First report of Alternaria leaf spot on gerbera (Gerbera jamesonii L.) in North of Iran.Introduction
Gerbera is one of the top ten cut-flower of the world, It is a genus of ornamental plants from the sunflower Asteracea family with great commercial value, successfully grown under different condition in several areas of the world . Because of its extensive cultivation and commercial importance keeping quality and quantity is serious so diagnose and control of diseases are necessary.
There are some published reports about disease on gerbera: In Argentina in autumn 2007, a downy mildew infection was observed on plants of gerbera var. Avant Garde cultivated in greenhouses. Symptoms began as irregularly shaped yellow discoloration on the upper surface of the leaf, later turning light to dark brown . Powdery mildew symptoms caused by Podosphaera sp. were observed on the leaves of Gerbera jamesonii during April 2008 in an experimental field of India . Powdery mildew caused by Golovinomyces cichoracearum was observed on potted gerbera growing in a private garden in Italy . In October, 1995, in North America, white erumpent sori were detected on the undersurfaces of leaves of a bed of Gerbera jamesonii that diagnose as white rust . Tomato spotted wilt virus reported on Gerbera hybrida in Serbia. The infected plants displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves .
Alternaria leaf spot was reported in Bulgaria. The symptoms of the disease on the leaves were characterized by the development of brown, small, scattered dots, which gradually enlarged and coalesced to form large, oval, circular or irregular, brown to black lesions with concentric rings . Also Alternaria sp. on Barberis thunbergii cuase stem spot showed dark brown, eye-like elongated spots on stems and red and yellow spots on the leaves . Alternaria alternata cause a stem spot disease on Gymnocalycium mihanovichii . In some greenhouses in north of Iran leaf spots with the same symptoms have been observed on Gerbera jamesonii. The objectives of this study were to describe the symptomatology of this new leaf spot disease of Gerbera jamesonii and to isolate and identify the causal organism and confirm its pathogenicity on healthy gerbara in greenhouses.
Materials and Methods
Sampling and Isolation:
Gerbera leaves with leaf spot were collected from five greenhouses in the north of Iran, they were transported to the labrotary for isolating pathogenic fungi. Leaves with spots were cut into small piece, surface sterilized with 1% NaOCl for 1 minute rinsed thoroughly in sterile distilled water and dried with sterile filter paper. The leaf pieces were placed on to potato dextrose agar (PDA) medium. The cultures incubated at 25 [+ or -] 1 [degrees]C for 10 days to promote fungal growth and sporulation. The growth fungi was isolated, and pure culture were obtained from single spores and hyphal tips and maintained on potato dextrose agar medium in plates.
The morphological and culture characteristic of the isolated organism were studied also conidia and conidiophores ditails were considered.
Conidial suspension of the isolate was prepared from fungi which growed for 10 days on PDA cultures, and the conidial concentration was adjusted to 2 x 105/ml using hemacytometer. The gerbera plants were cultivated at regular intervals in pots with sterile soil mixture and they were spray-inoculated with 100 ml of the spore suspension per pot. Inoculated plants were placed in a humid chamber with 100% relative humidity at 25[degrees]C for 24 hours. The plants were covered tightly with a transparent nylon foil, after 24 hours plants were uncovered and Constant humidity was maintained by spraying tap water. The plants were grown in a greenhouse (25-30[degrees]C) for 2 weeks for symptom development. After 2 weeks typical symptoms were produced on the inoculated leaves. The pathogen from the infected leaves was re-isolated on PDA medium as described above. The morphological and cultural characteristics of the re-isolated organism were compared with the original pathogen and they were the same.
[FIGURE 1 OMITTED]
The symptoms of the disease on the leaves were appearing by brown, small, scattered spots on the leaves that gradually become round or irregular, brown to black lesions, concentrically zonate with a dark brown margin. In later stages of infection affected leaves discolored to yellowing and finally dried up. The infection appear from the older, lower leaves and spread to upware. The percentage of diseases incidence was 22.6% (7 spotty leaves in 31 healthy leaves). Necrotic spots were often numerous and enlarged quickly to blight the leaf tissue Circular, necrotic and sunken dark brown spots on both side of the leaf, ranging from less than 20 mm to an average diameter of 30 mm were typical symptoms of disease (Fig1).
The fungus produced flat, downy to woolly and grey colonies with olive green peripheries. The Alternaria grew well on PDA and formed an olivaceous black colony with dark olive margins, reverse black at 25 [+ or -] 1 [degrees]C after 10 days. The surface was greyish white at the beginning which later darkens and became greenish black or olive brown.
