Garlic Isolate Research Articles

Host-specific adaptation drove the coevolution of leek yellow stripe virus and Allium plants.

Host adaptation plays a crucial role in virus evolution and is a consequence of long-term interactions between virus and host in a complex arms race between host RNA silencing and viral RNA silencing suppressor (RSS) as counterdefense. Leek yellow stripe virus (LYSV), a potyvirus causing yield loss of garlic, infects several species of Allium plants. The unexpected discovery of an interspecific hybrid of garlic, leek, and great-headed (GH) garlic motivated us to explore the host-adaptive evolution of LYSV. Here, using Bayesian phylogenetic comparative methods and a functional assay of viral RSS activity, we show that the evolutionary context of LYSV has been shaped by the host adaptation of the virus during its coevolution with Allium plants. Our phylogenetic analysis revealed that LYSV isolates from leek and their taxonomic relatives (Allium ampeloprasum complex; AAC) formed a distinct monophyletic clade separate from garlic isolates and are likely to be uniquely adapted to AAC. Our comparative studies on viral accumulation indicated that LYSV accumulated at a low level in leek, whereas LYSVs were abundant in other Allium species such as garlic and its relatives. When RSS activity of the viral P1 and HC-Pro of leek LYSV isolate was analyzed, significant synergism in RSS activity between the two proteins was observed in leek but not in other species, suggesting that viral RSS activity may be important for the viral host-specific adaptation. We thus consider that LYSV may have undergone host-specific evolution at least in leek, which must be driven by speciation of its Allium hosts. IMPORTANCE Potyviruses are the most abundant plant RNA viruses and are extremely diversified in terms of their wide host range. Due to frequent host switching during their evolution, host-specific adaptation of potyviruses may have been shaped by numerous host factors. However, any critical determinants for viral host range remain largely unknown, possibly because of the repeated gain and loss of virus infectivity of plants. Leek yellow stripe virus (LYSV) is a species of the genus Potyvirus, which has a relatively narrow host range, generally limited to hosts in the genus Allium. Our investigations on leek and leek relatives (Allium ampeloprasum complex), which must have been generated through interspecies hybridization, revealed that LYSV accumulation remained low in leek as a result of viral host adaptation in competition with host resistance such as RNA silencing. This study presents LYSV as an ideal model to study the process of host-adaptive evolution and virus-host coevolution.

Identification and molecular characterization of onion yellow dwarf virus (OYDV) in Allium cepa crops in Poland

Onion yellow dwarf virus is distributed worldwide significantly reducing yield of crops from the Allium genus. The aim of the study was the detection and molecular characterization of newly identified OYDV isolates infecting onions in Poland. The virus was detected by transmission electron microscopy and RT-PCR techniques using two pairs of diagnostic primers: OYDV-NibCPF1/R1 and OYDV-CPF2/R2. The specificity of obtained RT-PCR products was confirmed by Sanger sequencing and received viral coat protein sequence was used for phylogenetic analysis. The phylogenetic analysis was carried out using CP sequences of the new Polish onion isolate obtained in this study and 37 other sequences of OYDV retrieved from GenBank. The analysis revealed that the Polish OYDV isolate is the most similar to the OYDV isolates derived from onions from Argentina and Germany, which may indicate their common origin. Moreover, it was observed that the Polish onion and garlic isolates are very diverse and belong to different phylogroups.

FIRST REPORT OF GARLIC COMMON LATENT VIRUS IN GARLIC FROM NIGERIA

Garlic (Allium sativum L.) is one of the oldest known horticultural crops of the world. Several viruses belonging to the genera Potyvirus, Carlavirus, and Allexivirus are known to infect garlic and reduce their yield worldwide (Dijk, 1994; Walkey and Antill, 1989). This study was undertaken to investigate the status of viral infection in Nigerian garlic. Ten garlic bulbs collected in March of 2015 from two fields in Kano and Sokoto region of Nigeria were tested by direct antigen coated (DAC)-ELISA with antisera to Garlic common latent virus (GarCLV) (Bioreba, Reinach, Switzerland). All the samples were positive for GarCLV. To confirm the presence of GarCLV, reverse transcription (RT)-PCR was performed using primers published earlier (Majumder and Baranwal, 2014) and total RNA extracted from 100 mg of leaves with the RNeasy Plant Mini kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's instructions. Expected amplicons of ca. 450 bp were obtained from all the samples tested. Direct sequencing of the PCR products from one sample produced a 418-bp long nucleotide sequence. It showed 95% identity with a garlic isolate from The Netherlands (GenBank accession No. AB004804). To our knowledge, this is the first report of GarCLV on garlic in Nigeria.

