Characterization of Pythium spp. associated with root rot of tobacco ...

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Received: 10 April 2017    Accepted: 17 July 2017 DOI: 10.1111/jph.12613

ORIGINAL ARTICLE

Characterization of Pythium spp. associated with root rot of tobacco seedlings produced using the float tray system in Zimbabwe Fortunate Mufunda1 | Norman Muzhinji1

 | Tertu Sigobodhla2 | 

Mike Marunda2 | Cleopas Chenayi Chinheya2 | Susan Dimbi2 1 Molecular Biology Services Division, Tobacco Research Board, Harare, Zimbabwe

Abstract

2

The study was undertaken to identify and characterize Pythium isolates associated

Plant Health Services Division, Tobacco Research Board, Harare, Zimbabwe Correspondence N. Muzhinji, Molecular Biology Services Division, Tobacco Research Board, Harare, Zimbabwe. Email: [email protected]

with root rot disease of tobacco seedlings as a first step towards developing management strategies for the pathogen. A total of 85 Pythium isolates were collected from diseased tobacco seedlings during 2015–2016 tobacco growing season. The isolates were identified to species level using sequencing of the internal transcribed spacer region. Thereafter, a subset of the isolates was tested for sensitivity to the commonly used fungicides, metalaxyl, azoxystrobin and a combination of fenamidone/propamocarbby growing isolates on Potato Dextrose Agar plates amended with the fungicides. The sequence analysis of the ITS-­rDNA identified Pythium myriotylum as the dominant Pythium species associated with the root rot of tobacco seedlings in Zimbabwe. Pythium aphanidermatum and P. insidiosum were also identified albeit at lower frequencies. Phylogenetic analyses of the ITS region of the P. myriotylum isolates showed little sequence diversity giving rise to one distinct clade. The fungicide sensitivity tests showed that metalaxyl provided the best control of P. myriotylum in vitro, as compared to other fungicides. To the best of our knowledge, this is the first comprehensive study to determine and characterize Pythium species associated with root rot of tobacco in the float seedling production system in Zimbabwe. KEYWORDS

float tray seedling production, fungicide sensitivity, ITSrDNA, Pythium spp., root rot, sequencing, tobacco

1 |  INTRODUCTION

Martin & Loper, 1999). Several species including P. aphanidermatum, P. ultimum, P. irregulare, P. spinosum, P. oligandrum, P. splendens and

The fungus Pythium belongs to the phylum Oomycota and is a diverse

P. myriotylum have been reported on tobacco worldwide (Cartwright,

genus believed to comprise more than 300 recorded species (Webster &

Spurr, & Shew, 1995; Moorman and Kim 2004; Gutiérrez, Melton, &

Weber, 2007; www.mycobank.org). These species exhibit a wide range

Mila, 2012).

of habits, from aggressive plant pathogens, saprophytes, weak root

Ribosomal DNA gene amplification is mainly used for identifica-

pathogens as well as biocontrol agents (Le, Aitken, & Smith, 2015; Martin

tion of organisms showing both intraspecies and interspecies varia-

& Loper, 1999). As plant pathogens, Pythium spp. are able to infect a wide

tion because ribosomal DNA comprises highly conserved coding and

diversity of plants, causing seed, root and lower stem rot, as well as seed-

variable non-­coding regions (Kageyama, 2015). Identification and

ling damping-­off of many crops (Broders, Lipps, Paul, & Dorrance, 2007;

characterisation using conventional methods based on morphology

Journal of Phytopathology. 2017;1–9.

wileyonlinelibrary.com/journal/jph   © 2017 Blackwell Verlag GmbH |  1

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MUFUNDA et al.

