Pythiaceous Fungi associated with Citrus Decline in Iran^)

Phytopath. Z., 74, 153—160 (1972) © 1972 Verlag Paul Parey, Berlin und Hamburg From the Department of Plant Protection, CoUege of Agriculture, Pahlav...
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Phytopath. Z., 74, 153—160 (1972) © 1972 Verlag Paul Parey, Berlin und Hamburg

From the Department of Plant Protection, CoUege of Agriculture, Pahlavi University, Shiraz, Iran

Pythiaceous Fungi associated with Citrus Decline in Iran^) By J. FATEMI Received June 21, 1971

Introduction Citrus decline has been reported from various citrus growing regions of the world. Any conditions that deprive the plant of an adequate root system can cause decline of the trees. Many causes of citrus decline has been proposed including nutrient deficiencies, excess of nutrients, compactness of soil, viruses, nematodes, ozone injuries, and fungi from the family Pythiaceae. PETRI (1929) reported that root rot caused by Phytophthora parasitica, and not associated with gummosis was the prevalent form. WAGER (1942) isolated Pythium ultimum, Phytophthora citrophthora, Pythium vexans, P. debaryanum, P. rostratum, and Phytophthora parasitica from fibrous roots of orange and lemon trees showing decline. FRASER (1942) reported a root rot of sweet orange root stock in New South Wales which was more severe in heavy soils. He was able to demonstrate the presence of Phytophthora citrophthora in a few cases showing initial decay of the fibrous roots in the field. KLOTZ and SOKOLOFF (1943) isolated Phytophthora citrophthora, and P.parasitica from feeder roots of sour orange stock and Washington Naval Orange trees that had wilted. Greenhouse experiments by the authors showed that these fungi are able to destroy feeder roots of sweet and sour orange seedlings at temperatures ranging from 10—30 °C. KLOTZ and FAWCETT (1944) in an investigation conducted to determine the potential of Phytophthora species to cause root rot found that, in order of resistance of the fibrous roots to Phytophthora species Sampson Tangelo was first, sour orange second, and Valencia orange third. CALAVAN (1949) found that P. ultimum was common on roots of trees showing decline. SLEETH (1953) reported a decline of citrus trees in Texas. The disease first affected a single tree and then spread outwards. The symptoms consisted of a dull green appearance of the leaves followed by gradual defoliation, and die-back. He isolated P. ultimum, and indicated that the fungus was partly responsible for the disease. BURKE (1956) reported that Phytophthora parasitica was widespread in old grapefruit orchards in Jamaica and was causing death of orange seedlings. KLOZ et al. (1958) investigated the severity of root rot in sweet orange seedlings caused by Phytophthora parasitica in disinfested inoculated soil; and found that it can be increased by excess of water. In addition they found in the greenhouse study that Phytophthora citrophthora was necessary to induce rotting. TsAO and GARBER (1960) stated that P. parasitica and P. citrophthora were two of the main causal fungi of citrus 1) This investigation was supported in part by a grant from Pahlavi University.

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fibrous root rot in California. STOLZY et al. (1965) by artificial inoculation discovered that saturated soil was required for P. parasitica and P. citrophthora to be effective in causing root decay.

Citrus decline is a disease that has recently attracted growers attention in Southern Iran. It is prevalent in newly established as well as old orchards, and has caused considerable damage. Symptoms associated with the disease include leaf chlorosis, retarded growth, partial defoliation, necrosis of the roots, and finally severe root rot and death of the trees. Preliminary investigations by ABIVARDI et al. (1970) have shown that nematodes are associated with the disease. The purpose of this investigation was to determine the association with, and the involvement of Phytophthora and Pythium species in the disease.

Materials and Methods Isolation Soil and root samples from infected groves with trees showing typical symptoms located m Khafre, Jahrom, De-Gonbadan, and Firooz Abad (table) were collected. They were placed in plastic bags in contact with ice during sampling. In the laboratory they were kept at 4 °G and processed gradually. Table Source of cultures used in this investigation Isolate No. 10O4 1005-4 1006-1 1008 1017-6 1018 1019 1026 1027 1029 1030 1034 1092

Host Unknown Tangerine Sweet lime Sweet lime .Sweet lime Sweet orange""'') Sweet orange Sweet orange Sweet lime Citron Sweet lime Sweet lime Lime

Location*)

Khafre (K. bala) Khafre (Babe-anar) Khafre (K. bala) Khafre (K. bala) Jahrom Jahrom Jahrom Jahrom De Gonbadan De Gonbadan De Gonbadan Jahrom Firooz Abad.

