IRANIAN JOURNAL OF PLANT PATHOLOGY
Year:2012 | Volume:48 | Issue:2 (190)|Papers Count:21

Phytophthora inundatahas been recently described and depicted as a ‘difficult’ taxon for identification due to its morphology and unusually high upper temperature limit for growth which has been mistaken for other non-papillate and high temperature tolerant plant pathogenic Phytophthoraspecies. A simple as well as a nested-PCR based method was developed for the identification ofP. inundata. A collection of isolates from different hosts representing diversity of species were examined for unique regions of coding as well as non-coding gene sequences. Based on internal transcribed spacers 1, 2 and 5.8S gene of rDNA (ITS), heat shock protein 90 gene (HSP), triosephosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase fusion protein (TIG), and 60S ribosomal protein L10 gene (RPL) ten PCR primers specific for P. inundata were designed. Annealing temperatures and extension times were optimized for each set of primers for maximum specificity and efficiency. To evaluate the specificity of the method, 28 morphologically and molecularly characterizedPhytophthora species were tested. In most cases neither set of primers amplified purified DNA from these non-homologousPhytophthora species. Analysis showed that the best candidate for identification of P. inundata isolates is ITS-I1 set which is a combination of ITS-IF1 and ITS-IR1. The optimized annealing temperature for this set was 69 °C and the best extension time was 40 sec. It seems that nested-PCR by ITS-I1 set together with universal ITS6 and ITS4 as external primers is at least 50 times more sensitive than conventional PCR.

During growing seasons 2005-2007 samples of grapevine with esca symptoms were collected from different vineyards in Bojnourd (North Khorassan province). Isolation was made from affected wood tissues from branches and trunks on PDA, MEA and OA media. In this study, 64 fungi isolates were recovered and identified as Phaeoacremonium parasiticum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium sp. and Phoma sp. Pathogenesity tests were carried out for Pm. Parasiticum, P. chlamydospora and F. mediterranea under field and greenhouse conditions. Within 6 months after inoculation of cuttings, symptoms developed as sever defoliating and wilting under greenhouse conditions.F. mediterranea caused white wood decay while Pm. parasiticumand P. chlamydospora produced wood discoloration in inoculated grapevines. In the field the pathogens were re-isolated and identified from inoculated grapevines after symptoms observed.

Alternative measures to chemical control of plant parasitic nematodes were evaluated by using plant extracts and (or) plant residues of five species includingBunium persicum, Cuminum cyminum, Carum copticum, Foeniculum vulgare and Eugenia caryophyllata. Tomato seedlings were either dipped in water extracts of these plants at different concentrations before planting in soil infested with Meloidogyne javanica, or infested soil was amended with plant residues before the tomato seedlings were planted. At 60 and 90 days after planting, growth response of tomato, root infection by nematodes, and reproductive factor of nematode in pot cultures were evaluated. The results indicated that root dipping in the aqueous extract of C. cyminum was the most effective to decrease second stage juveniles and lower the reproductive factor. The extracts of F. vulgare and B. persicum were also effective to reduce the gall indices on tomato. Amending soil with plant residue of C. cyminum (10% w/w) controlled M. javanica comparable to Caduzafos nematicide. Furthermore, amending soil with plant residues of C. copticum at 1, 5, and 10% w/w levels were effective against root knot nematode.Plants treatment with Caduzafos nematicide and residues of C. cyminum (10% w/w) was indicated the best control. Improving soil with C. copticum residues shown a favorable effect against root knot nematode (M. javanica) at all tested rates (1%, 5%, 10% w/w).

