Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.
Trang 1SUMMARY OF DOCTORAL THESIS
SPECIALITY: PLANT PROTECTION
Speciality ID: 62620112
DANG NGUYET QUE
RESEARCH AND APPLICATION OF ACTINOMYCETES STRAINS FOR THE MANAGEMENT OF RICE BLAST DISEASE
CAUSED BY Pyricularia oryzae Cav
IN THE SALT-AFFECTED SOILS AREAS
Trang 2THE THESIS WAS COMPLETED AT
CAN THO UNIVERSITY
Scientific supervisors 1: PGS.TS Le Minh Tuong
Scientific supervisors 2: PGS.TS Tran Thi Thu Thuy
Reviewer 1: Assoc Prof, Ph.D Pham Van Du
Reviewer 2: Assoc Prof, Ph.D Nguyen Thi Thu Nga
The thesis is defended in front of the Doctoral Thesis Examination Council at Can Tho University
Time: 8:00 am
Date: September 3th, 2022
Further information of the thesis could be found at:
1 Learning Resource Center of Can Tho University
2 National library of Vietnam
Trang 31 Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy,
2019 Determination of characterictic of actinomycetes
isolates on Pyricularia oryzae causing rice blast disease on salt afected soils Science and Technology Journal of
Agriculture and Rural Development Ministry of
Agriculture and Rural Development, Vietnam May, 2019, 81-86
2 Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy,
2019 Identification of actinomycetes as promissing biocontrol against rice blast disease in salt affected soils
Journal of Vietnam Agriculture Science and Technology
Vietnam Academy of Agricultural Sciences Vol 6
(103)/2019, 125-129
3 Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy,
2021 Determination biocontrol of actinomycetes isolates
on rice blast disease on salt affected soils in the net house condition Đánh giá khả năng phòng trị bệnh đạo ôn hại lúa canh tác vùng đất nhiễm mặn của một số chủng xạ
khuẩn trong điều kiện nhà lưới Journal of Plant
Protection Vol 5 (298)/2021, 21-27
Trang 41
Chapter 1: INTRODUCTION
1.1 The urgency of thesis
Blast disease caused by the fungus Pyricularia oryzae (P oryzae) is one
of the three major diseases that significantly affect the yield and quality of
rice worldwide (Dean et al., 2012; Gao et al., 2019; Chen et al., 2021) Because the fungus P oryzae attacks all parts of the rice plant, it causes
losses of up to hundreds of millions of tons of rice each year (Wang, 2009)
In Bac Lieu, rice blasts caused widespread damage in all seasons with varying degrees of severity Especially in salt-affected rice-shrimp fields, blast disease occurs relatively early and spreads quickly if not sprayed in time Meanwhile, chemical drug use also affects the giant freshwater shrimp intercropping in rice fields and the next shrimp rotation crop To maintain and develop an effective and sustainable rice-shrimp farming model and to adapt to climate change towards safe rice and shrimp production that meets export standards, it is necessary to research and find out biological solutions These must effectively manage rice blast disease, progressing to developing
an integrated rice blast disease process on saline soil in the rice-shrimp farming system
Regarding biological measures, the past has shown that the study of the application of actinomycetes is considered a promising solution and has great potential to replace fungicide to prevent blast disease towards safe agriculture and sustainability Many valuable scientific works on the application of actinomycetes to control rice blast disease have been published
in national and international journals (Tuong & Em, 2014; Khalil et al., 2014; Lan and ctv., 2015, 2016; Awla et al., 2016, 2017; Law et al., 2017; Gao et al., 2019) However, most of the studies about actinomycetes were conducted
in the laboratory Some studies were carried out in net houses and the field However, there is no research on the application of actinomycetes to prevent and treat rice blast disease in saline soil Therefore, the thesis "Research and application of actinomycetes strains for the management of rice blast disease