*) All locations except De Gonbadan are in Fars Province; De Gonbadan is in Boir Ahmadi. **) On lime rootstock. For isolation from soil a method reported by BANIHASHEMI (1970) was used. To isolate fungi from roots, infected roots were washed thoroughly, cut into small pieces, and surface sterilized m a 10% Clorox solution (containing 5.25% Sodium hypochlorite), then placed in petri dishes containing Corn Meal Agar (CMA) or Lima Bean Agar (LBA).

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Morphological Studies To induce oospore formation, all isolates were grown on Potato Dextrose Agar (PDA), Lima Bean Agar (LBA), and Cowpea Extract Agar (CPEA, extract of 100 g Cowpea + 15 g Agar/liter) and incubated at 25 °C or 30 °C. To produce sporangia, when production on solid media was unsuccessful, the fungi were grown on LBA or CPEA. When mycelia covered the plates, boiled and cooled hemp seeds were placed on mycelial surface and 24 hours later, the seeds were transferred to sterile petri solution (Calcium nitrate, 0.4 g; Magnesium sulfate, 0.15 g; Potassium acid phosphate, 0.15 g; Potassium chloride, 0.06 g; Distilled water, 1 liter) or sterile distilled water, and incubated on laboratory shelves near the window (15—25 °C). Twenty four hours later they were checked for the presence of sporangia and zoospores. In some instances when sporangia were not produced within twenty four hours, the solution was replaced with fresh petri solution or distilled water and incubated for another twenty four hours.

Pathogenicity Trials After isolation and partial identification, isolates were tested for pathogenicity on citrus twigs, fruits, and seedlings. 1. Pathogenicity test on twigs Green and apparently healthy twigs were cut from tangerine {Citrus reticula Blanco), sour orange [C.aurantium), and sweet orange {C. sinensis) trees. They were cut into 15 cm pieces and then inoculated as follows: A part of the bark plus cambium was removed aseptically in such a manner that the removed part remained intact. A block of the culture medium plus the fungus was cut by a No. 3 cork borer from a fresh culture, and placed aseptically on the wood surface. The removed part was then put bade, and sealed with scotch tape to keep the inoculum well in contact. The inoculated twigs were placed in large sterile test tubes (25 X 250 mm) containing sterile distilled water in such a manner that a part of the inoculated twig remained submerged in water. To reduce partial contamination, and in the meantime allow aeration, the tubes' mouths were covered with muslin cloth. They were then placed at 20 °C. Isolates used were 1017-6, 1029, 1004, 1018, 1006-1, 1005-4,. 1026, 1092, and 1027. Each treatment was replicated twice. For checks only a block of the culture medium was placed under the removed bark. 2. Pathogenicity test on lime fruits Young and apparently healthy lime (C. aurantifolia) fruits were prepared, and surface sterilized with alcohol. Then a part of the rind was removed superficially with a flamed scalpel, and 2 blocks cut from a fresh culture on LBA were placed on the fruit surface. Noninoculated blocks were used for checks. The inoculated fruits were placed in sterile glass jars. To keep the R. H. high inside the jars during the experiment, a sheet of sterile filter paper dipped in sterile distilled water was put at the bottom of the jar. Another sheet was placed inside the lid and then it was tightened and incubated at 20 °C. Isolates 1004, 1005-4, 1006-1, 1008, 1017-6, 1018, 1019, 1026, 1027 and 1092 were used in this experiment. Each treatment had four replicates. 3. Pathogenicity test on citrus seedlings in petri solution Five-month old seedlings of tangerine and sour orange (local varieties) grown in sterilized soil in pots in the greenhouse, were removed carefully, washed gently, and then placed in sterile glass jars containing sterile petri solution. A zoospore suspension of isolates: 1005-4, 1027, 1017-6, 1092, and 1006 produced on hemp seeds suspended in petri solution was added to the jars (25 cc/jar), and incubated on laboratory shelves at room temperature (20—27 °C). Each

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treatment was replicated 2 times, and each replicate had three seedlings. Nothing was added to the controls.