Control of citrus green mold caused by Penicillium digitatum, was evaluated on artificially inoculated oranges and tangerines immersed in 3 and 4% sodium bicarbonate (SB) (wt/vol) and potassium bicarbonate (PB) (wt/vol), with or without 200 ppm sodium hypoclorite for 60 or 180s; in 3 and 4% sodium carbonate (SC) (wt/vol) and potassium carbonate (PC) (wt/vol) at 25, 37, 45 or 53oC for 60, 180 or 300s; and kept in hot air (37oC) for 24, 48 or 72h. Hot air and water experiments were done in two sets of before and after treatment inoculations. After 3 weeks of storage at 10oC and 80% RH, the incidence of decayed fruits was determined. Results revealed that SB 4 and 3% for 180s reduced decay in Tompson Navel by 67, 65.11% and Shahsavar oranges by 80.16 and 76.04% respectively. SB4% and SB3%+NaClO 200ppm for 60s reduced decay in tagerines by 59.84 and 58.43% respectively. Control treatments caused 100%, 97-98%, and 89-90.5% decay respectively. In before treatment inoculations, PC and SC 4% at 53°C for 180s, PC3% at 45°C for 300s, PC and SC 3 and 4% at 53°C for 300s reduced decay in Tompson Navel by 100%.SC3% at 53°C for 60 and 180s, PC3% at 45°C for 180s, PC4% and SC 3 and 4% at 53°C for 300s reduced decay in Shahsavar oranges by 100%. PC and SC 4% at 53°c for 180s, PC 4 and 3% and SC4% at 53°c for 300s and PC3% at 37°C for 180s reduced decay in tangerines by 85, 82.5, 80, 77.5, 75, and 75% respectively. Control treatments caused 92%, 100%, and 80% decay respectively.In before treatment inoculations, Hot air for 72, 48 and 24h in Tompson Navel; for 72, 24 and 48h in Shahsavar oranges; and for 48, 72 and 24h in tangerines reduced decay by 95, 95 and 92%; 100, 85.14 and 66.22%; and 100, 95 and 33% respectively. Control treatments caused 100%, 74%, and 100% decay respectively. However, some damages observed on fruits treated with hot air for 72h.

Bacterial wilt of potato, a sever and devastating disease caused by Ralstonia solanacearum occurs in tropical and subtropical area. Using resistant cultivars is important in control of this disease.Discovery of plant resistant mechanisms and induced plant responses against pathogen may provide valuable information for improved resistant cultivars. In this direction, the expression pattern of some important defense genes against R. solanacearum in potato commercial cultivars like, Marfona (susceptible), Els (tolerant) of Solanum tuberosum and resistant genotype of Solanum phurejawas evaluated over times after inoculation the level of expression of evaluated defense genes including Chitinase A, Chitinase B, Glucanase, PR-10a in the resistant genotype of S. phureja was higher than the other two commercial cultivars. Results show expression of Chitinase A and PR-10a in S. phureja and tolerant cultivar, Els have increase compared to susceptible cultivar Marfona. Expression of defense protein and antimicrobial compounds were also evaluated based on the growth rate of the fungus Fusarium solani on culture media containing extract of induced plants. The growth rate of the fungus decreased on the culture containing extracts from the resistant genotype, compared that of susceptible cultivar.

Huanglongbing (HLB) has been previously reported in southern Iran. In the present study, additional information is given on the distribution of HLB agent and its vector. The psyllid Diaphorina citri, vector of ‘ Candidatus Liberibactor asiaticus’ was found in all surveyed citrus growing areas of Sistan- Baluchistan, Hormozgan, Kerman (Jiroft and Kahnooj) and Fars (Darab and Lar) provinces. Using direct and nested PCR with specific primer pairs 103 out of 140 psyllid samples from Ghasre Ghand (Sistan- Baluchistan province), Minab and Roodan (Hormozgan province) were positive for Asian form of HLB. By the same PCR methods positive reactions were obtained from 22 Valencia and two local sweet orange and 1 mandarin trees from Nikshhar and Sarbaz (Sistan- Baluchistan province) and 16 Valencia sweet orange from Roodan and Senderk (Hormozgan province). Using PCR with designed primers a 909 bp segment ofopm gene of two HLB isolates from Sistan- Baluchistan and Hormozgan was amplified, cloned and sequenced (Acc. No. HQ267229 and HQ267230, respectively). The two isolates showed 100% sequence identity and were identified as Ca. L. asiaticus but not Ca. L. africanus. Graft inoculation of grapefruit (Citrus paradis) and Valencia sweet orange seedlings with scions from a symptomatic Valencia sweet orange from Sarbaz caused blotchy mottle, a specific symptom of HLB.D. citri is reported for the first time from Fars province. Based on the results of the present study HLB disease appears to be widely distributed in citrus growing regions of Sistan- Baluchistan and Hormozgan provinces.