2      

(b) (a) (c)

of antheridia, oogonia and sporangia, which vary under different cultural conditions is time-­consuming and laborious, sometimes re-

F I G U R E   1   Healthy tobacco seedlings (a), Stunting, and damping-­off associated with Pythium root rot in tobacco floatbeds (b), tobacco seedlings infected with Pythium root rot in float seed beds (c)

2.2 |  Sample collection and Pythium isolation

sulting in misidentification (Dick, 1990; Uzuhashi, Tojo, & Kakishima,

Tobacco seedlings showing typical Pythium root rot symptoms

2010). Hence, molecular approaches have increasingly been adopted

(Figure 1a-­c) were collected randomly from the float seedbed at

for accurate identification and characterization of Pythium isolates

Kutsaga and Banket commercial seedling production sites with a his-

(Schroeder et al., 2013). In Zimbabwe, Pythium root rot is a problem in tobacco and became

tory of Pythium root rot infections. Samples brought by growers to the Tobacco Research Board’s Plant Clinic at Kutsaga and diagnosed

particularly serious in the float tray seedling production system. The

with Pythium root rot were also included in this study. Disease symp-

float tray hydroponic production system provides favourable condi-

toms on sampled plants included slight stunting and root rot ranging

tions for the growth of the fungus (Sigobodhla, Dimbi, & Masuka,

from mild to severe leading to seedling death. Upon sample receipt,

2010). Pythium is known to cause extensive root and stem rot which

all information on fungicide application history was recorded. The to-

results in reduced seedling vigour and subsequent poor plant growth

bacco seedlings were washed under running tap water, disinfected

and field performance in transplanted plants (Sigobodhla et al.,

with 10% hypochlorite and dried with absorbent paper. Root sections,

2010). In Zimbabwe, the species P. aphanidermatum, P. debaryanum,

4 mm long, were cut using a sterile scalpel and plated on solidified PDA

P. myriotylum and P. ultimum have been occasionally isolated from to-

(Biolab), amended with 50 mg/L of chloramphenicol (Sigma-­Aldrich

bacco seedlings brought to the Tobacco Research Board’s Plant Clinic

Co.). Plates were incubated in the dark at 25°C ± 0.1°C (Gallenkamp,

(Sigobodhla et al., 2010). However, studies have not been carried out

Cooled Incubator INF-­750) for 24 to 72 hr. Hyphal tips of myce-

to characterize Pythium spp. associated with tobacco seedling pro-

lia characteristic of genus Pythium as described by van der Plaats-­

duction in Zimbabwe. In order to device an effective management

Niterink (1981) and Dick (1990) were transferred to 90-­mm-­diameter

of Pythium root rot, it is important to have basic information on the

plates containing oatmeal agar (OMA) (Biolab) to obtain pure cultures.

composition of Pythium species present. The present study was un-

Purified cultures were maintained on sterile water at 25°C for further

dertaken to identify and characterize the Pythium species associated

studies.

with root rot of tobacco seedlings in Zimbabwe. In addition, in vitro sensitivity of the Pythium isolates to a few commonly used fungicides was tested.

2.3 |  DNA extraction and PCR amplification Actively growing Pythium mycelia were scrapped from the surface of

2 |  MATERIALS AND METHODS 2.1 |  Study location

OMA and macerated under liquid nitrogen. DNA was extracted using the modified CTAB protocol by Doyle and Doyle (1987). In brief, 200 mg mycelia were ground using liquid nitrogen in pestle and mortar and extraction buffer (100 mm Tris-­Cl, pH 8.0; 20 mm EDTA, pH 8.0;

The study was carried out at Tobacco Research Board (Kutsaga

1.4 m NaCl, 3% CTAB) was used to break up the cells. The tubes were

Research Station) located 15 km East of Harare at latitude 17055′S

centrifuged at 12,000 × g for 10 min, and the supernatant contain-

and longitude 31008′E with an altitude of 1,479 m above sea level.

ing DNA was transferred to a clean Eppendorf tube. DNA was then

The average temperatures were 32°C and 18°C during summer

cleaned with chloroform twice, pelleted with isopropanol and cen-

and winter, respectively, with annual rainfall between 800 and

trifuged at 12,000 × g for 5 min. DNA was quantified using BioDrop

1,000 mm.