FIRST REPORT OF ASSOCIATION OF SCLEROTIUM CEPIVORUM WITH WHITE ROT OF GARLIC IN OMAN

In the spring of 2014, a disease of garlic (Allium sativum) was observed in more than 30 plots in Jabal Akhdar (Sultanate of Oman), which was characterized by premature yellowing and wilting of the leaves followed by plant death. Upon uprooting, white cottony mycelium and black sclerotia were observed on the outer layer of the bulbs, roots and surrounding soil. The infection appeared to progress from the outer layers of the bulbs to the inner ones so as to involve the whole organ. Disease incidence ranged from 20 to 100%. On potato dextrose agar (PDA), fungal isolates with whitish/grayish mycelium and small-sized sclerotia typical of Sclerotium cepivorum were consistently recovered from symptomatic bulbs and roots. The sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA (Al-Sa'di et al., 2007) of three fungal isolates using ITS1 and ITS4 primers produced in all cases a nucleotide sequence 510 bp long (accession Nos GCC3471-73) which, when compared with sequences from database revealed 99.8% nucleotide similarity to a previously published sequence (accession No. FJ460433) of the garlic isolate H-292 of S. cepivorum from Hungary. The disease was reproduced in healthy garlic bulbs planted in inoculated soil with an incidence ranging from 30 to more than 65%. To the best of our knowledge, this is the first report of the association of S. cepivorum with white rot of garlic in Oman.

Fusarium proliferatum from rainwater and rooted garlic show genetic and pathogenicity differences

Fusarium proliferatum (Matsushima) Nirenberg is one of the most important Fusarium species considering its worldwide distribution, its pathogenic ability in a wide range of crops, and its mycotoxigenic potential. F. proliferatum conidia are easily carried by rainwater and, in this work, five isolates from rainwater were evaluated as inoculum sources to produce garlic rot and their pathogenic ability were compared with six F. proliferatum isolates from diseased garlic. A phylogenetic analysis was also performed to unravel possible genetic differences among the isolates. Pathogenicity tests were performed in three garlic varieties (purple, white and purple Chinese) and the results revealed that the isolates from rainwater were pathogenic to each of them. However, in all cases, their pathogenic ability was lower than in the case of isolates obtained from rooted garlic. The phylogenetic analysis based on parsimony carried out with the hypervariable region of the intergenic transcribed spacer (IGS) in F. proliferatum showed genetic differences depending on the origin of the isolates. Isolates from rainwater were definitely separated in an independent branch from garlic isolates. This paper shows an interesting approach to the ecological significance of F. proliferatum as a fungal pathogen on garlic. Some degree of relation between genetic variability and pathogenicity was also demonstrated.

LEEK YELLOW STRIPE VIRUS ISOLATES FROM BRAZIL FORM A DISTINCT CLADE BASED ON THE P1 GENE

The complete genome sequence of a garlic isolate of Leek yellow stripe virus (LYSV) from Brazil (LYSV-MG) was determined and its phylogenetic relationships with other LYSV isolates from other parts of the world were inferred. The LYSV-MG genome consists of 10,341 nucleotides and encodes a polyprotein of 3,221 amino acids. Brazilian LYSV isolates are more closely related to S-type than to N-type isolates but do not have a deletion in the P1 gene. Partial nucleotide sequence analysis of the P1 gene revealed 97-99% identity among Brazilian isolates, and 51-64% identity between Brazilian isolates and isolates from other countries for which sequence information is available in Genbank. The Brazilian isolates formed a monophyletic group closest to the S-type isolates and one isolate from Okinawa (O-type). These data suggest that Brazilian LYSV isolates are derived from an ancestral source of the Okinawa and S-type isolates, prior to the occurrence of the P1 deletion and divergence of the S-type isolates.