Many plant and animal viruses overcome their host defense by encoding protein that suppress posttranscriptional gene silencing (PTGS). Protein 2b in cucumber mosaic virus (CMV) is regarded as a strong suppressor of PTGS. In the present study the 2b gene of CMV was used to transform of tobacco plant. Possibility of induction of resistance to CMV in the tobacco plants was investigated.Two different constructs, S1 and S2, were used to study PTGS of CMV. The designated S2 construct contained a sequence of sense and antisense of coding region of 2b gene, expected a hairpin-like structure transcribed. Control construct (S1) consisted of the same construct without any sequence of 2b gene. These constructs were first made in pHANNIBALL and then ligated into pART27, a plant specific vector.Agrobacterium tumefactions strain GV3101 was used for stable transformation of Nicotiana tabaccum Var xanthi. Forty regenerated plants were transferred to soil and challenged by CMV inoculation. Thirty three percent of plants showed resistance to CMV while 30% showed delayed in symptom development. Resistance of plants to CMV was confirmed by ELISA. The present study demonstrated that 2b- derived PTGS is an effective plant defense mechanism against CMV and can be used in breeding programs.

Armillaria mellea the causal agent of root and crown rot of trees has a universal distribution and causes extensive economic disease on a broad host range of trees in gardens, forests and urban environments. The pathogen can survive under the bark as inoculum and there is no effective control method against the disease, so pathogen detection from soil and wood is important to predict disease severity and prevent disease dispersal from one tree to another. To achieve a rapid and sensitive detection method for A. mellea in soil and wood, sampling was performed from fruiting bodies, soil and wood and after isolation of pathogen on culture media, comparison was done among different detection methods using a semi-selective medium, baiting and molecular methods.Inoculum was prepared for two isolates of A. mellea and was inoculated to soil. Pathogen detection was done with soil direct culture method, baiting and nested PCR simultaneously. The culture medium was not effective to detect pathogen in long-term period. In baiting method, Pelargonium hortorumwas used as a baiting plant that required long time to detect pathogen. The results revealed that nested PCR is an efficient method for detection of A. mellea.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is one of the rapeseed important diseases in the world and Iran. In order to study the infection status of this disease in rapeseed fields of Mazandaran province, 120 fields in five different regions of Mazandaran (including Sari, Babolsar, Dashte Naz, Behshahr and Galogah) selected during years 2010 and 2011. From the beginning of disease symptoms appearance, weekly surveys were done from fields and amount of disease was recorded during the infection period. Based on the highest disease incidence on leaves, there were no significant differences between regions and fields in first year, but in second and combined two years, there were significant differences (P<0.001) between regions and years. The infection in first year (35%) was higher than second year (24%) and the region Galogah had the highest infection (42%) and Sari lowest infection (20.8%). Based on the final disease incidence on stems, there were significant differences between regions (P<0.001), but no significant differences between years.Based on disease severity, there were significant differences between regions (P<0.001) and years (P<0.05). Disease incidence was 21% in both two years and disease severity in first year (15%) was more than second year (11.5%). In combined two years, Galogah with disease incidence 32% and severity 21.2% had the highest infection and Sari with disease incidence 13.75% and severity 8.25 % had the lowest infection. Based on areas under disease progress curves, there were significant differences between regions and years, that these factors in first year were more than second year and in regions Galogah and Behshar were more than other regions.

During 2009 and 2010 growing seasons, different irrigated fields were surveyed in Gorgan region.Infected plants with symptoms such as damping off, crown and root rot, and foliar blight were collected. Samples were washed in running tap water and cultured on CMA+PARPH. Isolates were identified on the basis of morphological and some physiological characteristics. 50 isolates belonging to four species were identified. The species were: Phytophthora citrophthora from persimmon fruits (Diospyros kaki); Phytophthora cryptogea from kiwi trees (Actinidia chinensis) with root and crown rot sympotoms, crown and root of potato (Solanum tuberosum) and gerbera (Gerbera jamesonii), Phytophthora nicotoanae from tomato (Lycopersicum esculentum) root and stem, Phytophthora palmivora from root and crown of olive (Olea europae) seedlings and Phytophthora inundatafrom root and stem of broad bean (Vicia faba). All of Phytophthora isolates were found pathogen on their host at greenhouse conditions. Based on our knowledge isolation of P. cryptogeagroup II from kiwi and potato and Phytophthora palmivora from olive trees were new for Iran, also P. inundata from broad bean was new for Iran and the world.