μLITE UV/VIS spectrophotometer. Amplification of the ITS-­rDNA was

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MUFUNDA et al.

performed using primers ITS1 (5′-­TCCGTAGGTGAACCTGCGC-­3′)

a.i./ml. The fungicides were first dissolved in sterile deionized water

and ITS 4 (5′-­TCCTCCGCTTATTGATATGC-­3′) (White, Bruns, Lee,

to achieve the proper concentration of active ingredients. Thereafter,

& Taylor, 1990). Amplification was conducted in 25 μl reaction mix-

they were added to autoclaved media. This was performed under

tures containing 4 ng of template DNA; 250 mm each dATP, dTTP,

laminar airflow to prevent contamination. After the media had been

dGTP and dCTP (Fermentas); 10 × PCR reaction buffer, consist-

left to cool down to approximately 40°C, 15 ml of the agar-­fungicide

ing of 160 mm (NH4)2SO4, 670 mm Tris–HCl at pH8.8, and 100 mm

mixture was poured in 90 mm Petri dishes. A set of Petri dishes with

KCl (GeneDireX); 0.25 U of Taq DNA polymerase (GeneDireX) and

non-­amended PDA were included as the control treatments for all

0.2 mm each primer (Inqaba Biotechnical Industries, South Africa).

fungicides.

Amplification was carried out in a thermal cycler (9700 GeneAmp)

For the fungicide sensitivity trial, a 5-­mm-­diameter plug from the

with the following conditions: an initial step at 95°C for 3 min; fol-

edge of a five-­day-­old culture of each Pythium isolate was transferred

lowed by 35 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 45 s;

to the centre of each plate having PDA amended with the fungicides.

and a final extension of 72°C for 5 min. A 5 μl aliquot of each poly-

Plates were incubated at 25°C for 5 days. The fungal colony diameter

merase chain reaction (PCR) product was separated by electrophore-

was measured after 24, 48, 72 and 96 hr, at perpendicular angles,

sis on 1.5% (wt/vol) agarose (Lonza) stained with ethidium bromide

and the average of the two measurements was used for data analysis.

solution (0.1 mg/L) and visualized using a UV transilluminator (UVitec

The per cent growth inhibition was calculated for each concentra-

U.K). When the bands of the appropriate size (700 bp) were ob-

tion by dividing the average colony diameter of the isolate, minus

served, the remaining PCR product was purified using Sephadex spin

the 5 mm for the agar plug, by the average colony diameter of the

column (Sigma Aldrich Co.) (5 g of Sephadex G-­ 50 powder dissolved

non-­fungicide-­amended media, multiplied by 100. A 6 × 4 factorial

in 75 ml of sterile water) and the resulting amplicons were submit-

systematic design in three blocks was used and the experiment was

ted to Inqaba Biotechnical Industries (South Africa) for sequencing.

conducted twice.

The DNA sequences obtained were edited and consensus sequences

The sensitivity results were interpreted as follows: Sensitive—

created from both the forward and reverse sequences using BioEdit

when there was no growth, intermediate sensitivity—with hyphal

v 7.1.3 (Hall, 1999). Sequences generated in this study have been

growth of 40% of growth on the unamended plate (Olson & Benson, 2011). The variation in sensitivity among isolates to the three

2.4 |  Pythium species identification and phylogenetic analysis

different fungicides was analysed using analysis of variance (ANOVA) Genstat18th Edition (VSN International 2015), and significant differences were determined by Fisher’s LSD test at the 5% level.

The consensus sequences of each isolate were compared with reference sequences (Table 2) in the nucleotide database GenBank, available through the National Centre for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) algorithm. The BLAST parameters used for the sequence identification were e-­values

3 | RESULTS 3.1 |  Identification of Pythium spp.

of 0.0, a maximum identity match of 98% or greater, and a query cov-

Eighty-­three of the 85 isolates were identified as P. myriotylum while

erage of 98% or greater. Multiple sequence alignments were gener-

one was identified as P. aphanidermatum and another as P. insidiosum

ated using ClustalW (2.0.12) in BioEdit (7.0.9.0). Gaps were treated

(Table 3).