Molecular characterization and recombination analysis of an Indian isolate of Onion yellow dwarf virus

This study presents the first whole genome sequence of Onion yellow dwarf virus (OYDV isolate RR1) from onion (Allium cepa) in India along with phylogenetic and recombination studies. We examined the sequence variability, distribution of simple sequence repeats (SSRs), and recombination breakpoints of different OYDV geographical isolates. The P1 and P3 regions of OYDV show a higher rate of sequence variability in amino acid and nucleotide sequences than other genomic regions. Entropy peaks of deduced amino acid sequences were higher in both regions (P1 and P3) of different OYDVs. The observed frequency of microsatellites was also higher in the P3 region of all OYDV genomes. The Indian isolate RR1 showed 75–98 % similarity with the other OYDV isolates in nucleotide and amino acid sequences and has 43 microsatellites and two compound microsatellites. It was most closely related to garlic isolate MS/SW1 from Australia. Isolate RR1 contained six recombination breakpoints in different genomic regions with the major parent related to the MS/SW1 (Australian) and SG1 (Spanish) OYDV isolates. The phylogenetic and recombination study demonstrated the divergence of Indian isolate RR1 from OYDV isolate from Australia.

Genetic diversity and evidence of recombination in the coat protein gene of Onion yellow dwarf virus

The coat protein (CP) coding region of seven Polish Onion yellow dwarf virus (OYDV) isolates was sequenced. It was 771 bp long in all the seven isolates, and encoded a protein of 257 amino acids. Comparative nucleotide sequence analysis revealed diversity of 3 % among the Polish isolates and of 20 % among all garlic isolates worldwide. The comparison of CP sequences of 15 OYDV onion isolates, retrieved from GenBank, revealed an identity of 88–100 and 94–100 % at nt and aa level, respectively. Phylogenetic analysis clustered Polish OYDV isolates in the Subgroup I with isolates from Italy, Argentina and Japan. GARD and SBP algorithms in Datamonkey server and RDP program were employed to detect recombination events in the genome. Recombination analysis provided strong evidence for recombination events in 12 of the sequences analyzed. As regards selection inference, six algorithms (SLAC, FEL, IFEL, REL, MEME and PARRIS) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of OYDV capsid. The ratio of nonsynonymous and synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. This is the first report of recombination in OYDV isolates.

Molecular analysis of the 3' terminal region of Onion yellow dwarf virus from onion in southern Italy

Onion yellow dwarf virus (OYDV) is an economically important pathogen causing severe disease in garlic, onion and other Allium crops. Eleven isolates of OYDV, all from onion originating from Calabria, southern Italy, were genetically analyzed. An OYDV onion isolate from Sudan was also included in this study. The 3’ terminal region of about 2.5 kb of the twelve isolates were sequenced and the sequences comprising a part of the nuclear inclusion a (NIa-Pro), the complete nuclear inclusion b (NIb) and coat protein (CP) genes and the 3’ untranslated region (3’UTR), were compared to each other and to corresponding sequences of other OYDV isolates from different countries and Allium hosts. The within-population nucleotide identity of the Italian OYDV onion isolates was very high (more than 99.3%), whereas nucleotide identity between them and OYDV onion isolates from Germany was 94%, Argentina 92% and Sudan 87%. Recombination analysis among the complete 3’ terminal sequences showed putative recombination breakpoints in the NIb region of the Argentine isolate, with the minor parent related to the Sudanese isolate. Comparison between OYDV isolates from onion and isolates from garlic produced identities of 77-78% for the complete nucleotide region. When the 3’ terminal nucleotide sequence and the complete NIb protein were analyzed, the phylogenetic analysis generated rooted trees with high bootstrap values (100%), showing a genetic grouping into two well separated clades distinctive for onion and garlic isolates of OYDV. Phylogenetic analysis of CP protein and 3’UTR showed lower bootstrap separation values and no distinct sub-grouping of the OYDV isolates from the two major Allium species.

First Report of Iris yellow spot virus Infecting Allium tuberosum in India.