caused by Pyricularia oryzae in the salt-affected soils area" is essential
1.2 Research objectives and requirements
(i) Selecting salt-tolerant actinomycete strains’s ability against pathogen
P oryzae in the laboratory
Trang 5(ii) Study on the antagonistic ability of actinomycetes through the ability
to secrete chitinase and β-1,3-glucanase enzymes
(iii) Identifying the species of promising actinomycetes strains
(iv) Evaluation capable of managing rice blast disease in the net house conditions
(v) Evaluating the effectiveness of managing rice blast disease of promising salt-tolerant actinomycetes strains in field conditions
1.3 Scientific and practical significance of the thesis
The topic has high scientific and practical significance; the project's results are to find actinomycetes strains capable of managing rice blast
disease caused by the fungus P oryzae on salt-affected soils The results of
this study serve as a premise for the following studies to find probiotics from salt-tolerant actinomycetes and apply them to the integrated management of rice blast disease on salt-affected soils Both are effectively and environmentally friendly, contributing to developing a sustainable rice-shrimp farming model
1.4 The new results of the dissertation
- Three salt-tolerant actinomycetes isolates, including S06-MBL, MBL and S17-MBL, have been identified to prevent rice blast disease under
S09-net house conditions Two strains of Streptomyces bikinensis (S09-MBL) and Streptomyces lavendulate (S17-MBL) could prevent rice blast disease in
saline soil field conditions in Bac Lieu
- The biocontrol ability of 3 actinomycetes S06-MBL, S09-MBL and
S17-MBL against rice blast disease caused by P oryzae was related to the
ability to secrete chitinase and β-1,3-glucanase enzymes
- Species of 3 actinomycete strains have been identified: S06-MBL is
Streptomyces fradiae, S09-MBL is Streptomyces bikiniensis, and S17-MBL
is Streptomyces lavendulae These species differ from previous actinomycete
species studied in managing blast disease in Mekong Delta
Chapter 2: RESEARCH METHODS 2.1 Time and place of the thesis
- Time: from October 2015 to February 2019
Trang 63
- Place: the laboratory and the nethouse of the Plant Protection
Department, Can Tho University; Bạc Liêu University và saline rice fields
of farmers in Hồng Dân district, Bạc Liêu province
2.2 Experimental Materials: fungus P oryzae causing rice blast disease
and actinomycetes
2.3 Experimental methods
2.3.1 Content 1: Collect, isolate and evaluate the pathogenicity of P
oryzae strains causing rice blast disease
- Collect and isolate P oryzae causing rice blast disease in the Mekong
Delta salt-affected soils area: Investigate and collect rice samples with blast disease in, rice-shrimp fields in Bac Lieu, Ca Mau, Kien Giang and Hau
Giang provinces; Isolation and culture of P oryzae fungus were carried out
at the Plant Diseases laboratory of Plant Protection Department, Can Tho
University according to the method of Burgess et al (2009) Identification
of P oryzae fungus was based on morphological characteristics of fungal strains compared with those of P oryzae described by Agrios (2005) and Zhang et al (2014)
- Evaluation of pathogenicity of P oryzae isolates under the nethouse
condition: In the period of 18 days after planting, the rice plants were
artificially infected Spraying a suspension of P oryzae fungi spores evenly
wet the foliage with a spore density of 105 spores/ml in the fantastic afternoon; each spraying pot is 10 ml Pots of rice after artificial inoculation were placed in the incubation room and covered in darkness for 24 hours (the temperature at 260C and humidity at 96-98%) After that, the pot of rice was transferred to the net house with a shading system to reduce 50% of light conditions, misted to create the right humidity for fungal growth (incubation net house) The recorded indicators were the ratio of infected leaf area and disease index at the time of 4, 6, 8, 10, 12 and 14 days after artificial
