Z Score,T Score, Percential Rank and Box Plot Graph
Powerpoint Presentation (FFV).pptx
1. Cultural, Morphological and Pathological Characterization
of Alternaria porri Isolates on Onion (Allium cepa L.) in
Central and Eastern Ethiopia.
Seminar Paper Presentation
Abu Jambo Yae
(PhD in Plant Pathology)
February 2021
HU, Haramaya
2. PRESENTATION OUTLINE
This presentation consists of the following four major components.
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS AND RECOMMENDATIONS
3. 1. INTRODUCTION
Onion (Allium cepa L.) is among the most important vegetable
crops grown in Ethiopia.
The national average bulb yield of onion in Ethiopia under farmers
conditions is remarkably lower (9 t ha-1) (CSA, 2019) than its
potential yield (30 t ha-1) due to several biotic and abiotic factors
(Lemma and Shimeles, 2003).
One of the major contributing biotic factors to this low yield is
fungal diseases.
Purple blotch (Alternaria porri), downy mildew (Peronospora
destructor) and basal rot (Fusarium oxysporum f.sp. cepae), are the
three major economically important fungal diseases of onion in
Ethiopia (Wondirad et al., 2009; Sintayehu et al., 2011).
4. INTRODUCTION …
Among these diseases, purple blotch (PB) is the most predominant
and serious disease in nearly all onion-growing areas of the
country.
In Ethiopia, onion bulb yield loss due to PB was estimated to reach
50% of the total production (Lemma and Shimeles, 2003).
Onion PB management options include cultivation of resistant
varieties (Yadav et al., 2017) and fungicide applications as seed
treatments and foliar sprays (Jhala and Mali, 2017).
The preference of past research on onion PB management strategy
in Ethiopia was mainly towards application of foliar fungicides
(Olani and Fikre, 2010).
5. INTRODUCTION …
However, continuous use of fungicides alone leads to development
of fungicide resistant pathogen races and accumulation of toxic
residues in produce and environment (Chethana et al., 2015;
Ahmed et al., 2018).
Understanding of pathogen variability in a population on the bases
of CMP phenotypes may be helpful to develop successful crop
disease management strategies (Priya et al., 2018).
In spite of the importance of PB in Ethiopia, no study has been
conducted to characterize the CMP variability of the pathogen
isolates on onion.
The objective of this study was to determine the CMP variation
among A. porri isolates of onion PB from central and eastern
Ethiopia.
6. 2. MATERIALS AND METHODS
2.1. Description of the Study Areas
Collection of PB infected onion leaf samples were made in seven
districts of central and eastern Ethiopia (Fig. 1) on smallholder
farmers’ fields during the 2016 main cropping season.
The districts are located in the East Shewa and East Hararghe Zones
of Oromia Region, and lies between 07°58´ and 09°26’ N latitudes,
038°43’ and 041°57´ E longitudes, with altitudes ranging from 1543
to 1997 m above sea level.
The mean annual temperature and rainfall of the study areas are in
the range of 17 to 22 oC and 600 to 1700 mm, respectively.
7. MATERIALS AND METHODS …
Figure 1. Map of central and eastern Ethiopia showing the onion purple blotch survey districts in East
Shewa and East Hararghe Zones of Oromia Region during the 2016 main cropping season.
8. 2. MATERIALS AND METHODS …
2.2. Sampling Process and Sample Size
In each of the sampling districts, onion fields were randomly taken
at intervals of 1-2 km along the main roads.
Within each of the selected fields, 3-5 onion leaf samples exhibiting
typical PB symptoms (Fig. 2A) were collected at 10 m intervals
along the diagonals of the field.
A total of 70 samples were collected from 70 different onion fields
and taken to Plant Protection Laboratory, HU, for isolation and
identification of the pathogen.
9. 2.3. Isolation, Identification and Maintenance of the Pathogen
Tissue plating method was used to isolate the pathogen from the
infected leaf samples.
Fungal colonies developed from tissue pieces (Fig. 2B) were
purified by transferring hyphal-tip sample to fresh PDA plates and
incubated at 25 oC for 7 days.
Identification of the fungal isolates was done from pure cultures
based on microscopic examination (Fig. 3A) as described by Ellis
(1971).
Pure cultures (n = 18) confirmed to be A. porri were transferred to
fresh PDA medium slants in culture tubes (Fig. 3B) and incubated
at 25 ºC till full growth, then maintained in refrigerator at 4 °C for
further analysis.
MATERIALS AND METHODS …
10. MATERIALS AND METHODS …
A B
D
C
Figure 2. Isolation of A. porri from onion leaf samples on potato dextrose agar medium: A) Collected
leaf samples; B) Handling of the leaf tissue; C) Unpurified cultures; D) Purified cultures
11. MATERIALS AND METHODS …
A B
Figure 3. Microscopic structure (A) and maintained pure cultures (B) of A. porri isolates
from central and eastern Ethiopia on potato dextrose agar medium.
12. 2.4. Cultural and Morphological Characterization
The cultural (Fig. 4A) and morphological (Fig. 4B) characteristics
of the 18 isolates were studied on PDA medium.