Allium tuberosum L., commonly known as garlic chives, is an important spice in northeastern India as well as in many other parts of the world. Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) is an important pathogen of onion (4) and other related Alliums such as garlic (3) and leek (2). During April 2013, symptoms potentially induced by IYSV such as chlorotic and straw-colored spindle-like lesions were observed on leaves of A. tuberosum accession Hanzong Winter (CGN 20779) plants in the wild species garden at the Directorate of Onion and Garlic Research (DOGR), Rajgurunagar, Pune, Maharashtra, India. Ten plant samples of A. tuberosum were randomly collected from the wild species garden and the upper, middle, and lower portions of the leaves were pooled and tested by double-antibody sandwich (DAS)-ELISA using a commercially available kit (Agdia Inc., Elkhart, IN) for IYSV. All of them showed positive results for IYSV incidence. Total RNA from the ELISA positive leaf samples of A. tuberosum was extracted using the RNeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany). The primer pair IYSV-F (5'-TCAGAAATCGAGAAACTT-3') and IYSV-R (5'-TAATTATATCTATCTTTCTTGG-3') (1) was used for RT-PCR. The primer pair was specific to amplify 797 bp of the nucleocapsid (N) gene of IYSV. The amplified product derived from A. tuberosum isolate was purified by QIAquick PCR Purification Kit (Qiagen) and cloned using the vector pDrive (Qiagen). The recombinant clone was sequenced (Accession No. KF624624). Sequence analysis performed on CLC Main Workbench Version 6.8.4 confirmed that the fragment was of IYSV. Nucleotide sequence comparison of our virus with other IYSV isolates revealed that the highest nucleotide identity (99%) was with the IYSV garlic isolate (HM173691) from India. Further, maximum 96% protein identity was with IYSV onion isolate (ACA09432) and garlic isolate (ADK56108) from India. To our knowledge, this is the first report of IYSV naturally occurring on A. tuberosum in India. It is evident from previous studies that IYSV causes significant losses in onions (1) and from this study, that its symptoms have direct impact on quality of garlic chives. Further detailed studies are required to assess the magnitude of the impact of IYSV infection on yield and quality of A. tuberosum.

Striking differences in the biological and molecular properties of onion and garlic isolates of onion yellow dwarf virus

Complete nucleotide (nt) and deduced amino acid sequences of two onion yellow dwarf virus (OYDV) isolates showing mild and severe symptoms in onion but being unable to infect garlic were determined. The genomes consisted of 10,459 and 10,461 nt (without the 3' poly(A) tail) and were 92.2 % identical. Comparison of their whole genomes, polyproteins and P1, HC-Pro, P3, CI, VPg and NIa-Pro regions with those of garlic isolates previously identified as OYDV gave percentage values below that proposed as the molecular threshold for potyvirus species demarcation. This and the striking differences in host range between onion and garlic isolates suggest that they represent different virus species.

First Report of Tomato spotted wilt virus Infecting Onion and Garlic in Serbia

In June 2011, extensive bleaching and numerous small whitish spots on leaves were observed in an onion (Allium cepa) seed crop as well as chlorotic spots and streaks in the neighboring garlic (A. sativum) bulb crop in the Aleksandrovo locality (Central Banat District, Serbia). Affected plants occurred throughout the field and disease incidence was estimated at 60% in the onion and 40% in the garlic crop. A high population of Thrips tabaci that was found in both crops, and local necrotic spots on Petunia × hybrida mechanically inoculated with infected onion or garlic sap by a chilled 0.01 M phosphate buffer, pH 7.0, containing 0.1% sodium sulfite (1), suggested the presence of a Tospovirus. For these reasons, sampled symptomatic onion and garlic plants were tested for the presence of Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) using commercial double-antibody sandwich-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy onion and garlic tissue were included in each ELISA. Of the 18 onion and 10 garlic plants tested, 16 and 7 samples, respectively, were positive for TSWV, and all were negative for IYSV. The identity of TSWV was further confirmed by conventional reverse transcription (RT)-PCR analysis. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using TSWV-specific forward (5'-GGTTAAGCTCACTAAGAAARCA-3') and reverse primers (5'-TTTAACYCCRAACATTTCATAGA-3'), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs obtained from plants infected with a Serbian isolate of TSWV (GenBank Accession No. GQ373173) and healthy onion garlic plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from the 16 onion and 7 garlic ELISA-positive plants, but not from healthy controls. The amplified products derived from the two selected isolates, 114-11 from onion and 115-11 from garlic, were sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen); the sequences obtained were allocated GenBank Accession Nos. JQ619234 and JQ619235, respectively. Sequence analysis of the partial N gene, conducted with MEGA5 software (4), revealed 99.9% nucleotide identity (100% amino acid identity) between the two Serbian Allium isolates. Serbian onion and garlic isolates showed the highest nucleotide identities of 100% and 99.9% with Serbian summer squash isolate (JF303081) and tobacco isolate from Montenegro (GU369729), respectively. Well-established in many European countries, TSWV has been reported as an important constraint to the production of tomato, pepper, tobacco, and ornamentals (2), but the information on TSWV naturally infecting Allium spp. is limited. The presence of TSWV on onion and garlic in Serbia revealed that its known host range has expanded in Europe. To our knowledge, other than Marchoux's unpublished data (3), there are no other reports of garlic as a natural host of TSWV. The TSWV presence on Allium spp. represents a serious threat for these crops in Serbia, considering that it is prevalent in other crops in the area and its vectors are widespread.