infection
2.3.2 Content 2: Collect, isolate and evaluate the antagonistic ability of
actinomycete isolates derived from rice saline soil against the fungus P
oryzae strains causing rice blast disease under laboratory condition
- Collecting and isolating actinomycetes from rice saline soil: Soil samples were taken around the root zone of rice, 10-25 cm from the surface
Trang 7and isolated actinomycetes at the laboratory of the Department of Plant Protection Can Tho University according to the method of Hsu and Lockwood (1975)
- Evaluation of the antagonistic ability of actinomycetes against P oryzae causing rice blast disease under laboratory condition: Put the sterile
absorbent paper rolls into the actinomycete suspension solution (108cfu/ml), shook the solution well Use specialized clamps to put the absorbent paper
on the wall, let it dry Place the blotting paper (Ø = 5 mm) impregnated with
actinomycetes opposite the P oryzae and 1 cm from the sides of the plate In
the control treatment, the paper strips impregnated with actinomycetes were replaced with ones impregnated with sterile distilled water The follow-up indicator was to measure the radiuses of inhibition zones and calculate the antagonistic efficiency at the time of 3, 5, 7 and 9 days after inoculation (DAI)
- Investigation of the antagonistic ability of promising actinomycete
strains against P oryzae under the conditions of addition of NaCl salt:
perform the same experiment as above, except that the experimental medium
is supplemented with NaCl salt (2g/l) The follow-up indicator was to measure the radiuses of inhibition zones and calculate the antagonistic efficiency at the time of 3, 5, 7, 9 and 11 days after inoculation (DAI)
- Investigation of the ability to inhibit the growth of spores of P
oryzae of promising actinomycetes: Treatment of actinomycetes: Add 500
µl of actinomycete suspension (108 cfu/ml) + 500 µl of fungal suspension (5x105 spores/ml) into eppendorft tubes and keep at 28oC, each treatment with 4 times of replicationt Control treatment: Put 500 µl of sterile distilled water + 500 µl of fungal spore suspension (5x105 spores/ml) into eppendorft tube and keep at 25oC Recording indicators: The percentage of spores sprouting at the time of 6, 12 and 24 hours after treatment (HAT) by
observation under the microscope
2.3.3 Content 3: Investigate a number of mechanisms related to the antagonistic ability of promising actinomycetes
- Investigate the ability to secrete chitinase-degrading enzyme
chitinase of actinomycete strains on agar: The experiment was arranged
in a completely randomized design with one promising actinomycete strain
Trang 8randomized design with 5 replications and performed according to the
method of Dai et al (2011) and Ha (2012)
- Investigation of the ability to secrete β-1,3-glucanase enzyme to degrade β-1,3-glucan of actinomycete strains on agar: The experiment
was arranged in a completely randomized design with each treatment being
a promising actinomycete strain with 4 replicates The experiment was
carried out according to the method of Renwich et al., (1991)
- Determination of the secreted β-1,3-glucanase enzyme content of actinomycete strains: The experiment was arranged in a completely
randomized design with one factor, the number of treatments was the number
of actinomycete strains tested with 4 replicates and was performed according
to the method of Renwich et al (1991)
2.3.4 Content 4: Identify promising actinomycetes species by observation biological characteristics methods and molecular biology methods
- Identification of actinomycetes based on culture, morphological and biochemical characteristics
