Arbuscular mycorrhizal fungi spore propagation using single spore as starter inoculum and a plant host.

AIMS: The propagation of pure cultures of arbuscular mycorrhizal fungal (AMF) is an essential requirement for their large-scale agricultural application and commercialization as biofertilizers. The present study aimed to propagate AMF using the single-spore inoculation technique and compare their propagation ability with the known reference spores. METHODS AND RESULTS: Arbuscular mycorrhizal fungal spores were collected from salt-affected Saemangeum reclaimed soil in South Korea. The technique involved inoculation of sorghum-sudangrass (Sorghum bicolor L.) seedlings with single, healthy spores on filter paper followed by the transfer of successfully colonized seedlings to 1-kg capacity pots containing sterilized soil. After the first plant cycle, the contents were transferred to 2·5-kg capacity pots containing sterilized soil. Among the 150 inoculated seedlings, only 27 seedlings were colonized by AMF spores. After 240 days, among the 27 seedlings, five inoculants resulted in the production of over 500 spores. The 18S rDNA sequencing of spores revealed that the spores produced through single-spore inoculation method belonged to Gigaspora margarita, Claroideoglomus lamellosum and Funneliformis mosseae. Furthermore, indigenous spore F. mosseae M-1 reported a higher spore count than the reference spores. CONCLUSIONS: The AMF spores produced using the single-spore inoculation technique may serve as potential bio-inoculants with an advantage of being more readily adopted by farmers due to the lack of requirement of a skilled technique in spore propagation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study describe the feasible and cost-effective method to mass produce AMF spores for large-scale application. The AMF spores obtained from this method can effectively colonize plant roots and may be easily introduced to the new environment.

First Report of Freesia sneak virus in Commercial Freesia hybrida Cultivars in Korea.

In order to investigate the virus infection rate of commercial freesia cultivars in early February 2013, 19 freesia cultivars showing necrotic purple speckles or streaks on leaves, purple streaks parallel to the midrib, and necrotic speckles on leaves were collected from three different regions (Suwon and Icheon in Gyeonggi Province and Jeonju in North Jeonla Province) and used for virus detection. Nucleic acid extracts were analyzed for detection of major freesia-infecting viruses including Freesia sneak virus (FreSV) by reverse transcription (RT)-PCR with specific primer pairs. The FreSV CP gene was amplified using primer pair FreSV-F (5'-TTAGATAGTGAATCCATAAGCTGC-3') and FreSV-R (5'-ATGTCTGGAAAATACTCCGTCCAA-3'). The approximately 1.3-kb fragment of the FreSV amplified product was cloned and sequenced (GenBank Accession No. KC771891 to 98). The nucleotide sequences of CP gene of FreSV korean isolates showed 99.2 to 99.8% similarity to other FreSV isolates DQ885455, FJ807730, and GU071089, which are registered in GenBank. FreSV was detected from 71.7% of 138 plants tested while the infection rate of Freesia mosaic virus (FreMV) was 34.8%. Neither Bean yellow mosaic virus (BYMV) nor Tobacco rattle virus (TRV) were detected from any plants tested in this study. In certain cultivars, such as 'Bluebau' (II) and 'Pretty women,' most plants planted in the field showed purple streak symptoms on the leaves. In conclusion, FreSV was detected from some symptomatic freesia cultivars showing purple streak or speckles on leaves with or without necrotic spots and necrotic speckles on leaves. FreSV is currently widespread in Korea and some freesia plants were mixed infected with FreMV. FreSV has been occurring in the Netherlands for over 40 years (2). It is a plant virus in the family Ophioviridae and Ophiovirus genus. Once it occurs in freesia plantation fields, eradication is almost impossible because FreSV is transmitted by zoospores of Olpidium brassicae, which is a soilborne root-infecting fungus (3). Resting spores of O. brassicae can remain dormant in the soil and can be infective for 20 years (1). To produce virus-free freesia plants, growers should consider whether or not their fields are contaminated with O. brassicae carrying FreSV. To our knowledge, this is the first report of FreSV in freesia plants in Korea. References: (1) R. N. Campbell. Can. J. Bot. 63:2288, 1985. (2) Y. Koot et al. Tijdschrift over Plantenziekten 60:157, 1954. (3) H. J. M. van Dorst. Neth. J. Plant Pathol. 81:45, 1975.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Papaya in Korea.