First Report of Embellisia allii Causing Skin Blotch and Bulb Canker on Garlic in California

In April 2011, commercial garlic (Allium sativum) in Monterey County, CA showed symptoms of an undocumented disease. Bulb and stem sheaths were dark, decayed, and sloughing off the plants. Dissection of diseased sheaths revealed black hyphae between layers. Lower leaves wilted, turned tan, and dried up. Disease occurred in small patches scattered in two fields. In the patches, disease incidence was as much as 50%; however, overall field incidence was less than 1%. Isolations from 80% (16 of 20 plants) of collected plants resulted in the recovery of a dark olivaceous black fungus. Conidiophores were geniculate and brown and conidia were borne singly, brown, and ellipsoidal to cylindrical. Conidia had two to five but mostly three transverse septa. Longitudinal septa were infrequent and apical cells were rounded. Conidia measured (19.0-) 26.3 to 36.6 (-42.8) × (6.7-) 9.2 to 9.9 (-12.9) μm. Dark, intercalary chlamydospores were observed as colonies aged. DNA sequencing of the internal transcribed spacer (ITS) regions of four, single-spored isolates was completed with primers ITS1 and ITS4 (3). Sequences of all isolates (GenBank Nos. JN588614 to JN588617) were identical and 100% similar to Embellisia allii (AY278840). On the basis of morphological and molecular data, the fungus was identified as E. allii (Campanile) Simmons (1). Pathogenicity of four of the sequenced E. allii isolates and one additional E. allii isolate was tested using inoculum grown on acidified potato dextrose agar and garlic (cv. California Late) planted into 15-cm pots. A transverse incision was made at a point 2 cm above the garlic bulb so that a colonized agar plug could be inserted between the second and third sheath layer. The stem was then wrapped with Parafilm. Ten plants per isolate were inoculated and kept in a greenhouse (24 to 26°C). Seven to eight days after inoculation, the tissue around the incision turned tan and dark fungal growth was observed. Fourteen days after inoculation, the inoculated area was necrotic and dark fungal growth developed between stem layers. E. allii was reisolated from all inoculated plants and matched the morphological characteristics of the original isolates. Control plants, inoculated with uncolonized agar plugs, developed no symptoms. This experiment was repeated with similar results. In addition, one isolate was used to inoculate leek (A. porrum cv. Lancelot) and onion (A. cepa cv. Evergreen). Similar symptoms developed on these two species and E. allii was reisolated from all plants. To our knowledge, this is the first documentation of skin blotch and bulb canker caused by E. allii on garlic in California. Affected plants were of poor quality and could not be harvested. Our findings that garlic isolates of E. allii can infect leek and onion provide preliminary evidence that this pathogen is not restricted to garlic; this information may be useful to growers when considering crop rotations. E. allii has been reported on garlic in a number of places in Africa, Asia, Europe, the Middle East, and North and South America (2). The sequenced E. allii isolates are deposited in the fungal collection at the CDFA Plant Pest Diagnostics Lab (CDFA798-801).

Molecular Characterization of Onion Yellow Dwarf Virus (Garlic Isolate) with Production of Virus-free Plantlets

Molecular Characterization of Onion Yellow Dwarf Virus (Garlic Isolate) with Production of Virus-free Plantlets

First Report of Iris yellow spot virus on Garlic in India.