* Observation of the color of biogas, substrate fibers and soluble pigments which were conducted according to the method of Shirling and Gottlieb (1966)
* Observation of spore stalk and spore surface shape which were
conducted according to the method of Tresner et al (1961)
* Protease secretion ability of actinomycete strains: was performed according to the method of Mitra and Chakrabartty (2005)
* The ability to secrete lipase enzyme of actinomycete strains: was
performed according to the method of Ertuğrul et al., (2007)
* The ability to secrete amylase enzyme of actinomycete strains: was performed according to the method of Santos (2012)
* The melanin pigment formation of actinomycete strains: was performed according to the method of Shirling and Gottlieb (1966)
Trang 9- Identification to species of actinomycete strains by molecular biology method
DNA extraction of actinomycetes was performed according to the
method of Weisburg et al., (1991) The primer pairs were used to amplify
16S-rRNA gene segments of actinomycete strains in the study were: 1492R:
27:5'-AGAGTTTGATCCTGGCTC-3' (Weisburg et al., 1991) Analytical samples
were sequenced on the ABI 3130XL system The results were anylized by using sequecing analysis 6.0 software and compared with the results on the gene bank to determine the name of actinomycetes
2.3.5 Content 5: Evaluation of the ability to control rice blast disease with promising actinomycetes strains under nethouse condition
- Location: the experiment was carried out at the nethouse of the Plant Protection Department, Bac Lieu University
- Experimental arrangement: The experiment was arranged in completely randomized design with 14 treatments (12 treatments using actinomycetes and 2 controls) with 4 replications
- Monitoring criteria: count the number of leaf blast lesions according
to Pinnschmidt et al (1993), measure length and width leaf, and calculate the
percentage of infected leaf area, disease index and disease suppression efficiency at the time of 4, 6, 8, 10, 12 and 14 days after artificial infection (DAAI)
2.3.6 Content 6: Evaluation of the ability to control rice blast disease with promising actinomycetes strains under the field condition
Field experiment was carried out in 2 seasons: 2017 season in farmer's fields in Ninh Thanh Loi commune, Hong Dan District, Bac Lieu province from September 2017 to December 2017 with an area of 2,200 m2, block layout completely random, 1 factor, 3 repetitions The 2018 season was carried out in Ninh Hoa Commune, Hong Dan District, Bac Lieu province from October 2018 to January 2019 with an area of 2,700 m2, arranged like the 2017 province, with 4 repetitions Each experimental unit has a size of
30 m2 (6 m x 5 m) Experimental units are arranged 0.5 m apart to limit the interaction when handling actinomycetes The experimental field is 5 m away
Trang 107
from the farmer's field in order to limit the influence of fertilizers and pesticides of next rice fields Experimental treatments as Table 3.5
ò Record disease parameters: Each experimental plot placed five
frames (40x50) cm fixed on two diagonal lines On each frame, the indicators
of leaf blast disease (27, 34, 41, 48 and 55 days after sowing) and blast disease were recorded collar blast (67, 74 and 81 days after sowing), then calculate the following indicators: (1) Disease rate; (2) Disease index; (3): Reduce disease severity
ò Record productivity targets
+ The number of panicle /m2: in each experimental plot of 30 m2, count all panicle in 5 plots to record the criteria (40x50 cm2), from which the number of panicle/m2 is calculated
+ The number fill grain/ panicle: randomly select 50 flowers in the target recording box (40x50 cm2) Count the number fill grain in each panicle, and then calculate the average number of fill grain for each of the 50 panicles + Actual yield (ton/ha): harvest all rice within a frame of 5m2 Weigh and measure grain moisture at the time of weighing, then convert to 14% moisture From there calculate the actual yield
