidoplichko N.M. 1977. Fungi are parasites of cultural plants. Keys. Kiev: Naukova Dumka, Vol. 2. 299 p. (In
Russian).

Yakutkin V.I.
Picture is taken from Peresypkin V.F. 1987. Atlas of diseases of field cultures. Kiev: Urozhai. Table 88.