Garlic (Allium sativum) is a spice crop of prime importance in India as well as other parts of the world. Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) is an important pathogen of onion bulb and seed crops in many parts of the world (3). The virus is also known to infect garlic and other Allium spp. (2-4). IYSV infection of garlic was reported from Reunion Island (4) and the United States (1). In February 2010, straw-colored, spindle-shaped spots with poorly defined ends were observed on the leaves of a garlic crop at the research farm of the Directorate of Onion and Garlic Research in the Pune District of Maharashtra State, India, 105 days after planting. The spots coalesced to form larger patches on the leaves, suggesting possible IYSV infection. Symptoms were visible on older leaves and more prevalent on cv. G-41, G-282, AC50, AC200, AC283, and Godavari than on other cultivars. The incidence of symptomatic plants was estimated at 5% for G-41 and AC-200, 8% for G-282 and AC283, and 10% for AC50. Leaves were sampled from 40 symptomatic plants per cultivar with each sample composited from young, middle, and older (basal) leaves of the plant. Samples were assayed by double-antibody sandwich-ELISA (Loewe Biochemica GmbH, Sauerlach, Germany) and each tested positive for the virus. Total RNA was extracted from the leaves of ELISA-positive plants using the RNAeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany) and tested by reverse transcription-PCR assay using primers IYSV-F (5'-TCAGAAATCGAGAAACTT-3') and IYSV-R (5'-TAATTATATCTATCTTTCTTGG-3') (2) designed to amplify 797 bp of the nucleocapsid (N) gene of IYSV. Amplicons of expected size were obtained and cloned into a pDrive vector (Qiagen GmbH). The recombinant clone was sequenced (GenBank Accession No. HM173691). Sequence comparisons showed 98 to 100% nt identity with other IYSV N gene sequences in GenBank (Nos. EU310294 and EU310286). A phylogenetic analysis of the deduced amino acid sequences of the N gene showed that the garlic isolate of IYSV grouped most closely with onion IYSV isolates from India (GenBank Nos. EU310294, EU310286, EU310300, and EU310296). To our knowledge, this is the first report of natural infection of garlic by IYSV in India. Additional surveys and evaluations are needed to obtain a better understanding of the potential impact of IYSV on garlic production in India.

First Report of Shallot latent virus in Garlic in Argentina.

Because of exclusively agamic propagation, garlic is commonly infected with a virus complex mainly composed of species within the genera Potyvirus, Allexivirus, and Carlavirus. This virus complex causes leaf striping that ranges from various shades of green to yellow and results in yield losses (2,4). Onion yellow dwarf virus, Leek yellow stripe virus (potyviruses), Garlic virus A, Garlic virus C (allexiviruses), and Garlic common latent virus (carlavirus) have been detected in Argentina previously (1,2). Recently, Shallot latent virus (SLV; another carlavirus) was detected in 25 of 30 garlic plants (cv. Morado) growing in four different fields near Córdoba, Argentina by double-antibody sandwich (DAS)-ELISA using BIOREBA (Reinach, Switzerland) antibodies. To confirm the presence of the virus, DAS-ELISA-positive plants were also analyzed by one-step reverse transcription (RT)-PCR using the Access RT-PCR system (Promega, Madison, WI) with specific primers reported by Tsuneyoshi et al. (3). RNA extractions were performed from 100 mg of leaves with the Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Primers used were Car-V1 (5'-AAACCTTTTGGTTCACTTTAGG-3'); Car-V2 (5'-AGGTGCATTGTTATCATTACTGG-3'); and Car-Cp3 (5'-GCGTGCTATATTTAAGTTGCATAC-3'). Primer pairs Car-V1/Car-Cp3 and Car-V2/Car-Cp3 were used for the amplification of the coat protein (CP) gene of SLV and an isolate of SLV formerly known as Garlic latent virus, respectively. Fragments of 992 bp and 1,079 bp were amplified with these primer pairs, respectively. The RT-PCR products were cloned with the TOPO TA Cloning Kit in the 3.9-kb pCR-TOPO vector (Qiagen). The nucleotide sequences of both fragments were determined and were found to be identical (GenBank No. GU355922) showing 94.2% nt sequence identity with the CP gene of an isolate of SLV from Indonesian garlic (GenBank No. AB004686) formerly referred to as Garlic latent virus (3). Consequently, the Argentinean virus is now considered a garlic isolate of SLV.

