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ITS Sequence Analysis of Brazilian Stenocarpella Macrospora and

ITS Sequence Analysis of Brazilian Stenocarpella Macrospora and Stenocarpella Maydis Isolates Anania Fessehaie , Elcio Alves

Stenocarpella Maydis Isolates

Anania Fessehaie 1 , Elcio Alves 2 , and Gary Munkvold 1

ITS Sequence Analysis of Brazilian Stenocarpella Macrospora and Stenocarpella Maydis Isolates Anania Fessehaie , Elcio Alves

1 Iowa State University Seed Science Center, Ames IA 50011, and 2 USDA-ARS-MWA, Ames IA 50011; 2 Pioneer Sementes Ltda., Rodovia GO 210 km 04, Itumbiara, GO, Brazil, CP1014-CEP 75503-971

ABSTRACT

Stenocarpella macrospora and S. maydis cause ear rot, stalk rot, and leaf diseases in maize. These diseases can cause considerable yield losses in maize in warm, humid environments. The objective of this work is to assess the occurrence and distribution of Stenocarpella species in maize producing regions of Brazil and evaluate variability among isolates based on geographic origin. Stenocarpella species were isolated from maize kernels and leaf tissues obtained from representative locations, with two independent isolations made from each kernel and leaf sample. Pure cultures were identified based on morphological characteristics and the analyses of nucleotide sequence of the rDNA ITS regions. Stenocarpella species were isolated from 100% (12 of 12) and 37.5% (5 of 16) of the kernel and leaf samples, respectively. S. macrospora occurred in nine of the twelve kernel samples as a sole contaminant. Two samples were infested by both species and only one sample was infested by S. maydis alone. From maize leaf samples, only S. macrospora was recovered. The sizes of the rDNA ITS of S. macrospora and S. maydis were 493 and 495bp in length. Phylogenetic analyses based on the rDNA ITS sequence data revealed that S. macrospora isolates formed a homogenous cluster regardless of geographic origin. S. maydis isolates grouped in a separate distinct cluster. S. macrospora was more prevalent as a pathogen of maize in Brazil. S. maydis occurred only in samples from southern Brazil.

INTRODUCTION

Stenocarpella macrospora and S. maydis cause ear rot, stalk rot, and leaf diseases in maize. These diseases can cause considerable yield losses in maize in warm, humid environments. Maize debris and infected seed are the primary inoculum source. In Brazil, the pathogens are found in all maize growing regions and the damage caused exclusively by Stenocarpella has not yet been determined (Casa et al. 2006). Under severe and favorable environmental conditions the disease may cause yield losses reaching to 85% in major maize production areas of South Africa (Rossouw et al. 2002). Recent studies from South Africa reported that few inbred and hybrids showed promising Stenocarpella ear rot resistance and increased grain yields (Moremoholo et al. 2010). The objective of this work was to assess the occurrence and distribution of Stenocarpella species in maize producing regions of Brazil and evaluate variability among isolates based on geographic origin by comparing the nucleotides sequences of the internal transcribe spacer regions between the 18S and 28S rRNA genes.

Fig. 1. Map of Brazil showing the origin of the maize samples. Location of samples is
Fig. 1. Map of Brazil showing the origin of the maize samples.
Location of samples is marked with an asterisk.

MATERIAL AND METHODS

Maize samples. 12 seed lots and 16 leaf samples were collected in 2008-2009 from representative maize-growing regions of Brazil. Samples were sent to the Iowa State University Seed Science Center for analyses.

Isolation and identification. Stenocarpella species were isolated from maize kernels and leaf tissues with two independent isolations made from each seed lot and leaf sample. Locations of the samples are marked with an asterisk on the map (Fig. 1). To isolate Stenocarpella species, infested kernels and leaf tissues were surface-sterilized by soaking in 5% bleach plus 0.2% Tween 20 for 90 s, air-dried on filter paper. Seed and leaf tissues were incubated on PDA at room temperature. Single conidia were transferred to PDA and purity was determined further by morphological analysis. The identity of each isolate was confirmed by PCR (Xia and Achar, 2001). Illinois isolates of S. macrospora were collected in 2009 by Dr. Carl A. Bradley, University of Illinois.