as missing data in the subsequent analyses. Phylogenetic analysis was performed based on maximum likelihood (ML) implemented in MEGA (7.0.14) using the Tamura-­Nei model (Tamura, Stecher, Peterson,

3.2 |  ITS sequencing and phylogeny

Filipski, & Kumar, 2013). Bootstrap analyses were performed to de-

Amplification of the ITS region of Pythium isolates using ITS 1 and ITS

termine branching point confidence intervals (1,000 replicates) gener-

4 universal primers generated a sequence length that varied between

ated for each data set. The ITS sequence of Aphanomyces euteiches

700 bp and 800 bp for the ITS1, 5.8 S and ITS2 regions. The identity

Drechs was used as the out-­group (Figure 2).

of these species was confirmed with a pairwise comparison of the ITS sequences of the study isolates with reference sequences from the

2.5 |  Fungicide sensitivity of Pythium isolates

GenBank database. A BLAST search from the GenBank database resulted in very high identity levels, with over 99% to 100% homology

Fungicide sensitivity tests were carried out in vitro using five Pythium

to well-­known reference isolates, so multiple gene sequencing was

myriotylum isolates (Py26, Py57, Py93, Py94 and Py99) and one

not required.

P. insidiosum (Py100) isolate. The fungicides metalaxyl (Acomil 68%

Maximum likelihood of the ITS region grouped Pythium isolates

WP; Technical Services Africa), azoxystrobin (Azoxystrobin 25 SC,

including the references into two distinct well separated clades I and

Technical Services Africa) and fenamidone/propamocarb (Consento

II (Figure 2). The clades were further separated into distinct phyloge-

450 SC, Bayer) all reported as effective for Pythium control were used

netic subclades each corresponding to different Pythium species with

in this study. All three fungicides were evaluated at the rate of 10 μg

branching patterns supported with high bootstrap values (Figure 2).

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MUFUNDA et al.

4      

T A B L E   1   Pythium species used in the study

T A B L E   1   (Continued)

