الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Oranges (Citrus sinensis L.) are the most important fruit crop grown in Egypt, with a cultivated area of more than 129860 hectares, a total production of more than 3 million tons, and an exportation value of more than 714 million US$ in 2021.
However, Navel orange fruit is susceptible to infection by pathogenic fungi between harvest and consumption. The most common and serious postharvest disease of citrus in all production areas of the world is green mould caused by Penicillium digitatum (Pers.: Fr.) Sacc. especially in production areas that have low summer rainfall.
This study aimed to isolate the pathogen of citrus green mould from different regions in Egypt and identify these isolates by morphological and molecular methods. Meantime, throwing light on the control of green mould disease by several measures. The obtained results could be summarized as follows:
1- Nine Penicillium sp. isolates were isolated from naturally infected citrus fruits with green mould symptoms collected from orchards and markets located in 9 different areas of 5 different governorates in Egypt.
2- The nine isolates of Penicillium sp. were tested as a pathogenic agent for causing green mould on the Navel orange fruit, and the inoculated fruit developed symptoms identical to the green mould although their differences in disease virulence.
3- Macro-morphology identification of the Penicillium isolates was carried out on two standard media (malt extract agar and Czapek-yeast agar) while micromorphology was achieved by observed microscopic features i.e., stipes, branches, metulae, phialides, conidia measurements, and type of conidiophores.
4- Molecular identification of the nine Penicillium isolates was done by amplification of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers, and nucleotide sequences DNA of the ITS region of the Penicillium isolates.
5- Morphological identification and phylogenetic analysis of ITS sequences revealed that all nine isolates belong to a single species i.e., P. digitatum.
6. The sequences were submitted to respective GenBank nucleotide databases with accession No. OR198852, OR198853, OR198854, OR198855, OR198856, OR198857, OR198858, OR198859 and OR198860.
7. Sixty-eight yeast isolates were isolated and purified from the surface of citrus fruits. The citrus fruits were collected from orchards in 6 different areas of 5 different governorates. The primary screening of yeast isolates was done according to their capability to control green mould on Navel orange fruit.
8. Of 68 yeast isolates, only three isolates (LaK06, LbK08, and LbK13) have completely protected wounded navel orange fruit from infection by P. digitatum. Meantime, the yeast isolates LaK04, KK01, MG11, and MB06 reduced the disease development by more than 96%.
9. All seven promising isolates selected for secondary screening at a high dose (2x108 cfu /ml) prevented the development of green mould on wounded Navel orange fruit. While the isolates LbK08, LbK13, and LaK06 at the concentration of 6.6x107 cfu/ml completely inhibited the development of green mould on wounds of fruit. In addition, complete protection from green mould infection on wounded orange treated by the yeast isolate LaK06 at the lowest tested concentration of washed cells of a yeast isolate, i.e., 5x107 cfu /ml against P. digitatum 1x104 conidia /ml was achieved.
10- The antifungal activity of ten ethanolic extracts was tested on spore germination of P. digitatum and against orange green mould. Harmal and cinnamon extract showed the best results, completely inhibiting the conidial germination of the fungus at all tested concentrations. Meantime, pomegranate peel extract had good efficacy in reducing spore germination by more than 80% and 98% at concentrations of 1% and 2% respectively.
11. The pomegranate peel ethanolic extract exhibited the highest significant antifungal activity at all tested concentrations (2%, 4%, and 8%), at a concentration of 8% it completely protected wounded navel orange fruit from infection by P. digitatum. Meanwhile, the ethanolic extracts of cinnamon exhibited a significant antifungal activity, 54.3% and 79.8% inhibition of the infected area at a concentration of 4%, and 8% respectively. Meantime, there were no significant differences in the infected area (cm2) between treatments by ethanolic extracts of cinnamon and lantana at all tested concentrations. However, the ethanolic extracts of thyme, ginger, hot chili, and harmal cannot control the green mould of orange at all tested concentrations.
12. The antifungal activity of three aqueous extracts (Pomegranate, Lantana, and Cinnamon) was tested on spore germination of P. digitatum. The cinnamon aqueous extract completely inhibited the conidial germination of the fungus at 2% and 4% concentrations. Meanwhile, pomegranate peel aqueous extract showed complete inhibition of the fungus spore germination at the higher used concentration of 4%.
13. The aqueous pomegranate peel extract exhibited significant antifungal activity at all tested concentrations, and at a concentration of 8%, it completely protected wounded navel orange fruit from infection by P. digitatum. However, the aqueous extract of Lantana leaves did not have an inhibitory effect at all tested concentrations.