MATERIALS AND METHODS cont'd

DNA isolation. Fungal mycelia for DNA extraction were produced by growing single spores of S. macrospora and S. maydis on PDA. Genomic DNA of pure fungal cultures was extracted from harvested mycelia and spores using the PrepMan TM Ultra DNA Kit (Applied Biosystems, Foster City, CA).

Amplification and sequencing of internal transcribed spacer (ITS) region. The regions between the 18S-28S rRNA genes, comprising ITS1, the 5.8 S rRNA gene, and ITS2 were amplified by PCR using the universal set of primers ITS1 and ITS4 (White et al. 1990). PCR was conducted in a reaction mixture with a total volume

of 50 µl reaction with NH4 Buffer: 16 mM (NH4)2 SO4, 67 mM Tris HCl pH 8.8;

0.01% Tween-20; 2 mM MgCl2; 200 each µM dNTP; 0.4 mM each primer;

1.25

units of Taq Polymerase (BioTaq, Bioline, USA), 5 ng genomic DNA with the following thermal cycling parameters: 95ºC for 3 min followed by 30 cycles of 94ºC for 30 s, 60ºC for 1 min and 72ºC and a final extension at 72ºC for 10 min.

PCR products were fractionated by electrophoresis at 100 V for 45 min on a 1.5% agarose gel in 1x TBE buffer and stained with ethidium bromide and gel image was documented using BIO RAD Gel Doc (Bio RAD Laboratory Inc., Hercules, CA). PCR Products were purified with a PCR Clean-Up Kit (Promega, Corp., Madison, WI) and sequenced using the universal primers ITS 1 and ITS 4 (White et al. 1990) following a standard BigDye terminator protocol (Applied Biosystems, Foster City, CA). Sequencing was conducted at the DNA Sequencing Facility of Iowa State University.

Phylogenetic analysis. ITS sequences were edited using the Gene Codes Sequencher 4.10.1 program (Gene Codes Corp., Ann Arbor, MI). Assembled sequences were used to probe the GenBank database to retrieve similar sequences for inclusion in the phylogenetic analysis. ITS sequences were aligned using the ClustalX2 program (Thompson et al., 1997). Phylogenetic analysis for maximum parsimony was performed using PAUP 4.0b10 (Swofford, 2005). Alignments gaps were treated as missing data. Maximum parsimony method was used to create phylogenetic trees. Parsimony trees were obtained using heuristic searches with 1000 random addition sequence replicates, tree-bisection-reconstruction, branch swapping, and the MULTREES options. The tree was rooted with D. phaseolorum var. meridionalis gi209979571 and Diaporthe sp. gi83034959 as the out-group. To evaluate clade support, bootstrap analyses were performed for 1000 replicates.

Nucleotide sequence accession numbers. Nucleotide sequences of the nuclear rRNA internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and short stretches of the adjacent 18S and 28S rRNA gene coding regions were deposited in GenBank.