Results Morphological observations 1. Pythium aphanidermatum (Edson) Fitz

Isolates 1017-6, 1005-4, 1004, 1008, 1027, and 1018 showed similar morphological characteristics (with minor differences) such as those described for P. aphanidermatum (MIDDLETON 1943). Sporangia were produced 24—48 hours after transferring hemp seeds covered with hyphae to petri solution or water. They were lobate or digitate, and produced vesicles abundantly from which zoospores were released. Oospores were produced on LBA, and other media tested 7—10 days following inoculation. They were mostly aplerotic with average diameters of 21, 20, 20.8,17.36,19.5,17.6,«; for isolates 1017-6,101 8-5, 1005-4,1004, 1008, and 1027 respectively. Antheridia were mainly spherical, monoclinous and paragynous; produced 1—2 per oogonium. Optimum temperature for growth was 30 °C. As it can be seen all these isolates show similar morphological characteristics corresponding to those of Pythium aphanidermatum. 2. Isolate 1026 {Phytophthora parasitica Dastur)

Sporangia were produced on hemp seeds suspended in sterile distilled water or petri solution within 24—48 hours. They were mostly pear shaped, papillate, with average dimensions of 35.6 X 32.20/*. Zoospores were formed in a vesicle at the end of exit tube. In some cases encysted oospores could be seen inside the vesicle. Oogonia and antheridia were not produced on solid or liquid media. Optimum temperature for growth was 30 °C. These criteria correspond to those of P. parasitica Dastur (WATERHOUSE 1956 and 1963). However, oosporeformation and measurement are needed to make accurate identification. 3. Isolate 1006 (Phytophthora sp.) This isolate had many features similar to those of 1026; however some differences also existed. As in isolate 1026 sporangia were produced on hemp seeds suspended in sterile distilled water or sterile petri solution. They were mostly papillate with average dimensions of 32.0 X 24.8 ,u. Zoospores were produced in a vesicle at the end of exit tube. Oospores were produced in single strain cultures originated from hyphal tips, five days following inoculation. They were aplerotic and had an average diameter of 19.2//. Antheridia were spherical or coiled, 80 % of which were paragynous and 20 % amphigynous. Optimum temperature for growth was 30 °C. Most of these criteria correspond to those of Phytophthora parasitica Dastur; however this

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species does not produce oospores in single strain cultures, and has mostly amphigynous antheridia. In addition in P. parasitica sporangia are larger than in isolate 1006. 4. Isolates: 1030, 1092, Phytophthora a7ropj!»/j!)ora. (Smith & Smith) Leonian

These 2 isolates showed similar morphological and cultural characteristics. Neither produced oospores on solid or liquid media. Sporangia were produced on hemp seeds covered with mycelia suspended in sterile petri solution or sterile distilled water as in other isolates. Sporangial shape was very variable and some had papillae. Average sporangial dimensions were 48.4 X 36, and 46.8 X 35.4/t for isolates 1030 and 1092 respectively. Exit pore was less than 7/t. In the case of isolate 1092 encysted zoospores could be seen inside sporangia. These criteria correspond to those of P. citrophthora (Smith & Smith) Leonian. (WATERHOUSE 1956, 1963). 5. Isolates: 1019,1029 and 1034

All attempts to induce sporangial production in these isolates failed. However, oospores were produced readilly on all media tested. Average oospore diameters were 19.0, 16.4 and 16.32 for isolates 1019, 1029 and 1034 respectively. These isolates have not been identified hitherto. Studies on Pathogenicity 1. On twigs

