diagnosis of fungal bean root rot pathogens using
TRANSCRIPT
Common Bean Disease Workshop on Angular Leaf Spot and Root Rots Protea Hotel, Skukusa, South Africa
July 20-24, 2015
Diagnosis of Fungal Bean Root Rot Pathogens using Molecular and Culture Methods Graciela Godoy-Lutz, Chikoti Mukuma, J. R. Steadman , Suzana Fernandes-
University of Nebraska-Lincoln Kennedy Muimui- Misamfu Regional Research Center, Kasama, Zambia
Celestina Jochua- Instituto de Investigação Agrária de Moçambique
Introduction
• Root/Stem/Crown Rot (RSCR) is a disease complex
• Many fungi and Oomycetes are associated with symptoms on dry beans in the LAC region and Africa
• RSCR has emerged as a limiting factor in dry bean production in Sub-Saharan, Eastern and Southern Africa and is reaching epidemic proportions in the Great Lakes region
Objective
1. Identify and characterize predominant pathogens associated
with plants showing RSCR symptoms in initial diverse germplasm screening nurseries in representative dry bean production areas each in Zambia and Mozambique.
Component of objective: a) To compare molecular (454 pyrosequencing/PCR/Sanger sequencing) and culture based methods to determine predominant taxa ( biomass/counts) associated with RSCR
Field sampling
# Locations
# Samples
Mozambique 5 200
Zambia 5 194
Local lab processing Local lab processing
UNL lab processing
Pipeline analysis
OTU sequences
454 Pyrosequencing
PCR
Gel electrophoresis
Binary matrix
Koch’s postulates
Microscopic ID
Sanger sequencing
Numerical data
Electrophenogram
Zambia Biodiversity indices 2014
Shannon-H
Simpson I-D
Misamfu Regional Research Center
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0.8
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1.2
1.4
1.6
Inde
x va
lue
Method
Index
Sampling sites- 2014
Chokwe
Chate
Chissano
Mozambique
F. solani
F. solani
F. solani
A. alternata
A. alternata
other
other
other
Fusarium sp.
Zambia
Sampling sites
Misamfu Regional Research Center
Landraces- Mt. Makulu Research Center
Mt. Makulu Research Center
F. solani
F. oxysporum
F. oxysporum F. oxysporum
F. oxysporum Fusarium sp.
Fusarium sp.
Fusarium sp.
A. alternata
A. alternata
A. alternata
Alternaria sp.
other
other
other
Fusarium spp.
Fusarium spp.
F. equiseti
Neighbor-joining trees depicting variability within taxonomic units of the same species. Samples from Misamfu , Zambia 2014
FTA cards Tissue
Isolates from diseased tissue varied in culture and morphology
F. solani
M. phaseolina
R. solani
P. ultimum
Straw test Stem test Detached leaf Test Cup test
Pathogens in culture
All isolates were tested for pathogenicy with the straw test method which performed the best in previous experiments
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9
1 2 3 5 6 7 8 9 10 11 12 16 17 18 19 20 21 22 23 24 25 26 28
Mea
n Le
sion
size
in m
m
OTU
Pathogenicity of isolates from culture- 2014. Misamfu, Zambia
Alternaria Fusarium Pythium Rhizoctonia
OTU Nursery ID
Variety NCBI GenBank closest match GenBank # of closest match
% Coverage/ Identity
1 RR123 CIMSUG07-ALS-2 Alternaria sp KM088044.1 96/95 2 RR123 CIMSUG07-ALS-2 Fusarium verticillioides KJ957786 97/99 3 RR205 CIMSUG07-ALS-5 Fusarium equiseti KC254029 96/98 4 RR205 CIMSUG07-ALS-5 Dothideales sp. CY148 HQ607988 95/96 5 Survey Survey Fusarium equiseti strain UOA/HCPF 47B isolate ISHAM KC254029 96/98 6 Survey Survey Pythium myriotylum isolate FJTP02 KJ162354 92/97 7 Survey Survey Fusarium equiseti strain HM5 KJ677236 91/97 8 Survey Survey Fusarium oxysporum strain DR2F54 KJ938019 94/98 9 Survey Survey Fusarium equiseti strain HM5 KJ677236 87/95
10 Survey Survey Fusarium equiseti strain HM5 KJ677236 90/97 11 Survey Survey Fusarium equiseti strain HM5 KJ677236 91/97 12 RR 226 ADP 517 Rhizoctonia solani isolate IQ40 KF372662 85/97 13 Survey Survey SEQUENCE NON RESPONSIVE IN BLAST 14 RR 261 ADP 629 Ampelomyces sp. A10 EU781667 100/96 15 RR 261 ADP 629 Curvularia trifolii strain KACC 42434 EF600959 87/94 16 RR 261 ADP 629 Fusarium equiseti isolate 50A KP067236 94/98 17 RR 261 ADP 629 Fusarium oxysporum strain DR2F54 KJ938019 96/96 18 RR 202 ADP 461 Fusarium oxysporum isolate CU2-97 KP119604 93/98 19 RR 202 ADP 461 Fusarium oxysporum isolate F37-SMVDA2t1 KC304797 95/97 20 RR 355 NE34-12-37 Pythium myriotylum isolate BRIP52426 FJ797577 79/89 21 Survey Survey Fusarium equiseti isolate s077 HQ649908 86/95 22 RR59 ADP 61 Fusarium equiseti isolate 50A KP067236 91/95 23 RR59 ADP 61 Fusarium oxysporum isolate HQ649815 82/95
24 RR 92 CIM SUG05-ALS-
33 Fusarium oxysporum isolate F12-BKR6 KC304817 79/96
25 Survey Survey Fusarium oxysporum isolate F37-SMVDA2t1 KC304797 91/97 26 RR 186 CIM SUG05-ALS-3 Pythium myriotylum isolate GPy 35 KM434129 83/93 27 RR 376 CIM-CLIMB03-48 Talaromyces cellulolyticus strain NFML_CH49_258 1 KM458826 61/94 28 RR 355 NE34-12-37 Fusarium equiseti isolate 07D KP067237 44/95
Cultu
re
Pyro
sequ
enci
ng
FTA
card
s
PCR
FTA
car
ds
PCR
Tiss
ue
Pyro
sequ
enci
ng
tissu
e
Culture Pyrosequencing
FTA cards 0.62
PCR FTA cards 0.66 0.85
PCR Tissue 0.45 0.89 0.49
Pyrosequencing tissue 0.55 0.64 0.76 0.29*
P < 0.001 *P= 0.03
Spearman’s and Polyserial rho correlation between methods for Fusarium species counts in 2014 Samples from Misamfu, Zambia
Summary of results
• Using combined approaches we detected similar fungal species mainly of Fusarium complex associated with RSCR in nine locations in two countries ( Zambia and Mozambique)
• Common ocurrence of a few fungal species from all locations and bean lines sampled warrants investigations on what triggers pathogenesis in the host
• New pathogenic species and abundance of a predominant pathogen were detected by 454 pyrosequencing over other methods
Summary of results
• Fusarium oxysporum , F. solani and other species of the Fusarium complex are the most abundant as OTU’s and biomass of 2013-2014 samples detected in DNA (FTA cards/tissue) or intact diseased tissue.
• Over 90% of culture isolates belong to genus Fusarium and were pathogenic to bean plants ( Zambia and Mozambique)
• Next step is finding the target pathogen for breeding for RR/CR resistance
Suzana Fernandes (Mozambique)
2016 M.Sc.
Graduate students at UNL responsible for the work in the project
Chikoti Mukuma ( Zambia) 2015 M.Sc