RESULTS AND DISCUSSION

Isolation and identification. Pure cultures of Stenocarpella species were identified based on morphological characteristics and the analyses of nucleotide sequence of the rDNA ITS regions. Stenocarpella species were isolated from 100% (12 of 12) and 37.5% (5 of 16) of the seed lots and leaf samples, respectively. S. macrospora occurred in nine of the twelve sampling locations (representative seed lots) as a sole contaminant. The seed lot from Xanxere, State of Santa Catarina (SC) was contaminated solely with S. maydis ; whereas the seed lots collected from the neighboring states of Rio Grande do Sul (Sarandi, RS) and Paraná (Ponta Grossa, PR2) were infested by both species. As expected, from maize leaf samples, only S. macrospora was recovered (TABLE 1). This survey showed that S. macrospora was more prevalent as a pathogen of maize in Brazil. On contrary, the occurrence of S. maydis was limited to the three southern state of Brazil. These findings are important information when determining the losses cause by Stenocarpella species in Brazil. In Illinois, Diplodia leaf streak, caused by the fungus Stenocarpella macrospora, was observed in research plots at the Dixon Springs Agricultural Center recently. Although this disease has been observed in Illinois in the past, its occurrence was characterized as “An uncommon disease of corn in Illinois” (Carl Bradley, 2008).

I

P

kb kb 25 23 21 19 17 15 13 11 7 9 5 3 1
kb
kb
25
23
21
19
17
15
13
11
7
9
5
3
1

Fig. 2. PCR amplicons produced by primers P1/P2 and ITS1/ITS4 using DNA of representative isolates of S. maydis and S macrospora. Both outer lanes: 1Kb Plus DNA Ladder, Lanes 1-6: PCR products generated with primer sets ITS1/ITS4 and P1/P2 from S. maydis isolates SMA K1, SMA K11, and SMA K12, respectively. Accordingly, products from isolates of S. macrospora SMAC K2-K12 were in lanes 7-26.

I = PCR product amplified by the primer pairs ITS1/ITS4; P = PCR product amplified by the primer pairs P1/P2

RESULTS AND DISCUSSION (cont’d)

Table 1. Stenocarpella species isolated from seed and leaf samples collected from maize grown regions of Brazil and S. macrospora isolates received from Illinois used in this study.

Sample ID

Origin

Species ID

K1

S. maydis

K11

Xanxere - SC

S. maydis

K12

Sarandi - RS

S. maydis

K2

Ponta Grossa - PR2

S. macrospora

K3

Santa Helena - GO2

S. macrospora

K4

Bom Jesus - GO1

S. macrospora

K5

Goiatuba - GO

S. macrospora

K6

Nova Punta - MG

S. macrospora

K7

Humbiara - GO

S. macrospora

K8

Sorriso - MT

S. macrospora

K9

Bom Jesus - GO

S. macrospora

K10

Santa Helena - GO1

S. macrospora

K11

Sao Mateus de Sol - PR

S. macrospora

K12

Sarandi - RS

S. macrospora

L13

Ponta Grossa - PR2

---

L14

Santa Helena - GO2

S. macrospora

L15

Panama - GO

---

L16

Panama - GO

S. macrospora

L17

Ponta Grossa - PR1

S. macrospora

L18

Ipameri - GO

---

L19

Morrinhos - GO

S. macrospora

L20

Ipameri - GO

---

L21

Planaltina - DF

---

L22

Toledo - PR

S. macrospora

L23

Palmeira - PR

---

L24

Faxinal dos Guedes - SC

---

L25

Goiatuba - GO

---

L26

Bom Jesus - GO2

S. macrospora

L27

Planaltina - DF

---

L28

Cristalina - GO

---

BF09184

Palmeira - PR

S. macrospora

BF09185

Vermilion/IL, USA

S. macrospora

BF09186

Vermilion/IL, USA

S. macrospora

BF09221

Vermilion/IL, USA

S. macrospora

BF09222

Ridgway/IL, USA

S. macrospora

BF09380

Ridgway/IL, USA Dixon Springs/IL, USA

S. macrospora

 

S.

maydis (gi241061838)

S.

maydis (gi32967058)

S.

maydis (gi32967065)

S.

maydis (gi32967060)

S.

macrospora (gi253970749)

S.

macrospora (gi241061840)

S.

macrospora (gi241061839)

Diaporthe sp. (gi83034959)

 

D.

phas.var. meridionalis (gi209979571)

K = Seed isolates ; L = Leaf isolates

The complete rDNA ITS sequences of

S. macrospora and S. maydis were 493

and

495

nucleotides

in

length,

respectively. Nucleotide sequences of all S. macrospora isolates from Brazil

and from Illinois were 100%

homologous.