The conidiophores were branched, straight, Pale brown to olive brown in colour, measuring 25-60 um long and 3-3.5 um thick. The conidia were pale brown to light brown in colour and produced in long branched chains, obclavate to obpyriform or ellipsoid in shape with short conical beak at the tip, or beakless. The size of conidia varied from 20-63 [micro]m in [micro]length and 9-18 [micro]m in width. Conidia had two to three transverse septa and usually several longitudinal septa (Fig2).
[FIGURE 2 OMITTED]
Pathogenicity tests conducted on healthy potted gerbera plants in a glasshouse showed as the same typical leaf spot symptoms of the first isolation after two weeks and enlarging necrotic areas developed on gerbera leaf following inoculation after two weeks.
These results, based on morphological characteristics and pathogenicity tests, confirm the identity of the fungus as Alternaria alternata. This is the first report of Alternaria alternata as leaf spot disease of gerbera in north of Iran.
In this report we describe the symptomatology of a new disease on gerbera at greenhouses in north of Iran. Small, circular, necrotic and dark brown spots on the leaves that gradually become larger and irrigular were the typical symptoms of Alternaria disease that it was agree with result of Long et al.,  depicted brown, circular or oval spots. These enlarged and linked together, extending until entire leaves withered. Bajwa et al.,  reported a leaf spot disease on Aloe vera plants that observed as small, circular to oval dark brown necrotic sunken spots on the leaves. Leaf spot of gerbera was reported in Bulgaria by Mirkova et al.,  that observed brown, small, scattered dots approximately 1 mm in diameter. They gradually enlarged and coalesced to form large (20-25 mm), oval, and circular to irregular brown to black lesions with concentric rings. Alternaria is a common plant pathogen, which cuase a variety of fruit and plant disease. Previously, Alternaria alternata reported in Iran from tangerine that brown necrotic lesions surrounded by yellow haloes, characteristic of Alternaria brown spot of citrus, were found on young leaves and fruits . Alternaria mali cuase apple leaf blotch that symptoms included black and dark brown leaf spots on leaves of apple trees . Alternaria alternata on wheat grains reported by Mirzadi Gohari et al.,  and Saberi Riseh et al., , and as a leaf spot pathogen on potato .
In this study we observed the pale brown to olive brown branched conidiophores with 25-60 um long and 3-3.5 um thick, that was the same with other results, the toxonomy of Alternaria alternata in our studying is based on the morphology and development of conidia and conidiophores and colony morphology. The Alternaria alternata colony grew well on PDA medium and formed an olivaceous black colony with dark olive margins. These morphological characters of the colony of the present isolate coincide with those of colonies described by Kamalakannan et al.,  and Siddiqui et al., .
Kamalakannan et al.,  reported Alternaria alternata as leaf spot on Aloe barbadensis, the fungus produced conidiophores with branched, straight, golden brown in colour, measuring 15 um long and 2-6 um thick. Alternaria alternata isolated from Chenopodium album produced conidiophores simple, olive-brown, septate, variable in length with terminal conidia, which are solitary or in short chains . The conidiophores of Alternaria alternata on grafted cactus were solitary or in small fascicles, commonly branched, pale brown, up to 60 [micro]m long, 3-4.5 [micro]m wide .
The conidia were pale brown to light brown in colour and produced in long branched chains, obclavate to obpyriform or ellipsoid in shape with short conical beak at the tip, or beakless. The size of conidia varied from 20-63 um in length and 9-18 um in width and two to three transverse septa conidia were observed. It is the same with report of Alternaria alternata on Chenopodium album, the fungus produced obclavate to obpyriform conidia with a short conical or cylindrical apical beak not exceeding one third of the conidial length, or beakless, smooth walled or verruculose, slightly constricted with 3-8 transverse septa, the lower part each portion had one or two longitudinal septa . Kamalakannan et al.,  reported the size of conidia of Alternaria alternata on Aloe barbadensis varied from 22.75 to 63.70 [micro]in length and 13.65 to 18.20 [micro]m in width with an average beak length of 7.73 [micro]m. Conidia had two to three transverse septa and usually several longitudinal septa, that was according to our result.
As a species of the genus Alternaria, Alternaria alternata belongs to the family of black pigmented molds Dematiaceae. Alternaria alternata has a wide host range, causing leaf spots and blights on many plant parts .
According to our studying, this is the first report of leaf spot disease, Alternaria alternata, of Gerbera jamesonii in north of Iran.
It has been concluded from present research that one of the most important factors which disappearing small, circular, necrotic and dark brown spots on the leaves of Gerbera jamesonii could be influence of Alternaria alternata which belongs to Dematiaceae family.
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Shokooh Farhood and Shervin Hadian
Young Researchers Club, Gorgan, Islamic Azad University, Gorgan, Iran.
Shokooh Farhood, Young Researchers Club, Gorgan, Islamic Azad University, Gorgan, Iran. E-mail: [Shokooh.Farhood@Yahoo.Com]