In March 2013, papaya (Carica papaya L. cv. Sunrise) plants growing in polyethylene-film-covered greenhouses in Agricultural Research Center for Climate Change located in Jeju City, Korea, were observed severely affected by a powdery mildew. Symptoms appeared as circular to irregular white patches on both sides of the leaves. As the disease progressed, the plants were covered with dense masses of the spores, eventually causing senescence and withering of leaves. Voucher specimens were deposited in the Korea University Herbarium (KUS). Hyphae were flexuous to straight, branched, septate, and 5 to 8 µm wide. Conidiophores were 110 to 250 × 10 to 12.5 µm and produced 2 to 5 immature conidia in chains with a crenate outline followed by 2 to 3 cells. Foot-cells of conidiophores were straight, cylindric, slightly constricted at the basal septum, and 55 to 110 µm long. Conidia were hyaline, ellipsoid-ovoid, measured 22 to 38 × 18 to 21 µm with a length/width ratio of 1.2 to 1.8, and had distinct fibrosin bodies. Chasmothecia were scattered or partly clustered, dark brown, spherical, 80 to 100 µm in diameter, and each contained a single ascus. Appendages were mycelioid, 1- to 5-septate, brown at the base and becoming paler. Asci were sessile, 72 to 87 × 52 to 68 µm, had a terminal oculus of 17 to 23 µm wide, and contained 8 ascospores, each 17 to 23 × 12.5 to 15 µm. The morphological characteristics and measurements were consistent with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (1). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA of KUS-F27269 was amplified with the primers ITS5/P3 and sequenced (3). The resulting 443 bp sequence was deposited in GenBank (Accession No. KF111806). The Korean isolate showed >99% similarity with those of many P. xanthii isolates including an isolate on papaya from Taiwan (GU358450). Pathogenicity was confirmed through inoculation tests by gently pressing a diseased leaf onto young leaves of three asymptomatic, potted seedlings (cv. Sunrise). Three non-inoculated seedlings were used as control. Inoculated plants were isolated from non-inoculated plants in separate rooms in a greenhouse at 26 to 30°C. Inoculated leaves developed symptoms after 7 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was identical morphologically to that observed on the original diseased leaves, fulfilling Koch's postulates. Powdery mildews of papaya caused by Podosphaera species including P. caricae-papayae have been reported in North America, South America, Hawaii, Africa, Ukraine, Australia, New Zealand, the Cook Islands, India, Thailand, Taiwan, and Japan (2,4). P. caricae-papayae is currently reduced to synonymy with P. xanthii (1). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on papaya in Korea. Though papaya is a minor crop in Korea, producing about 300 M/T annually in greenhouses, powdery mildew disease is a threat to safe production of the fruits. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11, CBS, Utrecht, 2012. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, retrieved April 9, 2013. (3) S. Takamatsu et al. Mycol. Res. 113:117, 2009. (4) J. G. Tsay et al. Plant Dis. 95:1188, 2011.

The complete genome sequence of a member of a new species of tobamovirus (rattail cactus necrosis-associated virus) isolated from Aporcactus flagelliformis.

In this study, we identified a new tobamovirus from diseased Aporcactus flagelliformis cactus plants, named it rattail cactus necrosis-associated virus (RCNaV), and determined its complete genome sequence. The full RCNaV genome consisted of 6,506 nucleotides and contained four open reading frames coding for proteins of M(r) 128 kDa (3,441 nt), 185 kDa (4,929 nt), 55 kDa (1452 nt), 36 kDa (1,005 nt) and 19 kDa (513 nt) from the 5' to 3' end, respectively. The overall similarities for the four ORFs of RCNaV were from 32.5% to 64.1% and from 17.0% to 67.3% to those of the other tobamoviruses, at the nucleotide and amino acid level, respectively. Comparison of the coding and non-coding regions of the virus with those of other tobamoviruses showed that RCNaV is the most closely related to cactus mild mottle virus.

First Report of Blueberry red ringspot virus Infecting Highbush Blueberry in Korea.