Table 3.5 Treatments used in the experiment (Days after treatment: DAT)
agent
Handling measures
Processing time
Dosage/ density
suspension:
10 8 cfu/ml
* Seed coating: Just enough (12 H)
* Folia Spraying + Tillering stage:
25 lít/1000 m 2
+ Flowering stage:
40 lít/1000 m 2
Seed coating + Folia Spraying S17-MBL
Trang 11S09-MBL-A,20,40,60 NT16 20, 40 & 60 NSS * Fungicide
spraying: as recommended
by the manufacturer
- Beam 75WP;
- AmistarTop 325SC
2.4 Data processing
The data were processed by Microsoft Excel program; monitoring indicators were entered, and data were processed by Excel software Statistical calculation of the data by SPSS software; using Duncan's test to compare the difference at 5% significance level
Chapter 3: RESULTS AND DISCUSSION
3.1 CONTENT 1: COLLECTED AND ISOLATED THE FUNGUS P
oryzae, CAUSING RICE BLAST DISSEASE, EVALUATION OF
PATHOGENICITY OF P oryzae UNDER THE NET HOUSE
CONDITION
3.1.1 Collect disease samples, isolate and identify P oryzae fungal strains
causing rice blast disease
From October 2015 to December 2015, nine strains of the fungus P oryzae
causing rice blast disease were collected and isolated in saline-affected rice fields in four provinces of the Mekong Delta (Ca Mau, Bac Lieu, Kien Giang and Hau Giang) Disease samples were collected from rice-shrimp fields with an area of over 1,000
m2 and field water with salinity > 0.5‰ The distribution of fungal strains by sampling location is presented in Table 3.1
Table 3.1 List of Pyricularia oryzae were collected in saline rice fields in
04 provinces of the Mekong Delta
No of strains Symbols Location of collected samples Rice seeds
1 Po1-CM Khanh Binh –Tran Van Thoi – Ca Mau OM5451
2 Po2-CM Tran Hoi – Tran Van Thoi – Ca Mau OM6976
3 Po3-BL Ninh Hoa – Hong Dan – Bac Lieu OM11735
4 Po4-BL Ninh Quoi A – Hong Dan – Bac Lieu OM5451
5 Po5-BL Ninh Thanh Loi – Hong Dan – Bac Lieu OM7347
6 Po6-HG Hoa Luu –Vi Thanh – Hau Giang OM4900
7 Po7-HG Vinh Vien – Long My – Hau Giang OM5451
8 Po8-KG Tay Yen – An Bien – Kien Giang OM9921
9 Po9-KG Đong Thanh – An Minh – Kien Giang OM5451
Note: Po: Pyricularia oryzae
Trang 129
* Symptoms of disease in the field: the disease is like a swallow's
eye with two pointed ends The middle part is enlarged, around the border is brown, and the centre is grey-white The disease on the panicle blast-neck blast is light brown, causing the rice grain to be flat
* Morphological characteristics of isolated strains of P oryzae:
Colonies grow slowly on PDA medium, white when the mycelium is young and turn black-grey when the mycelium is mature when viewed from the underside The disc is black or dark grey in the centre, fades to the edge of the mushroom, and the mycelium is thin
* Spore shape and mycelium: Observing the spores under an optical
microscope, the spores are pear-shaped, enlarged at the base and gradually smaller towards the top, usually with two septa, colourless or pale green
3.1.2 The ability to cause rice blast disease of P oryzae strains under net
house conditions
The pathogenicity of nine strains fungus of P oryzae causing rice blast
disease under net house conditions was shown disease rate (Table 3.2) and disease index (Table 3.3)
Table 3.2 Rate of rice leaf blast disease area caused by 9 strains of
P.oryzae under nethouse condition over the survey periods
Fungal
strains
Rate of rice leaf blast disease area (%) over the survey periods
4 DAI 6 DAI 8 DAI 10 DAI 12 DAI 14 DAI
Po1-CM 2.11a 4,49a 7.65a 9.69a 15.36a 18.42aPo2-CM 1.63ab 3.12bc 4.24bc 6.98b 7.75c 8.95dPo3-BL 1.60ab 2.45cd 3.13cd 4.48cd 6.90c 7.35ePo4-BL 0.72cd 2.32d 2.65d 3.87cde 4.25d 5.12fPo5-BL 0.86cd 2.48cd 3.06d 4.69c 6.051c 6.44ePo6-HG 1.13bc 3.81ab 5.41b 7.78b 10.12b 10.93cPo7-HG 0.07d 1.92d 2.24d 2.87e 3.74d 3.95gPo8-KG 1.87a 3.36b 4.95b 6.62b 11.22b 15.12bPo9-KG 1.15 bc 1.91d 2.36d 3.25de 4.92d 6.04ef