A 6 mm mycelia-disc was taken from a week-old actively-growing
pure culture of each isolate and placed in inverted position in the
centre of a 90 mm Petriplate containing 20 mL of PDA medium.
Three replications were maintained for each isolate, in a CRD and
then incubated at 25 oC.
Starting from 2-days after incubation, the colony diameter of each
isolate was measured at 24 hr interval as an average of two
perpendicular criss-cross diameters until full growth occurred on
the Petriplate.
MATERIALS AND METHODS …
13. Colony characters, such as color, texture, margin and zonation of
all the isolates, were recorded by visual observation on the culture
medium 10 days after incubation as described by Chowdappa et al.
(2012).
Fifteen-day-old pure cultures of all the isolates were studied for
morphological variations in size and septation of conidia.
The conidial size of each isolate was determined by measuring the
length and the width of 30 randomly taken conidia using ocular
micrometer on a stage micrometer calibrated microscope.
Number of transverse and longitudinal septa in the conidia were
counted for the respective isolate.
MATERIALS AND METHODS …
14. MATERIALS AND METHODS …
Figure 4. Tests for cultural (A) and morphological (B) characterization of A. porri isolates
from central and eastern Ethiopia on potato dextrose agar medium
A B
15. 2.5. Pathological Characterization
Pathogenicity test of all the isolates was conducted on onion
seedlings of the susceptible Adama-Red variety grown in 20 cm
pots containing a mixture of soil, sand and FYM at 2:1:1 ratio.
A fresh conidial suspension (5 x 104 mL-1) was prepared from a 10
day-old pure culture of each isolate using SDW (Fig. 5A) and
inoculated by spraying on 60-day-old seedlings (two per pot) at
five to six leaf growth stage.
Non-inoculated control seedlings (Fig. 5D) were treated with an
equal volume of SDW for comparison.
MATERIALS AND METHODS …
16. Immediately after inoculation, the seedlings were covered with
polyethylene bags (Fig. 5B) to maintain high relative humidity.
The treatments were arranged in a CRD in three replications in
glasshouse.
Disease severity of each isolate was assessed every three-days
begging from six-days after inoculation.
Severity was scored on all leaves of the two seedlings in each pot
using the 0 to 5 rating scales described by Sharma (1986).
The severity scores were converted into percentage severity index
(PSI) for analysis according to the following formula (Wheeler,
1969).
MATERIALS AND METHODS …
17. The AUDPC of each isolate was calculated from PSI data assessed
at different days after inoculation using the formula of Madden et
al. (2007):
Where, n is the total number of disease assessments, ti is the time
of the ith assessment in days from the first assessment date and xi is
the PSI of PB at the ith assessment.
The DPR of each isolate was obtained from regression of PSI data
fit to logistic model, ln (x/1-x) (Van der Plank, 1963) with dates of
assessments, where x is PB severity index in proportion.
The slope of the regression line estimated the DPR.
MATERIALS AND METHODS …
18. MATERIALS AND METHODS …
Figure 5. Testing for pathological characterization of A. porri isolates from central and eastern Ethiopia on
seedlings of onion variety Adama-Red grown in pots under glasshouse conditions: A) Overall view of conidial
suspensions; B) Overall view of experimental pots; C) Inoculated seedlings; D) Control seedlings
A B
C D
19. MATERIALS AND METHODS …
2.6. Data Analysis
Data for cultural, morphological and pathogenic characters of the
isolates were subjected to ANOVA.
The treatment means were separated using DMRT at 5%
significance level (Gomez and Gomez, 1984).
Regression analysis of diameters of colony growth against time
after incubations was performed and the slopes were used as
measures (mm per day) of colony growth rates for each isolate.
Both analysis of variance and regression were done using GLM
Procedure of the SAS software version 9.1 (SAS Institute, 2003).
21. RESULTS AND DISCUSSION …
Figure 6. Variability in colony characteristics of A. porri isolates from central and eastern Ethiopia on potato dextrose
agar medium 10 days after incubation at 25 oC
Dark-gray Whitish-gray
Cottony texture
Velvety texture
Irregular margin Circular margin
Concentric zonation No zonation
Reddish-brown Dark-gray
3.1.2. Other colony characters
24. RESULTS AND DISCUSSION …
The cultural, morphological and pathological characters observed
on the 18 A. porri isolates were not according to the geographical
locations of the isolates.
The variations occurring within the pathogen population might
have been attributed to strong selection pressure resulting from
widespread use of mono-cultured crops with little to no genetic
diversity and indiscriminate application of synthetic fungicides for
long period (Weber and Halterman, 2012; Sofi et al., 2013).
It has also been speculated that the dissemination of spores by
biotic and abiotic factors may contribute to the variability among
isolates examined (Chethana et al., 2018).
25. CONCLUSION AND RECOMMENDATION
The results of the present study indicated that the A. porri isolates
of onion PB from central and eastern Ethiopia were highly variable
in their CMP characteristics.
Based on these results, extensive variability studies with inclusion
of molecular characterization are recommended to:
Recognize the overall nature of the Ethiopian A. porri isolates; and
Focus efforts in developing effective and sustainable onion PB
management options.
