Identification of Phaeoacremonium aleophillum Phyt

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Journal of Agricultural Science and Technology A 2 (2012) 983-987

Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

Identification of Phaeoacremonium aleophillum Phytotoxin Decline Platanus Trees Platanus occidentalies L.

Khalid Hassan Taha

Department of Plant Protection, College of Agriculture and Forestry, University of Mosul, Iraq

Received: April 11, 2012 / Published: August 20, 2012.

Abstract: The genus Phaeoacremonium is associated die-back of various woody hosts. In this study, Phaeoacremonium specie was isolated from necrotic woody tissue of Platanus occidentalies L. (Platanus). Morphological and cultural characteristics were ud to identify the species, which known, and being described as Phaeoacremonium aleophillum. This is the first report on identifying Phaeoacremonium aleophillum from Platanus in Iraq, previously reports accommodate Cephalosporium acremonium from Platanus trees. This specie is able to produce toxic metabolites. Applications of P. aleophillum crude and cell-free filtrates induced vere chlorosis and necrosis on

Platanus leaves after 24 hours of treatment with P. aleophillum culture filtrate. A novel phytotoxin was isolated from P. aleophillum and culture filtrate was identified as galactouronic acid.

Key words: Phaeoacremonium aleophillum, Platanus, phytotoxin, galactouronic acid.

1. Introduction

Wood decline dia caud by fungi is similar to Cephalosporium types is reported for more than half a century on oak trees in the state of Texas [1] since then the dia was reported in many species of wooden trees. In Iraq, the dia has been recorded on the palm and saved in the CMI under the name of Phialophora parasitica [2]. On eucalyptus [3] poplar [4] Platanus [5] was identified as C. acremonium. Classification of genus Cephalosporium has been subjected to re-classification. As a result, three Genera were named Acremonium, Phialophora and Phaeoacremonium,the latest was recently described as intermediate genus between Phialophora and Acremonium [6, 7], since that time, this genus registration was in progress where 14 species were receded in stone nut trees [2], 13 species in Grapes [8, 9] and various other species of Phaeoacremonium can

Corresponding author: Khalid Hassan Taha, Ph.D., rearch fields: plant pathology, biological control, fungal taxonomy.E-mail:**********************.be found on, including olive, eucalyptus and cyp

ress [10, 11]. The fungus caus gradual decline symptoms on the wooden trees as stunting, wilting, branches death, cankers and death is localized to specific areas between the veins in the leaves. The internal symptoms are wood streaking or browning spots the wood of the trunk infected trees will eventually die [12-16]. The goal of this rearch was to re-identify the genus and specimen of Platanus decline agent in Mosul province and the in vitro extraction of biological activate compounds (phytotoxin) induced by P. aleophillum.

2. Materials and Methods

2.1 Pathogenisolation

Samples of infected Platanus branches collected from Mousal forestry branches showing typical symptoms of the dias approximately 2 mm were ud for the isolation of pathogen. The infected parts were surface sterilized with 6% sodium hypochlorite

solution for 3 minutes and washed rially in sterilized

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Identification of Phaeoacremonium aleophillum Phytotoxin

Decline Platanus Trees Platanus occidentalies L.

984

distilled water to remove the traces of sodium hypochlorite solution and then transferred to sterilized petri plates containing potato dextro agar PDA containing 10 µg/mL of tetracycline hydrochloride and 100 µg/mL of streptomycin sulfate. The petri plates were incubated at 25 ± 2 °C and obrved periodically for the growth of pure colonies. The pure colonies which developed from the bits were transferred to PDA slants and incubated at 27 ± 1 °C for 15 days. Then such slants were ud to study characters.

2.2 Extraction of Phytotoxic Metabolites

The fungus was grown in a customized liquid medium czapek-dox total of 500 mL of CZD medium placed in 1,000 mL Erlenmeyer ﬂasks and autoclaved for 20 min (1.5 Bar and 121 °C). After cooling, the medium was inoculated with four agar plugs of fungus. Inoculated ﬂasks were incubated in the d

ark at 27 ± 2 °C. After 15 days incubation, methanol 1:1 was added in equal amount, and kept at 4 °C for five hours. The mixture was centrifuged at 4500 g for 30 min. The supernatant was collected and concentration was done up to 1/10 volume at 40 to 45 °C with a rotary evaporator. Chloroform was added in equal amount to the supernatant, pooled and shaken, the aqueous layer was parated, the chloroform extract was dried over Na2SO4 and then subjected to flash evaporation to remove the solvent from 40 to 45 °C, the residue was dissolved with 2 mL of water and subjected to a leaf bioassay. The aqueous extract was concentrated to 20 mL and subjected to a leaf bioassay. For partial purification of toxins aqueous extract, butanol was added in equal amount to aqueous extract, pooled and shaken, the aqueous layer was parated and subjected to a leaf bioassay [17] butanol extract was dried over Na2SO4 and then subjected to flash evaporation to remove the solvent at 40 to 45 °C, the residue was dissolved with 2 mL of water and subjected to a leaf bioassay. The butanol extract was chromatographed by thin layer chromatography TLC [18]. Preparative analytical silica gel plates 0.25 were ud and developed in butanol: acetic acid, distillate water 5:1:4. The spots were developed by exposing the TLC plates to iodine vapor [19].