Blueberry red ringspot virus (BRRSV) of the Soymovirus genus in the family Caulimovididae causes red ringspot diseases in highbush blueberry (Vaccinium corymbosum L.) on leaves, stems, and fruits. The virus has been identified in the United States, Japan, Czech Republic, Slovenia, and Poland (1). In July 2010, highbush blueberry with red ringspots on leaves and circular blotches on ripening fruits was found in one plant of cv. Duke in Pyeongtaek, Korea. The symptoms were similar to red ringspot disease caused by BRRSV (3), although stems did not show any characteristic symptoms. Red ringspots on the upper surface of leaves were the most visible symptom and became more prominent as leaves matured in August through October. Leaves of the symptomatic plant were collected and tested for BRRSV infection by PCR, and were also embedded for electron microscopy. DNA was extracted from leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Primer pairs BR1512F/BR2377R (5'-ACAGGACGATTAGAAGATGG-3'/5'-CCTTTAGGGCAATATTTCTG-3', amplifying a fragment of the coat protein region with an expected size of 865 bp) and BR2961F/BR3726R (5'-ACCGATACATCACAGTTCAC-3'/5'-TGGTTGTGATAAGATGATTCC-3', amplifying a fragment of the reverse transcriptase region with an expected size of 766 bp) were used to amplify the indicated region of BRRV in PCR. Primers were designed on the basis of the BRRSV isolate from New Jersey (GenBank Accession No. AF404509). DNA fragments of the expected sizes were obtained from the symptomatic plant, while no amplification products were obtained from highbush blueberry without symptoms. The PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of obtained fragments revealed 91 to 98% nucleotide sequence identity with the coat protein gene (GenBank Accession No. JQ706341) and 96 to 98% nucleotide sequence identity with the reverse transcriptase gene (GenBank Accession No. JQ706340) of known BRRV isolates. Electron microscopy of thin sections revealed particles approximately 50 nm diameter within electron-dense inclusion bodies, characteristic of BRRSV (2) To our knowledge, this is the first report of BRRSV infection of highbush blueberry in Korea. Highbush blueberries are usually propagated by cutting, so BRRSV suspicious plants should be tested with PCR before they are propagated. References: (1) E. Kalinowska et al. Virus Genes. DOI 10.1007/s11262-011-0679-4, 2011. (2) K. S. Kim et al. Phytopathology 71:673, 1981. (3) M. Isogai et al. J. Gen. Plant Pathol. 75:140, 2009.

First Report of Stolbur Phytoplasma Infection in Commercial Freesia hybrida Cultivars.

In January 2012, disease symptoms including chlorosis, leaf crinkle, leaf curving and stunting of whole plants, virescence, and curving and necrosis of flower stalks were observed in Freesia hybrida cvs. Evone, Honey Moon, Golden Gem, and Pallas in Icheon and Suwon (Gyeonggi Province in Korea). To determine a possible phytoplasma infection, the symptomatic freesia plants were examined for the presence of phytoplasma 16S rDNA fragment by PCR with the primer pair P1/P6 (2) and R16F1/R16R1 (in nested PCR), which amplifies phytoplasma 16S rDNA regions (4). An expected PCR product of ~1,096 bp was obtained from the symptomatic freesia plants, and they were designated as FreLN, Fre-phy-Ev4, Fre-phy-Ev6, Fre-phy-GG, Fre-phy-HM, and Fre-phy-Pal. The PCR products were sequenced and registered as GenkBank accessions AB695174 and AB709951-55. The sequence corresponding to symptomatic freesia had 98.8 to 99.4% identity with Stolbur phytoplasma strains in the 16S rDNA region, and it had only 95.7 to 96.3% identity with AY phytoplasma strains. In the ultra-thin sections of the leaf midribs, globous phytoplasmal bodies 54 to 214 nm in size were observed in sieve tube elements of phloem tissue. Fre-Phy-Ev6 and Fre-Phy-HM were doube-infected with Stolbur phytoplasma and Freesia mosaic virus (FreMV). Fre-Phy-Ev6 and Fre-Phy-HM revealed necrosis of flower stalks and flower color breaking besides curving of flower stalks. Therefore, flower color breaking and flower stalk necrosis were assumed to be caused by FreMV (1). Symptoms of chlorosis and stunting of whole plants shown in FreLN and virescence of Fre-phy-GG were typical symptoms of phytoplasmal diseases, while leaf crinkle, leaf curving, and curving of flower stalks appeared to be unique symptoms in F. hybrida. Stolbur phytoplasma was abundant in commercial freesia cultivation fields. Some of the cultivars, such as cv. Pallas, showed only curving of leaf and flower stalks without any typical symptom of phytoplasmal diseases. A phytoplasmal disease was reported in Poland in 2001 from F. hybrida exhibiting leaf chlorotic and necrotic spots, and classified as AY I-B based on RFLP analysis of PCR products (3). To our knowledge, this is the first report of Stolbur phytoplasma in F. hybrida. This result is significant because F. hybrida could be the infection source of Stolbur phytoplasma disease in floricultural crops. Interestingly, we found a prevalence of Stolbur phytoplasma in Petunia hybrida cultivars (GenBank Accession Nos. AB713757 to AB713758). High nucleotide sequence identity of 99.8% in the 16S rDNA region of Stolbur phytoplasma isolates from petunia and freesia support the inference that those Stolbur phytoplasma isolates could infect both floricultural crops. References: (1) A. A. Brunt. Freesia. Page 274 in: Virus and virus-like Diseases of Bulb and Flower Crops, John Wiley & Sons, Chichester, 1995. (2) S. Deng and C. Hiruki. J. Microbiol. Methods. 14:53, 1991. (3) M. Kaminska and H. Sliwa. Plant Dis. 85:336, 2001. (4) I. M. Lee et al. Phytopathology 84:559, 1994.