Significance
level * * * * * *
Note: in the same column numbers followed by the same letters are not statistically significant
in Duncan's test Data are converted to for statistical analysis *: statistically significant difference at 5% level; Days after Inoculation (DAI)
5 , 0 +
x
Trang 13The results presented in Table 3.2 and Table 3.3 show that all nine
strains of P oryzae are capable of causing rice blast disease with typical
symptoms of rice blast disease and exhibiting the potential to cause damage with various degrees of damage The difference between disease rates and disease indexes among fungal strains Which the Po1-CM strain (collected
in Khanh Binh commune, Tran Van Thoi district, Ca Mau province) showed the highest pathogenicity through the highest disease rate and highest disease index out of nine investigated fungal strains Therefore, strain Po1-CM was selected as the source of blast fungus for subsequent experiments
Table 3.3 Index of rice blast disease caused by 9 strains of P.oryzae
under nethouse condition over the survey periods
Fungal
strains
Disease index (%) over time of survey
Note: in the same column numbers followed by the same letters are not statistically significant
in Duncan's test Data are converted to for statistical analysis *: statistically significant difference at 5% level; Days after Inoculation (DAI)
3.2 CONTENT 2: COLLECT, ISOLATE AND EVALUATE THE ANTAGONISTIC ABILITY OF ACTINOMYCETE ISOLATES
DERIVED FROM RICE SALINE SOIL AGAINST THE FUNGUS P
oryzae STRAINS CAUSING RICE BLAST DISEASE UNDER
LABORATORY CONDITION
3.2.1 Isolation results of actinomycetes
From October 2015 to December 2015, 126 actinomycete strains were collected and isolated from saline rice fields in 6 provinces of the Mekong Delta, including Bac Lieu (38 strains), Ca Mau (33 strains), Hau Giang (14 strains), Kien Giang (13 strains), Soc Trang (10 strains), and Tra Vinh (18
5 , 0 +
x
Trang 14Table 3.4 List of actinomycetes isolated from soils of saline rice fields in 04 provinces of the Mekong Delta
Symbol Location Total
S01-MHG;…;S06-MHG Hoa Luu - Vi Thanh - Hau Giang 6 S07-MHG;…;S14-MHG Vinh Vien - Long My - Hau Giang 8 S01-MKG;…;S06-MKG Tay Yen -An Bien - Kien Giang 6 S07-MKG;…;S13-MKG Đong Thanh - An Minh - Kien Giang 7 S01-MBL;…;S09-MBL Ninh Thanh Loi - Hong Dan - Bac Lieu 11 S10-MBL;…;S20-MBL Ninh Quoi A - Hong Dan - Bac Lieu 15 S21-MBL;…;S29-MBL Ninh Hoa - Hong Dan - Bac Lieu 12 S01-MCM;…;S11-MCM Khanh Binh -Tran Van Thoi - Ca Mau 11 S12-MCM;…;S19-MCM Tran Hoi - Tran Van Thoi - Ca Mau 8 S20-MCM;…;S24-MCM Khanh An - U Minh - Ca Mau 5 S29-MCM;…;S37-MCM Tran Van Thoi - Tran Van Thoi - Ca Mau 9 S01-MST;…;S10-MST TT Tran De - Tran De - Soc Trang 10 S01-MTV;…;S10-MTV TT Tra Cu - Tra Cu -Tra Vinh 7 S11-MTV;…;S21-MTV Tap Son - Tra Cu - Tra Vinh 11
antagonism against P oryzae cause blast disease Results of evaluation of the
antagonistic ability of 23 promising actinomycete strains through the parameters of sterility radius (Table 3.5) and antagonistic efficiency (Table 3.6)
Trang 15The results of Table 3.5 and Table 3.6 show that there are 7 actinomycete strains S17-MBL, S31-MCM, S06-MBL, S09-MBL, S03-
MST, S18-MCM and S09-MTV which are resistant to P oryzae through a
high sterility ring radius and high antagonistic performance up to 9 days after experimental setup In which, three strains S17-MBL, S09-MBL and S06-MBL showed the highest antagonistic ability
Table 3.5 Antagonistic ability of 23 actinomycetes against P oryzae over the
survey times
Treatment Radiuses of inhibition zones over times (mm)
3 DAT 5 DAT 7 DAT 9 DAT
Note: in the same column numbers followed by the same letters are not statistically significant
in Duncan's test *: statistically significant difference at 5% level; Days after Testing (DAT)