Isolate

species

Location

GenBank Accession number

Py01

P. myriotylum

Kutsaga commercial site

KX671044

Py02

P. myriotylum

Kutsaga commercial site

KY019263

Py03

P. myriotylum

Kutsaga commercial site

KY019264

Py04

P. myriotylum

Kutsaga commercial site

KY019265

Py05

P. myriotylum

Kutsaga commercial site

KX671049

Py06

P. myriotylum

Kutsaga commercial site

KY019285

Py07

P. myriotylum

Kutsaga commercial site

KX671052

Py08

P. myriotylum

Kutsaga commercial site

KX671054

Py09

P. splendens

Kutsaga commercial site

KX671056

Py10

P. ultimum

Kutsaga commercial site

KX671058

Py11

P. irregulare

Kutsaga greenhouse

KY019267

Py12

P. myriotylum

Banket commercial site

KX671061

Py13

P. myriotylum

Banket commercial site

KX671063

Py14

P. myriotylum

Kutsaga greenhouse

KX671064

Py15

P. myriotylum

Banket commercial site

KX671066

Py17

P. myriotylum

Banket commercial site

KX671069

Py18

P. myriotylum

Kutsaga commercial site

KX671071

Py19

P. myriotylum

Kutsaga commercial site

KY019268

Py20

P. myriotylum

Kutsaga commercial site

KX671074

Py21

P. myriotylum

Kutsaga commercial site

KX671076

Py22

P. myriotylum

Kutsaga commercial site

KX671078

Py23

P. myriotylum

Banket commercial site

KX671079

Py23PHS

P. myriotylum

Kutsaga Plant clinic

KX671080

Py24

P. myriotylum

Kutsaga commercial site

KY019269

Py25

P. myriotylum

Banket commercial site

KY019270

Py26

P. myriotylum

Kutsaga greenhouse

KX671081

Py27

P. myriotylum

Banket commercial site

KY019271

Py28

P. myriotylum

Banket commercial site

KY019272

Py31

P. myriotylum

Kutsaga commercial site

KX671085

Py32

P. myriotylum

Banket commercial site

KX671087

Py33

P. myriotylum

Kutsaga greenhouse

KX671089

Py34

P. myriotylum

Kutsaga greenhouse

KX671091

Py35

P. myriotylum

Kutsaga commercial site

KY019273

Py36

P. myriotylum

Kutsaga commercial site

KX671094

Py37

P. myriotylum

Kutsaga commercial site

KX671092

Py38

P. myriotylum

Kutsaga commercial site

KY019274

Py39

P. myriotylum

Kutsaga commercial site

KX671096

Py40

P. myriotylum

Kutsaga commercial site

KX671097

Py41

P. myriotylum

Kutsaga commercial site

KX671098

Py42

P. myriotylum

Kutsaga commercial site

KX671099

Py43

P. myriotylum

Kutsaga greenhouse

KX671100

Py44

P. myriotylum

Kutsaga commercial site

KX671101

Location

GenBank Accession number

P. myriotylum

Banket commercial site

KX671104

P. myriotylum

Kutsaga greenhouse

KX671105

Py51

P. myriotylum

Kutsaga commercial site

KX671106

Py52

P. myriotylum

Kutsaga commercial site

KX671107

Isolate

species

Py48 Py50

Py53

P. myriotylum

Kutsaga commercial site

KX671045

Py54

P. myriotylum

Kutsaga greenhouse

KY019276

Py55

P. myriotylum

Banket commercial site

KY019277

Py57

P. myriotylum

Kutsaga commercial site

KY019278

Py60

P. myriotylum

Banket commercial site

KY019279

Py61

P. myriotylum

Banket commercial site

KX671046

Py62

P. myriotylum

Kutsaga commercial site

KX671047

Py63

P. myriotylum

Kutsaga commercial site

KX671048

Py64

P. myriotylum

Kutsaga commercial site

KY019280

Py65

P. myriotylum

Kutsaga commercial site

KX671050

Py66

P. myriotylum

Kutsaga commercial site

KX671053

Py67

P. myriotylum

Kutsaga greenhouse

KY019281

Py68

P. myriotylum

Kutsaga greenhouse

KX671057

Py69

P. myriotylum

Kutsaga greenhouse

KX671059

Py70

P. myriotylum

Kutsaga commercial site

KX671060

Py77

P. myriotylum

Banket commercial site

KX671062

Py79

P. myriotylum

Banket commercial site

KY019282

Py80

P. myriotylum

Kutsaga greenhouse

KX671065

Py81

P. myriotylum

Banket commercial site

KX671067

Py82

P. myriotylum

Kutsaga commercial site

KX671068

Py84PHS

P. myriotylum

Kutsaga Plant clinic

KX671069

Py86

P. myriotylum

-­

KX671070

Py87

P. myriotylum

Kutsaga greenhouse

KX671072

Py88

P. myriotylum

Banket commercial site

KX671077

Py89

P. myriotylum

Banket commercial site

KX671073

Py99

P. myriotylum

Banket commercial site

KX671080

Py100

P. aphanidermatum Banket commercial site

KY019283

Py101

P. insidiosum

Kutsaga commercial site

KY019284

Py102

P. myriotylum

Banket commercial site

KX671082

Py103

P. myriotylum

Banket commercial site

KX671083

Py108

P. myriotylum

Kutsaga commercial site

KY019285

Py110

P. myriotylum

Kutsaga commercial site

KX671084

Py113

P. myriotylum

Banket commercial site

KX671093

Py117

P. myriotylum

Banket commercial site

KX671086

Py26PHS

P. myriotylum

Kutsaga Plant clinic

KX671088

Py122

P. myriotylum

Banket commercial site

KY019286

Clade I consisted of P. myriotylum, P. aphanidermatum and P. insidiosum.

