Rice (
Rice (
In Morocco, the total area of rice crop reached 7,973 hectares with a production of 64,598 tons (FAOSTAT,
Rice crop is facing several threats, such as diseases caused by fungi, bacteria, and viruses, which affect grain production and quality (Ou,
Many studies have reported the presence of
The optimum temperature for
According to Lage (
Benkirane et al. (
The leaves of
The study was carried out during 2014–2016 in the Moroccan North-West (Gharb), including the Souk Tlet region (latitude 34°36′6.153″ N, longitude 6°10′27.687″ W) and the Merjas of Kenitra (latitude 34°30′54.838″ N, longitude 5°52′59.243″ W).
Infected
Bipolaris spores were then single-spored with a capillary tube, placed on agar medium (agar-agar: 15 g, distilled water: 1,000 mL), and subsequently transferred using a sterilized needle to the surface of a rice flour-based medium (rice flour: 14 g, agar-agar: 15 g, yeast extract: 4 g, distilled water: 1,000 mL). Four isolates of
The seeds of five rice varieties, namely, Elio, Taibonet, Arpa, Eurano, and Cererrer, were disinfected by immersion in 5% hypochlorite sodium solution for 2 min, followed by three rinses with sterile distilled water. They were then dried on a sterile filter paper and pre-germinated on Petri dishes 90 mm in diameter containing sterile cotton soaked with sterile distilled water. After 72 hr of incubation in the dark at 28 °C, the obtained plantlets were transplanted into pots filled with Mamora soil and then placed in a greenhouse. The seedlings were watered with tap water until they reached the stage required for inoculation, that is, when they had grown four to five true leaves.
Inoculum was prepared by independently growing each of the four isolates of
Afterward, the fungal suspension was filtered through a fine mesh cloth to separate the spores from the mycelial fragments. The concentration of the conidia was adjusted to 105 conidia/mL using Malassez slide by adding sterile distilled water supplemented with a drop of Tween 20 and 0.5% gelatin.
Healthy leaves were soaked in the conidial suspension of each
Healthy leaves were placed in 9-cm-diameter Petri dishes containing glass beads in the presence of sterile distilled water. They were then inoculated with the mycelial plug (5 mm in diameter) of each isolate: one mycelial plug was placed on the central part of leaf segment, and another mycelial plug was deposited near the leaf apex. Noninoculated leaves (treated with water agar discs only) served as a control. In both detached leaf assays, controls were treated with sterile distilled water containing 0.01% Tween 20. Inoculated and control leaves were kept at ambient temperature under a natural light/dark cycle in the laboratory.
Rice seedlings at the stage of five to six leaves were inoculated by foliar spraying of 60 mL of spore suspension at 105 conidia/mL concentration using a hand compressed spray. Control plants were sprayed with sterile distilled water containing Tween 20 and gelatin. After spraying, all plants were covered with a black plastic bag and placed in a greenhouse for the development of symptoms. The plastic bag was used for the first 48 hr to ensure 100% relative humidity during conidium germination and fungal penetration. The replication consisted of three pots with three plants per pot for each rice variety. The experiment was repeated three times.
The degree of leaf necrosis was evaluated on the seventh day after inoculation for the four rice varieties artificially inoculated with conidial suspension, and at 2 days later for the rice inoculated with mycelial plugs. Disease severity was assessed as the proportion of infected leaf area on randomly selected rice plants. It was estimated using a disease rating scale of 1–9, as suggested by Notteghem et al. (
Disease rating scale (Notteghem et al.,
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0 | 0.00 |
1 | 0.05 |
2 | 0.50 |
3 | 1.50 |
4 | 3.50 |
5 | 7.50 |
6 | 17.50 |
7 | 37.50 |
8 | 62.50 |
9 | 87.50 |
For analysis, the severity scale was converted into Percentage Severity Index (IS) using the following formula:
where
Sporulation was determined according to the technique of Hill and Nelson (
At 7 days after inoculation, rice leaves that showed lesions were removed, cut into four–five fragments, and then placed in Petri dishes containing filter paper moistened with sterile distilled water (one sheet per dish). The dishes were placed under continuous fluorescent light for 72 hr at 28 °C.
Each leaf segment was then collected in a test tube containing 1 mL of sterile distilled water. After that, the tubes were agitated in a vortex mixer for 2 min to detach the conidia from the mycelium. The conidia of the pathogen were counted using a Malassez slide under an optical microscope, with 10 counting for each sample.
The statistical analysis of data was conducted using analysis of variance after transformation of percentages to arcsin √P. Comparison between means was performed using the least significant difference (LSD) test at
On the rice flour-based medium, fungus isolates formed fluffy and cottony aerial mycelia. The colony was gray to dark greenish gray in color (
(
The size of the lesions induced by
The degree of lesion on
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Lesion size | 14.4 ab | 12.3 b | 15.8 a | 13.8 ab |
Two results on the same line are not significantly different at a 5% level (p.p.d.s test) if assigned with a common letter.