In sweet orange twigs the first symptoms of rotting occurred 3 days after Inoculation, indicating that sweet orange twigs are more susceptible. The primary symptoms consisted of a brown area near the inoculum that later advanced In all directions and within 10 days a great portion of the twigs turned brown and rotted. This was true for all isolates tested; except 1026, 1029 and 1092 in which one of the replicates was less affected. In the case of some isolates the twigs especially around the inoculated part became covered with mycelial tufts extending to both ends of the twig. In tangerine most of the isolates produced symptoms similar to those described for sweet orange twigs. However the degree and intensity of symptom expression seemed to be less. Twigs inoculated with 1004 did not show any symptoms. Regarding sour orange, all twigs except those Inoculated with 1006 remained symptomless. Isolate 1006 produced slight browning around the inoculum. 2. On lime fruits Five days after inoculation, the first symptoms began to show up in fruits inoculated with isolates 1004, 1005-4, 1006-1, 1008, 1017-6, 1018 and 1092. Symptoms consisted of browning of the tissues around the inoculum, that gradually advanced in all directions and within several days a great portion of the

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fruit rotted. All these isolates were recovered from rotton fruits. Isolates 1019, 1026 and 1027 did not produce any symptoms. In control blocks of agar placed on the fruit surface gradually dried up and no browning was observed. 3. On seedlings in petri solution

All sour orange and tangerine seedlings inoculated with isolate 1092 wilted and died within 10 days. However in the case of isolate 1027 only one of the sour orange seedlings showed disease symptoms. Seedlings inoculated with other isolates did not produce any symptoms ten days after inoculation. The experiment was continued for 35 days and during this period all sour orange seedlings inoculated with 1027 died (2 weeks after inoculation). The first symptoms on sour orange seedlings inoculated with 1006 showed up 3 weeks after the start of the experiment; these included dying of the leaves and wilting. Seedling inoculated with isolates 1005-4, and 1017-6 did not produce any symptoms during the experiment. However we were able to isolate 1027 from tangerine roots; 1005-4 from tangerine and sour orange roots; and 1017-6 from sour orange roots, non of which had shown any disease symptoms. Controls remained normal during the experiment. Discussion This investigation revealed that both Phytophthora and Pythium species are associated with citrus decline. Phytophthora species present were found to be P. parasitica Dastur, and P. citrophthora (Smith & Smith) Leonian, because they showed most of the characteristics described for these species. This is in agreement with the findings of other investigators in other countries. Both species plus isolate 1006 {Phytophthora sp.) were able to cause rots on sweet orange and tangerine twigs, under laboratory conditions. However sour orange twigs inoculated under the same conditions were not affected and might be resistant. Phytophthora sp. and P. citrophthora were also able to produce rot on lime fruits. In addition both sour orange and tangerine seedlings were killed within a relatively short time by P. citrophthora; however Phytophthora sp. killed only sour orange seedlings, and at a slower rate than the former species. This might mean that sour orange seedlings are more susceptible to P. citrophthora than to Phytophthora sp., under the conditions tested here. It also indicates that tangerine seedlings are resistant to Phytophthora sp. However the possibility that the rate at which the seedlings are killed might be due to differences in inoculum potential can not be overlooked. Pythium species found in this investigation was P. aphanidermatum (Edson) Fitz., and not P. rostratum, P. vexans, P. ullimum, and P. debaryanum, as reported by other investigators. This species produced rots on twigs of sweet orange and tangerine trees, but not those of sour orange. Furthermore lime fruits inoculated also rotted as in case of Phytophthora species. In addition, one of the