Also,

the

size

and

nucleotide

sequences

of

the

three

Brazilian

S.

maydis

isolates

were

identical. A total of 12 single

polymorphisms

nucleotide

and

two

insertion/deletions (indels) were found

between the ITS sequences of

Stenocarpella

species.

Of

the

12

(2.42%)

mismatched

nucleotides,

8

were located in ITS1 and 4 on ITS2. S.

maydis

ITS1

sequence

contained

an

extra “G” nucleotide at positions 88

and 160; these positions were deleted

on the S macrospora ITS1.

 
 

S.

S.

macrospora gi253970749

macrospora gi241061839

S.

macrospora gi241061840

SMACL26

SMAC2

SMAC3

SMAC4

SMAC5

SMACL22

SMAC7

SMAC6

SMACL19

 

100

SMAC8

     

SMAC10

SMAC9

SMACL17

S.

macrospora BF09222

SMACL16

S.

macrospora BF09380

SMACL14

100

S.

S.

macrospora BF09186

macrospora BF09221

 

S.

macrospora BF09185

 

S.

macrospora BF09184

SMAC12

SMAC11

SMA11

maydis AY332487

maydis AY332480

S.

S.

 

SMA1

maydis AY332482

S.

 

100

 

SMA12

   

S. maydis gi241061838

D. phaseolorum var. meridionalis gi209979571

 

Diaporthe sp. gi83034959

0.001 substitutions/site

Fig. 3. Phylogenetic analysis using maximum-

parsimony based on rDNA ITS sequences of 35 taxa showing the relationship within the Stenocarpella species. The tree was rooted with D. phaseolorum var. meridionalis gi209979571 and Diaporthe sp.

gi83034959 as the out-group. Bootstrap values greater

than or equal to 50% (1000 replicates) are shown at

branches.

Maximum parsimony analysis of aligned rDNA

ITS sequences of 35 taxa included the 17 S.

macrospora isolates from Brazil, Six from the

Illinois (USA), 3 S. maydis ITS sequences from

Brazil and nine other sequences of S. macrospora,

S. maydis and Diaporthe sp. retrieved from

GenBank. Parsimony tree based on the rDNA ITS

sequence data revealed that Stenocarpella species

grouped into two separate distinct clades. S.

macrospora isolates formed a homogenous

cluster regardless of geographic origin. Also, S.

maydis isolates were grouped in a separate distinct but phylogenetically related cluster. Grouping was supported by high bootstrap values (Fig. 3).

CONCLUSIONS

S. macrospora was found in almost all maize-growing regions of Brazil whereas the occurrence of S. maydis was limited to the states of Paraná and Rio Grand Do Sul and Santa Catarina in southern Brazil. Maximum parsimony tree revealed that Stenocarpella species grouped into two distinct but phylogenetically related clades corresponding to morphological species distinctions. S. macrospora isolates formed a homogenous cluster regardless of geographic origin.

REFERENCES

Bradley, C.A. 2008. Extension Bulletin No. 21 Article 5. Casa et al. 2006. Fitopatologia Brasileira 31: 427-439. Moremoholo et al. 2010. Euphytica 174:231–238. Rossouw et al. 2002. South African Journal of Plant and Soil 4, 182-187. Swofford, D.L. 2005. Sinauer Associates, Sunderland, MA. Thompson et al. 1997. Nucl. Acids Res. 24: 4876-4882. White et al. 1990. PCR Protocols: A guide to methods and applications. New York Academic Press, 315–322. Xia and Achar. 2001. J. Phytopathology 149: 35-44.

ACKNOWLEDGEMENTS

We thank Carl Bradley, Department of Crop Sciences, University of Illinois for

providing several S. macrospora isolates.