In order to identify fungi involving root and crown rot of wheat and to determine their distribution and frequency, wheat fields in different regions of Kermanshah province were sampled at seedling, tillering and heading stages during 2004-9. For isolation of fungi, some samples of infected roots, subcrown internode, crown and foot were surface sterilized and then placed on potato dextrose agar containing streptomycin sulfate. Pathogenicity test was performed by mixing of pasteurized soil with inoculum (colonized wheat seeds with an isolate) under greenhouse condition. Disease severity (D.S.) was evaluated by scoring from 1 to 5 (1=no symptoms, 5=extensive necrosis of the crown and stem base resulting in plant death). Besides other genera, 241 isolates of Fusarium spp. were isolated. Also the results revealed thatFusarium spp. exist in most parts of the province and their D.S. is from 1 to 2.9. AmongFusarium species Fusarium culmorum and F. pseudograminearum had the highest D.S., F. acuminatum, F.avenaceum, F. crookwellense, F. proliferatum and F. udumhad medium D.S. and that of F. nygamai, F. reticulatumand, F. merismoides was the least amount. The species that could not cause disease were F.equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. sambucinun, F. semitectum, F. solaniand F. trisinctum. This is the first report of pathogenicity of F. udum, F. nygamai and F. merismoides on wheat root in the world.

The symptoms of exocortis disease including bark scaling on trifoliate orange (Poncirus trifoliata) rootstock and stunting of citrus trees have been observed in some citrus orchards of Mazandaran province. To study the biological properties of Citrus exocortis viroid (CEVd) isolates, thirty samples with various degrees of severity in exocortis symptoms were collected from three large citrus orchards of Mazandaran in 2004. Having graft-inoculated into Arizona 861-S1 Etrog citron (Citrus medica cv. Etrog), the samples caused variable symptoms on the inoculated citron plants.The results of reverse transcription-polymerase chain reaction (RT-PCR) with specific primer pairs for CEVd showed that eighteen samples with the symptoms of the disease were infected with CEVd. In these samples, the related DNA bands were purified using High Pure PCR Product Purification Kit (Roche, Germany) and prepared for single–strand conformation polymorphism (SSCP) analysis. The DNA samples of the seven isolates were selected based on their differences in SSCP and cloned in pTZ57R/T (Fermentas), and sequenced at SeqLab Company (Germany).Sequence analysis revealed the existence of two or three nucleotide in the sequences of three isolates, while there were no differences in the sequences of the other four isolates despite the differences in the symptoms produced in citron. It seems that exocortis disease is caused by exocortis-like agents other than CEVd in Mazandaran. This is the first report on molecular characterization of CEVd in the infected citrus plants in Iran.

A field survey was conducted during spring and summer of 2010 from vineyards in different areas of Kerman province including Khanook, Mohy Abad, Qhavam Abad, Hamidiyeh, Mahan, Arab Abad, Sirjan, Sirch, Kerman, Bardsir, Joupar, Hootk, Chatroud, Golbaf, Dalfard and Rabor in order to determine the prevalence of esca and Petri diseases of grapevine in this province. Samples were taken from grapevines showing the following symptoms: interveinal chlorosis and leaf necrosis, reduced growth, wilting and wood and xylem discoloration in cross section. Isolations were made from affected wood tissues on MEA and PDA media. The isolates were identified based on morphological and cultural characteristics. A total of 216 fungal isolates were obtained from grapevines showing decline disease symptoms. The most important fungal isolates associated with grapevine showing decline symptoms in most vineyards were Phaeoacremonium aleophilum (=Pm. aleophilum) (20.83%), Phaeomoniella. chlamydospora (11.11%) and Pm. parasiticum (6.95%). In this study, esca and Petri disease symptoms were obsereved on 28.57% and 71.43% of visited vineyards, respectively. Moreover, the proportion of grapevines with Petri disease (76.75%) was much greater than those with esca (23.25%). Our results show the importance of Petri disease in most of the Kerman province viticultural areas.

During visit beech forests of Mazandaran province in May and June of 2010-2011, some infected twigs of Fagus sylvatica with signs of fungal infection were collected. Laboratory analysis revealed acervular fruiting bodies on infected twigs. Acervulus up to 500 mm in diameter and forming a black spot on main and secondary twigs. Conidia erumpent, form black masses over the acervulus.Conidia are spherical, brown to olive, with acropetal branched cells enclosed in a gelatinous sheath.Complex conidia are 12.5-22.5×25-45 mm, composed of sphaerical cells which are 1.25-3.75×2.5-7.5 mm. Conidiophores hyaline, up to 150 mm, septate, straight and cylindrical. The above morphological features fit well with the earlier descriptions ofCheirospora botryospora (Mont.) Berk. & Broome (Sutton 1980, The Coelomycetes). The species has been previously reported on Cornus alba, Hedera helix, Fagus gandifolia and Quercus rubra (Sutton 1980). This is the first report of the fungus from Iran.