14. The antifungal activity of ten salts at three concentrations (0.5%, 1.0%, and 1.5%) was tested on spore germination of P. digitatum. Complete inhibition of spore germination of the fungus was achieved at all tested concentrations of potassium bicarbonate, potassium carbonate, sodium bicarbonate, and sodium tetraborate. Meantime, the salts sodium carbonate, aluminum potassium sulfate, and potassium sorbate blocked the spore germination of P. digitatum at concentrations of 1.0% and 1.5%.
15. The antifungal activity of ten salts at three concentrations (1%, 2%, and 3%) was tested on the green mould of orange. No lesions were developed on the fruit surface treated with sodium tetraborate at all tested concentrations. However, at the highest tested concentration of 3%, sodium benzoate, potassium sorbate, and sodium bicarbonate completely inhibited rot formation. Meanwhile, at a concentration of 3%, the salts aluminum potassium sulfate and sodium carbonate had a significant antifungal activity of more than 99%. In addition, the salts potassium bicarbonate and potassium carbonate had a significant antifungal activity of more than 97%.
16. The fungicidal activity of eight fungicides at eight concentrations (0.25, 0.5, 1, 5, 10, 25, 50, and 100 ppm) was tested on spore germination and mycelial growth of P. digitatum. The fungicides imazalil, fludioxonil, and prochloraz completely prevented the conidial germination of the fungus at all tested concentrations. Apart from 0.25 ppm concentration, the fungicides difenoconazole, epoxiconazole, and pyraclostrobin also blocked the conidial germination at all rest-tested concentrations. On the other hand, the fungicide pyrimethanil reduced spore germination by more than 58.1%, and 97.3%, at 0.5 ppm and 1 ppm concentrations respectively, and no germinated conidia were detected at 5 ppm concentration or higher.
17. The fungicidal activity on the mycelial growth of P. digitatum was carried out on Potato dextrose agar medium amended with yeast extract (4g/L). Mycelial growth was not observed at all used concentrations of fungicides imazalil, prochloraz, fludioxonil, and difenoconazole. However, apart from 0.25 ppm concentration, the fungicide epoxiconazole stopped the mycelial growth at all rest tested concentrations.
18. Eight fungicides at different concentrations were used individually as a protective or curative application to study their capability to control the green mould of orange. Wounded Navel orange fruits were immersed for 45 seconds in the selected fungicide concentration. Wounded fruits of control were immersed with SDW. After 15 hr (protective) or before 15 hr (curative) from fungicide treatment, the fruit’s wounds were inoculated with 25 l 5x105 conidia/ml of P. digitatum. Then the fruits were stored at 212C and RH ≥ 93% for 21 days.
19.1 After 7 days no lesions were developed on the fruit surface treated protectively with the fungicides difenoconazole, epoxiconazole, fludioxonil, imazalil, and pyrimethanil at all tested concentrations. Meanwhile, the fungicides Prochloraz at a concentration of 500 ppm, and pyraclostrobin at a concentration of 800 ppm completely inhibited rot formation.
19.2 After 14 days, difenoconazole, epoxiconazole, and imazalil, as a protective application at 300 ppm and higher concentrations completely inhibited rot formation. Meanwhile, no lesions were developed on the fruit surface treated with the fungicides Prochloraz at a concentration of 500 ppm, pyrimethanil at a concentration of 750 ppm, Fludioxonil at a concentration of 900 ppm, and Pyraclostrobin at a concentration of 1000 ppm.
19.3 As a protective application after 21 days no lesions were developed on the fruit surface treated with the fungicides difenoconazole and imazalil, at all tested concentrations above 50 ppm. Also, epoxiconazole at a concentration of 200 ppm, pyrimethanil at a concentration of 1000 ppm, prochloraz at a concentration of 1100 ppm, Fludioxonil at a concentration of 1200 ppm completely inhibited rot formation.
20.1 As a curative application after 7 days no lesions were developed on the fruit surface treated with the fungicide imazalil at all tested concentrations. Also, the fungicides epoxiconazole at a concentration of 450 ppm, pyrimethanil at a concentration of 500 ppm, prochloraz at a concentration of 750 ppm, and fludioxonil at a concentration of 900 ppm completely inhibited rot formation.
20.2 As a curative application after 14 days imazalil at 50 ppm and higher concentrations completely inhibited rot formation. Meantime, no lesions were developed on the fruit surface treated with the fungicides epoxiconazole at a concentration of 450 ppm, and pyrimethanil at a concentration of 500 ppm.
20.3 As a curative application after 21 days no lesions were developed on the fruit surface treated with the fungicides imazalil, epoxiconazole, pyrimethanil, and prochloraz at concentrations of 100, 450, 1000, and 1100 ppm respectively. However, the fruit were completely rotted when treated as a curative application with the fungicides pyraclostrobin at a concentration of 800 ppm, prochloraz at a concentration of 250, fludioxonil at a concentration of 600 ppm, epoxiconazole at a concentration of 200 ppm and azoxystrobin at a concentration of 600 ppm.