Occurrence of Poinsettia Stem Flat Disease Caused by Phytoplasma in Korea.

In December 2009, commercially grown poinsettia (Euphorbia pulcherrima Willd cv. Ichibang) exhibited typical phytoplasma-like symptoms in 95% of an affected field in Yongin, Korea (Gyeonggi Province). Symptoms consisted of flat stems and fascicles and an abnormal number of apexes resulting in a cockscomb form of stem and flower bud proliferation. Leaf narrowing with curling of bracts was also associated with the disease. Symptomatic poinsettia plants were not marketable. Poinsettias, cv. Ichibang, without symptoms were obtained from a breeding collection in a glasshouse of the National Institute of Horticultural and Herbal Science in Suwon (Gyeonggi Province). The presence of phytoplasmas in symptomatic and healthy-looking flowers and stems of cv. Ichibang was demonstrated by PCR analysis with primer pair R16mF2/R16mR1 (1), which amplifies phytoplasma 16S rDNA regions. PCR products (~1,427 bp), were obtained from both symptomatic and healthy-looking plants, sequenced, and registered under the GenBank Accession Nos. of GU461275 and GU461277, respectively. Symptomatic poinsettia and healthy poinsettia sequences had 99.6 and 100% identity with U.S. PoiBI isolate FJ376625, indicating poinsettia stem flat disease is caused by PoiBI. A branch-inducing factor in poinsettia has been known for several decades, but only since 1997 was this graft transmissible factor identified as a PoiBI phytoplasma (1), belonging to the peach X-disease phytoplasma group (16S rRNA III) subgroup 16SrIII-H (2). Normally, phytoplasmas are associated with host quality loss, but abnormally, infection in poinsettia generates a desirable, free-branching growth habit. In this study we found that PoiBI could be detrimental to the quality of poinsettia depending on the cultivar and agronomic practice. Poinsettia stem flat disease presumably occurred because of increased levels of phytoplasma caused by successive stem cutting for commercial use. To our knowledge, this is the first report of PoiBI phytoplasma that affected marketability of poinsettia in Korea. References: (1) I. M. Lee et al. Nature Biotech. 15:178, 1997. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Cactus mild mottle virus is a new cactus-infecting tobamovirus.

A new cactus-infecting tobamovirus, Cactus mild mottle virus (CMMoV), was isolated from diseased grafted cactus, Gymnocalycium mihanovichii and its molecular properties were characterized. CMMoV is distantly related to known species of the genus Tobamovirus on the basis of serological and sequence analyses. Western blot analysis showed that CMMoV is serologically unrelated to Sammon's Opuntia virus, which is the only known species of the genus Tobamovirus found in cactus plants. The 3'-terminal 2,910 nucleotides of CMMoV have been sequenced. The coat protein (CP) and movement protein (MP) genes encode 161 and 306 amino acids residues, respectively, and the 3' untranslated region (UTR) consists of 229 nucleotides long. The nucleotide and amino acid sequences of the CP of CMMoV were 39.6% to 49.2% and 25.8% to 40.3% identical to other seventeen tobamoviruses, respectively. The MP shared 34.9% to 40.6% and 16.3% to 27.0% and 44.6% to 63.4% identities, respectively, at the amino acid and nucleotide levels with other members of the genus. Percentage identities of nucleotides of the 3' UTR ranged from 42.5% to 63.4%. Phylogenetic tree analyses of the CP and MP suggest the existence of the fifth cactus-infecting subgroup in the genus Tobamovirus. Sequence analyses of these two viral proteins revealed that the highest amino acid sequence identity between the virus and seventeen other tobamoviruses was 40.6%, supporting the view that CMMoV is a new definite species of the genus Tobamovirus.