Host range and phytotoxicity of Stemphylium solani, causing leaf blight of garlic (Allium sativum) in China

Since 2004, a new leaf blight disease on garlic of high severity has been observed in Dangyang County, Hubei province, China. Initial symptoms consisted of multiple, small, irregular to oval, white leaf spots, which enlarge to produce sunken purple lesions, sometimes surrounded by a bright yellow margin. As the disease progressed, lesions expanded and merged, resulting in withering of leaf tips. After isolation and pathogenicity testing, the causal agent of leaf blight of garlic was identified as Stemphylium solani from cultural and morphological characteristics, and subsequent analysis of the internal transcribed spacer region of ribosomal DNA. When fungal plugs of two S. solani isolates were inoculated onto 11 garlic cultivars and 20 other crop species, leaf spots appeared on all inoculated plants, but two garlic cultivars (Qingganruanye and Ruanruanye) and three crop species (Capsicum annuum, Brassica napus and Amaranthus mangostanus) showed the smallest leaf spots. In cross-inoculation experiments, no indications of host specificity were observed, but S. solani isolated from garlic was generally the most virulent on five plant species, while the isolate from leek (Allium odorum) was generally the least virulent. Toxicity testing of the crude culture filtrates indicated that garlic isolates produced toxin(s) that were not heat-labile and induced different levels of phytotoxicity toward various garlic cultivars and crops.

Complete nucleotide sequences of attenuated and severe isolates of Leek yellow stripe virus from garlic in northern Japan: Identification of three distinct virus types in garlic and leek world-wide

We have determined the complete nucleotide sequence of three isolates (two severe and one attenuated) of the potyvirus Leek yellow stripe virus (LYSV), the main causal agent of the garlic mosaic disease that is prevalent in Aomori prefecture, northern Japan. They contained 10,296-10,297 nucleotides and encoded a deduced polyprotein of 3,215 amino acids. Sequence variation among the three isolates was 1.5% at both the nucleotide and amino acid levels. Surprisingly, the sequences of all three isolates were quite different from that of recently described garlic isolate of LYSV from China. Sequence similarities among the 5'-UTR and P1 regions were only 56-57% and 52-53%, respectively, raising the possibility that these isolates may actually be distinct virus species. Phylogenetic analyses using 5'-UTR, P1 protein and coat protein revealed that all LYSV isolates described to date can be divided into three groups; i.e., those from leek, those from garlic in northern Japan, and those from garlic in southern Japan, China, and other parts of the world. The garlic isolates from northern Japan were more closely related to LYSV isolates from leek than to garlic isolates from southern Japan and China, suggesting a different origin for the garlic cultivars now cultivated in northern Japan.

Inhibitory effect of garlic on bacterial pathogens from spices

An unconventional technique for primary screening of bacterial susceptibility to garlic (Allium sativum Linn.), using a slice from its clove, was described. Aqueous extracts of garlic were found to possess a potent bacteriostatic principle against Gram-positive as well as Gram-negative foodborne bacterial pathogens. In agar medium, the minimum inhibitory concentrations (MICs) of garlic were 6–10 mg ml−1 for Bacillus cereus, 30–40 mg ml−1 for Staphylococcus aureus (excepting the isolate from garlic, where the MIC was 100 mg ml−1), 20–30 mg ml−1 for Clostridium perfringens, 10 mg ml−1 for Escherichia coli (30 mg ml−1 for the garlic isolate), 40–100 mg ml−1 for Salmonella, and 10–40 mg ml−1 for Shigella. It inhibited the growth of all these strains, which were resistant to some commonly used antibiotics. Most of the tested strains were resistant to penicillins, although sensitive to garlic. While the growth of B. cereus and Cl. perfringens was completely inhibited at 10 and 70 mg garlic, respectively, ml−1 test broth, their respective enterotoxin production ceased at 10 and 50 mg garlic ml−1.

First Report of Bulb Canker of Garlic Caused by Embellisia allii in Korea

Embellisia allii causing bulb canker of white garlic (Allium sativum) has been observed on harvested or stored garlic bulbs. Identification of the fungus was determined based on the symptoms and morphological characteristics on in vitro potato dextrose agar (PDA) and malt extract agar (MEA). Colonies were effuse, blackish brown to black, velvety or powdery. Conidia were solitary, almost straight, most commonly ellipsoidal (or subcylindrical) to ovoid, mid to dark brown, smooth, with very dark 1 to 6 transverse (commonly 3~4) and occasionally 1 or 2 oblique or longitudinal septa, and 30.4×13.4 μm (av.). Conidiophores were simple or branched, straight or flexuous, up to 80 μm long and 5.1~10.2 μm thick. Chlamydospores were developed through enlargement and repeated cellular division of several adjacent hyphal cells to form a complex of thickwalled cells. The conidial measurements of garlic isolates closely matched the previous description of E. allii.