A Leaf spot diseases caused by strains of Xanthomonas in wild primrose (Primula vulgaris) were reported in several forest locations of Mazandaran (Rahimian.1995.12th Iran. Plant Protec. Cong.P.278). Based on a genomic study of ITS area and 16S rRNA gene sequencing, it was reported that some isolates causing leaf spot of P. vulgaris probably belong to X. campestris (Rahimian et al. 2008. 18th Iran. Plant Protec. Cong. P.420). But there is insufficient information available on the taxonomical status of these isolates. The present study wase performed to evaluate the taxonomic position of the isolates obtained from several forest areas of Mazandaran province. Yellow pigmented٫ round and convexXanthomonas- like colonies were isolated from the diseased plants.Hypersensitive reaction (HR) after 24 hours injection of bacterial suspension to geranium leaves and pathogenicity on P. vulgaris leaves after 7 to 14 days were proven. Only slight differences were observed among the electrophoretic pattern of cell proteins of the strains. Strains were very similar phenotypically and differed mainly in utilization citrate as carbon soure for growth. Genomic DNA of isolates was extracted and PCR was done under previously described conditions (Parkinson et al. 2007. Int. J. Syst. Evol. MicrobioI.57: 2881-2887) reducing the annealing temperature. Products were electrophoresed in %1 gel and the gel was stained with gel red solution. Usingdnak and rpoD primers fragments 965 and 736 bp in size٫ were amplified in PCR٫ respectively. For differentiation of the isolates٫ the PCR products were digested with Bam HI and electrophoresed on acrylamid gel.Dendrograms were constructed using the data sets obtained after digestion ofdnak and rpoD amplified fragments with the restriction enzyme, using the Jaccard coefficient, the UPGMA algorithm and the NTSYS-PC program. Selected samples were sequenced based on RFLP results.The nucleotide sequence obtained٫ after multiple sequence alignment٫ were compared٫ with MEGA version 4.0٫ with other sequences to various Xanthomonas species present in the Gene Bank (NCBI). Results٫ showed that isolates resembled٫ at 98% similarity٫ with strains of Xanthomonas hortorumdeposited in GeneBank٫ but probably belong to a pathovar distinct from the existing ones in this spesies. This is the first report of Xanthomonas hortorum causing a leaf spot disease on Primula.

Rice sheath spot agent was first reported from California, Arkansas and Louisiana as Trichoderma sp. (Tullis, 1934). Then Ryker & Gooch introduced R. oryzae as the correct agent. Waitea circinata has WAG-O anastomosis group related to R. oryzae. The fungus was not reported from Iran.ARhizoctonia isolate was isolated from corn sheath bearing sheath blight symptoms. After fourteen days the colony was changed white to salmon and sclerotia were waxy, soft and often sunken in agar. Sclerotia were irregular and salmon in color. The species as pathogen has been reported from Japan, Cambodia, Thailand, Taiwan, Philippines, USA, West Africa and Brazil. Hoshioka and Mankino (1969) reported that this fungus was found in subtropical and tropical rice growing regions and regarding to this report, R. oryzae is confined to these climates due to its low cold tolerance threshold. Although sheath spot has observed in southeastern Australia which is outside the tropical and sub-tropical regions (Lanoiseletet al., 2001). The pathogen has been isolated from wheat, barley and rice (Ryker and Gooch, 1938). Also R. oryzae has been found with extensive distribution and high virulence on wheat, barley and pea fields (Paulitz, 2002). In Iran, the fungus has been previously reported as pathogen of corn, rice and wheat. Pathogenicity tests had positive results on roots and sheaths of corn and wheat seedlings. After DNA extraction, R. oryzae was subjected to amplification with ITS4 & 5 primer pairs with an annealing temperature of 60 oC. The PCR product on 1.5% agarose consisted of a 670 bp fragment. Comparison of the sequences with those present in NCBI led to identification of R.oryzae. There were no noticeable differences between characteristics of R. oryzae isolates in the present study with those described by Rayker and Gooch.Based on the morphological and molecular characteristics of the isolates WAG-O, It can be concluded that this and similar isolates described herein, are isolates of R. oryzae and the species is being reported for the first time on corn from the central region of Golestan province.