2.3 Leaf Bioassay

Excid Platanus leaves were ud to test the biological activities of condary metabolites. Excid

leaves were placed on moistened filter paper inside 9 cm diameter sterile Petri plates. The inocula of purified compounds were applied to the leaves with micropipettes. Amounts ud were 100 pL to adaxial were ud for each. Control leaves equivalent treatment with distilled water. The plates were aled with Parafilm and incubated under continuous or 12 h light. The phytotoxic effects on the treated excid leaves were evaluated visually for damage [20].

3. Results and Discussion

3.1 Phenotypical Characterization

Cultural characteristics of Colonies on PDA radius about 8-9 mm after 15 days is at 27 ± 2 °C (Fig. 1a). Colony honey-brown or beige, flat, wooly texture with entire edge areal structure hyphae is verruculo, medium to pale brown conidoiophore is mostly short and unbranched 17-46 µm long and 2-25 µm width phialides terminal or lateral, mostly monophialidic, smooth to verreculo, subhyline with three type of phialides (Fig. 1b). Type II and III are most common. Type I phialide are cylindrical occasionally wider at the ba 4-9 × 1-1.5 µm (Fig. 1c). Type II phialide is either elongate ampuilliform and attenuated at the ba or are navicular, tapering toward the apex 10-14 × 1.5-2.5 µm (Fig. 1d). Type III phialide is sub cylindrical or elongate ampuilliform and attenuated at the ba 1北京游

5-20 × 1.5-2 µm tapering gradually to long neck (Fig. 1e). Conidia are mostly oblong-ellipsoidal prcylindrical reniform 2.5-6 × 1-2 µm. This description was in agreement with the P. aleophilum characterization (Fig. 1f) [9, 15, 21, 22].

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Fig. 1 Cultu phialide; (d) T

3.2 Leaf Bio Only buta Platanus pr treatment, w 48 h. The ne margin at la produce nec ural characteris Type II phialide oassay

anol extract roduced chlo which enlarg ecrotic spots ater stages. W rotic spot.

(a)

(c)

(e) Identificat Decline

stics of Phaeoa es; (e) Type III p which appli orotic spots ed and turne were surroun While other ion

of Phaeo e Platanus Tre acremonium ale phialides; and (ied on leave after 24 h ed necrotic a nded by chlor extracts did acremonium ees Platanus eophilu m, (a) 1(f) Conidia on C s of h of

after

rotic d not 3.3B toxi spo sho and P. (b (d (aleophillum s occidentalie 5 day old colo Conidiophores.Extraction of

Bad on the ic metabolite ts were obr wed agreeme d xylo Tabl aleophillum b)

Phytotoxin

es L. ony on PDA; (b . Scale bar f Phytotoxic M e comparison e isolate fro rved with 0.1ent with gala le 1. Experim

butanol extr b) Arial structu 5 µm.

Metabolites n of the Rf m butanol e 15, 0.18 and 0actouronic ac mental results

ract induced 985

ure; (c) Type I value of the extract, three 0.2 Rf which id, arabino indicate that chlorosis as 5

e h e t s

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Identification of Phaeoacremonium aleophillum Phytotoxin

Decline Platanus Trees Platanus occidentalies L.

986

Table 1 Rf value of sugar isolateed from butanol extract.

Sugars Rf Color Sugars Rf Color

Gluco

0.12 Brown Ghlorounic

acid

0.12 Yellow-orange Galacto

0.12 Brown Glalagturinic

acid

0.15 Red Arabino

0.18 Red Ribo 0.27 Yellow Xiloze 0.20 Red Frecto 0.19 Yellow

Arrhemno 0.12 Yellow-red Manno 0.17 Pink

狂躁症的症状well as necrosis symptoms on leaves that indicate the involvement of toxins. This obrvations support the conclusions, demonstrating that the pathogen produces veral metabolites into culture filtrate, which were galactouronic acid, arabino and xylo, galactouronic acid can be considered as not pathogen-specific toxin vivotoxin which was produced by the pathogen during pathogenesis Identification of galactouronic acid by TLC could be regarded the first Identification of this compound from P. aleophillum rearch found that pallulans have the ability to cau the foliar symptous associated with dia due to its ability to reduce the chlorophyll content of the plant, hence causing chlorsis of the leaves, leading to a decrea in photosynthesis and therefore necrosis and stunted growth can occur [8, 22, 23]. Bruno et al. [21] and Cimmino et al. [24] found that P. aleophilum has been shown to produce lipophilic low molecular weight compounds and high molecular weight phytotoxin which non host to platanus appearing to be exo-polysaccharides EPSs but attempted to characterizes P. aleophilum (EPSs) has been mach no success, P. aleophilum (EPSs) maybe consist of glalagturinic acid. Lipophilic low molecular weight compounds did not characterize in the recent work due to the its neglecting fraction of culture filtrate which also caved necrotic local lesion on the platanus leaves. Preliminary method results showed that suitability of the

procedure and solvents for detection the mixed derived phytotoxic sugars, but not for Polykatides groups lipophilic low molecular weight [23, 25].

4. Conclusions

Our results indicate that P. aleophillum is the causal agent of Platanus in Iraq, leaf bioassay of P. aleophillum crude and cell-free filtrates resulting vere chlorosis and necrosis on Platanus leaves after 24 h of application. Authors identified galactouronic acid as a novel phytotoxin.

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