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three isolates of P. aphanidermatum tested for pathogenicity on seedlings, caused death of sour orange seedlings, indicating its potential to cause root rot. This investigation also revealed that susceptibility of the above ground parts and roots are controlled presumably by 2 different sets of factors. No general conclusion can be drawn regarding the isolates that did not produce sporangia. They might be Pythium or homothallic species of Phytophthora. This investigation in general showed that Phytophthora citrophthora, Phytophthora sp. (presumably a homathallic strain of P. parasitica) and Pythium aphanidermatum, in addition to causing twig and fruit rots, are potentially able to cause root rot and seedling decline; but this potential may vary from one species to another, and also from one host to another. However, more research is required to test this potential under more natural conditions. Along this line, investigation is in progress to test the pathogenicity of these fungi in soil in the greenhouse. Summary Citrus decline is a disease widespread, and causing considerable damage in southern Iran. It is prevalent in newly established as well as old orchards. Symptoms include leaf dilorosis, retarded growth, partial defoliation, root necrosis, and finally root rot and death of the trees. Phytophthora parasitica, P. citrophthora, Pythium aphanidermatum, and an unidentified species of Phytophthora {Phytophthora sp.) were found to be associated with the disease. All Phytophthora isolates tested, and one isolate of P. aphanidermatum caused death of the seedlings under laboratory conditions. In addition Phytophthora citrophthora and P. aphanidermatum produced fruit rots. All species also caused twig rots. Zusammenfassung Die Bedeutung versdiiedener Pythiaceen fur das ,,Citrus Decline" in Iran ,,Citrus Decline" ist in jungen und alten C i t r u s - Pflanzungen von Siidiran weit verbreitet und schadlich. Symptome: Blattchlorose, verzogertes Wachstum, teilweise Entblatterung, Wurzelnekrosen und schliefilich Wurzelfaule und Absterben der Baume. Aus Wurzeln kranker Baume und aus der Erde befallener Pflanzungen wurden Stamme von Phytophthora parasitica, P. citrophthora, Pythium aphanidermatum und einer nicht identifizierten Phytophthora-An isoliert. Alle Phytophthora-lsolierungen und ein Stamm von P. aphanidermatum brachten Keimpflanzen unter Laboratoriumsbedingungen zum Absterben. P. citrophthora und P. aphanidermatum verursachten auch Fruditfaule und alle Arten verursaditen Zweigfaule.

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ABIVARDI, C , K . IZADPANAH, A. SAFFARIAN, and M. SHARAFEH, 1970: Plant parasitic nema-

todes associated with citrus decline in southern Iran. Plant Dis. Reptr. 54, 339—342. BANIHASHEMI, Z., 1970: A new technique for isolation of Phytophthora and Pythium species from soil. Plant Dis. Reptr. 54, 261—262. BURKE, J. H., 1956: Citrus industry of British Honduras, Jamaica, Trinidad. Foreign Agric. Rep. 88, 77 P. CALAVAN, E. C , 1949: Lemon tree collopse. Phytopathology 39, 858—859 (Abstr.). FRASER, L., 1942: Phytophthora Root Rot of Citrus. J. Austral. Inst. Agric. Sci. 8, 101—105. KLOTZ, L. J., and V. P. SOKOLOFF, 1943: The possible relation of injury and death of small roots to decline and collapse of citrus and avocado. Calif, citrogr. 28, 86—87. , and H. S. FAWCETT, 1944: Progress report on decline of citrus. Calif. Citrogr. 29, 294—295. . T. A. DE WOLF, and P. P. WONG, 1958: Decay of fibrous roots of citrus. Phytopathology 48, 616—622. MIDDLETON, J. T., 1943: The taxonomy, host range, and geographic distribution of the genus Pythium. Mem. Torrey Bot. Club 20,1—171. PETRI, L., 1929: Methods for the control of root rot ol" citrus (in Italian). Boll. Staz. Pat. Veg. N.S. 9,255—272. SLEETH, B., 1953: Winter Haven decline of citrus. Plant Dis. Reptr. 37, 425—426. STOLZY, L. H . , J . LETEY, L . J. KLOTZ, and C. K. LABANANSKAS, 1965: Water and aeration as

factors in root decay of Citrus sinensis. Phytopathology 55,270—275. TsAO, P. M., and M. J. GARBER, 1960: Methods of soil infestation, watering, and assessing the degree of root infection for greenhouse in sito ecological studies with citrus Phytophthoras. Plant Dis. Reptr. 44, 710—715. WAGER, V. A., 1942: Phythiaceous fungi on citrus. Hilgardia 14, 535—548. WATERHOUSE, G. M., 1956: The genus Phytophthora. Comm. Mycol. Inst. Kew, Surrey, 120 P. . 1963: Key to the species of Phytophthora de Bary. Mycol. Papers No. 92, Kew, Surrey, 22 P. Author's address: Prof. J. FATEMI, Department of Plant Protection, Pahlavi University, Shiraz (Iran).

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