Py45

P. myriotylum

Banket commercial site

KX671102

Py46

P. myriotylum

Kutsaga commercial site

KY019275

Clade II consisted of P. irregulare, P. splendens and P. undulatum. The

Py47

P. myriotylum

Banket commercial site

KX671103

majority of the isolates in clade I, formed a single well-­supported sub-

(Continues)

clade IA (96% bootstrap support) containing the entire P. myriotylum

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MUFUNDA et al.

T A B L E   2   Reference GenBank Pythium sequences used in the study Pythium species

Country of Origin

Crop

GenBank accession number

Reference

P. myriotylum

South Africa

-­

FJ415904

McLeod et al., 2009

P. myriotylum

South Africa

-­

FJ415905

McLeod et al., 2009

P. myriotylum

USA

Irrigation water

KJ865239

Jones, Worobo, & Smart, 2014

P. myriotylum

Spain

Bean

AM396958

Serrano, Melero-­Vara, Guirado, Segundo, & Gomez, 2008

P. myriotylum

Australia

Ginger

FJ797576

Stirling, Turaganivalu, Stirling, Lomavatu, & Smith, 2009

P. myriotylum

Cameroon

Cocoyam

DQ222436

Tambong, Perneel, Hofte, & Levesque, 2004

P. myriotylum

Cameroon

Cocoyam

DQ222438

Tambong et al., 2004

P. myriotylum

Japan

Soybean

AB740139

Tomioka et al., 2013;

P. myriotylum

South Korea

Common Bean

KU882088

Y. H. Jeon, personal communication

P. myriotylum

Japan

Calla Lily

DQ298521

Al-­Sa’di, Drenth, Deadman, De Cock, & Aitken, 2007

P. myriotylum

India

-­

HQ643702

Robideau et al., 2011

P. myriotylum

Spain

Green beans

AM396958

Serrano et al., 2008

P. myriotylum

Japan

Glycyrrhiza uralensis

AB818457

Ishiguro et al., 2014

P. myriotylum

Canada

-­

HQ643704

Robideau et al., 2011

P. myriotylum

Canada

Cocoyam

DQ222437

Tambong et al., 2004

P. myriotylum

USA

Snap Beans

EU169137

Olson, Damicone, & Kahn, 2016

P. deliense

USA

Peanuts

KF500572

Parkunan, Brenneman, & Ji, 2014

P. insidiosum

Thailand

Environmental

EF016914

Kammarnjesadakul et al. 2011

P. irregulare

USA

-­.

AY907909

Garzon, Geiser, & Moorman, 2005

P. oligandrum

New Zealand

Mushroom

AF364536

Godfrey, Monds, Lash, & Marshall, 2003

P. splendens

Japan

Peanut field soil

AB796304

Ikeda et al., 2013

P. splendens

Japan

-­

AB796306

Ikeda et al., 2013

HQ643767

Robideau et al., 2011

P. ultimum

China

-­

JN695787

Zhao & Ding, 2011

P. zingiberis

Canada

-­

AY598679

Levesque & De cock, 2004

P. dimorphum

Canada

-­

AY598651

Levesque & De cock, 2004

P. aphanidermatum

Korea

Lampranthus spectabilis

KR095341

Park, Han, Kim, Park, & Shin, 2016;

P. aphanidermatum

Oman

Cucumber

DQ298521

Al-­Sa’di et al., 2007

P. rostratum

-­

-­

AJ233456

Matsumoto, Kageyama, Suga, & Hyakumachi, 2009

P. undulutum

Switzerland

Pine forest

EF055295

Chavarriaga, Bodles, Leifert, Belbahri, & Woodward, 2007

P. undulatum

Germany

Pseudostriga menziessi

AY436638

Weber, Sulzer, & Haarhaus, 2004

P. rostratum

species complex (Figure 2). Pythium myriotylum exhibited limited intraspecific variation in the sequences of the ITS region of the rDNA.

Py 26, 57, 94 and 99 but was ineffective against P. myriotylum isolate Py 93 (Figure 3). There was a varied response to azoxystrobin that ranged from sensitive (isolate Py 100) to intermediately sensitive (Py

3.3 |  Fungicides sensitivity There were significant differences (p