Symptoms developed on detached leaves of
The degree of lesion on
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Lesion size | 9.2 b | 16.7 a | 11.0 b | 20.8 a |
Two results on the same line are not significantly different at a 5% level (p.p.d.s test) if assigned with a common letter.
Symptoms of reddish-brown lesions developed on the leaves of
Through inoculation with either mycelial discs or spore suspensions,
Sporulation of
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Number of spores | 0.76 a | 0.83 a | 0.13 a | 0.2 a |
Two results on the same line are not significantly different at a 5% level (p.p.d.s test) if assigned with a common letter.
Sporulation of
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Number of spores | 0.43 a | 0.1 a | 0.6 a | 0.2 a |
Two results on the same line are not significantly different at a 5% level (p.p.d.s test) if assigned with a common letter.
After 7 days of inoculation, all the rice varieties tested displayed high sensitivity towards the four isolates of
The symptoms of rice leaves artificially inoculated with spore suspensions of
As shown in
The Cererrer and Elio varieties were more sensitive to the Hor2 isolate, with a severity index of 90.74% and 81.47%, respectively, which was superior to those of the Taibonet, Eurano, and Arpa varieties, which did not exceed 74.99%. The sensitivity of the Cererrer variety to the Hor3 isolate was marked by a high severity index of 90.73%, followed by the Elio, Eurano, Taibonet, and Arpa varieties, with respective indexes of 79.63%, 74.07%, 69.44%, and 62.03%. The Cererrer and Elio varieties were the most sensitive to the Hor4 isolate, showing the highest severity index (92.59%). However, the severity varied between 87.03% and 59.26% for the varieties Taibonet, Eurano, and Arpa.
Mean percentages of disease severity induced by
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Hor1 | 79.62 a | 85.18 a | 35.18 b | 79.63 a | 90.73 a |
Hor2 | 62.96 b | 74.99 a | 48.14 ab | 90.74 a | 81.47 a |
Hor3 | 74.07 ab | 69.44 a | 62.03 a | 90.73 a | 79.63 a |
Hor4 | 84.25 a | 87.03 a | 59.26 a | 92.59 a | 92.59 a |
Two results on the same column do not differ significantly at a 5% level (p.p.d.s test) if assigned with a common letter.
The Hor3 isolate was highly pathogenic to the Cererrer variety (90.73%) and less pathogenic on Elio and Eurano varieties, with respective disease indexes of 79.63% and 74.07%. The severity indexes for the Hor2 isolate were the highest in the Cererrer and Elio rice varieties (90.74% and 81.74%, respectively), but did not exceed 74.99% in the Eurano, Taibonet, and Arpa varieties. Hor1 was more pathogenic to the Elio and Taibonet varieties, with respective indexes of 90.73% and 85.18%.
Mean percentages of disease severity induced by
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Eurano | 79.62 a | 62.96 bc | 74.07 ab | 84.25 a |
Taibonet | 85.18 a | 74.99 ab | 69.44b | 87.03 a |
Arpa | 35.18 b | 48.14 c | 62.03b | 59.26 b |
Cererrer | 79.63 a | 90.74 a | 90.73 a | 92.59 a |
Elio | 90.73 a | 81.47 ab | 79.63 ab | 92.59 a |
Two results on the same column do not differ significantly at a 5% level (p.p.d.s test) if assigned with a common letter.
The sporulation ability of
The highest number of spores of the Hor2 isolate was 1.06 × 105 and 1.03 × 105 spores/cm2 in the varieties Elio and Arpa, respectively, followed by that in the Cererrer and Eurano varieties, with 0.23 × 105 and 0.13 × 105 spores/cm2, respectively. The Taibonet variety showed the lowest spore density, equal to 0.06 × 105 spores/cm2 (
The sporulation intensity of the Hor3 isolate on the leaves of the Arpa and Cererrer varieties was high (1.43 × 105 and 1.33 × 105 spores/cm2, respectively). In comparison, on the leaves of the Eurano and Elio varieties, the spore number was 0.7 × 105 and 0.63 × 105 spores/cm2, respectively. Moreover, no spore was found on the leaves of the Taibonet variety (
The maximum spore production of the Hor4 isolate was observed on the leaves of the Eurano variety, with 2.26 × 105 spores/cm2. However, no spore was found in the other varieties (Taibonet, Arpa, Cererrer, and Elio).
Sporulation of the four isolates of
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Hor1 | 1.16 ab | 0.16 a | 0.26 b | 0.06 b | 0.06 b |
Hor2 | 0.13 b | 0.06 ab | 1.03 a | 0.23 b | 1.06 a |
Hor3 | 0.70 b | 0.00 b | 1.43 a | 1.33 a | 0.63 c |
Hor4 | 2.26 a | 0.00 b | 0.00 b | 0.00 b | 0.00 b |
Two results on the same column do not differ significantly at a 5% level (p.p.d.s test) if assigned with a common letter.
In the pathogenicity tests, Moroccan isolates of
Regarding the sporulation ability, the tested isolates of