Almond Isolates Research Articles

Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Aggressiveness of Spanish Isolates of Xylella fastidiosa to Almond Plants of Different Cultivars Under Greenhouse Conditions.

The aggressiveness of Spanish isolates of Xylella fastidiosa, representing different sequence types, were studied in almond plants of several cultivars by means of the dynamics of the population levels and symptoms, colonization and spread, and dose-effect relationships. Pathogen dynamics in almond plants under greenhouse conditions showed doubling times of 2.1 to 2.5 days during the exponential growth phase, with a maximum population size of about 35 days postinoculation (dpi). Differences in patterns in population dynamics were observed between sap and xylem tissue after the exponential growth, as population levels in the xylem tissue remained stable while viable cells in sap decreased. Population levels were higher in two upward zones than in the downward zone with respect to the inoculation area. The first symptoms were observed between 20 and 60 dpi, and disease severity increased over time at doubling times of 30 days, with a maximum observed at 120 dpi. Strains tested showed differences in population levels in the cultivars studied and were able to spread with different intensity from contaminated plant parts to new growing shoots after pruning. Two almond isolates showed different performance in dose-effect relationships when inoculated in cultivar Avijor. Whereas IVIA 5387.2 reached high population levels but showed high median effective dose (ED50) and minimal infective dose (MID) values, IVIA 5901.2 showed low population levels and low ED50 and MID values. This study has implications for the epidemiology of X. fastidiosa in almond crops, estimating doubling times of the pathogen in planta and of symptom development and showing differences in aggressiveness between strains.

Molecular characterisation of little cherry virus 1 infecting apricots in the Czech Republic

Little cherry virus 1 is one of the pathogens associated with little cherry disease of cherries. The host range of the virus is widening and recently it has been detected in apricot trees in the Czech Republic, Hungary, and Morocco. To obtain a view about the importance of apricots in the epidemiology of the virus, a survey was conducted in Czech apricot-growing areas. Little cherry virus 1 was detected in seven out of 13 locations, and the infection was noted predominantly in old orchards, but its frequency was low. During the survey, an infected almond tree developing severe leaf mosaic was also detected. Using high-throughput sequencing and Sanger sequencing, five near-complete genome sequences and another 20 partial ORF8 sequences were obtained. Phylogenetic analysis showed a close relationship of most of the apricot isolates to the phylogenetic group G3 with UW2, UW1, and ITMAR isolates. Three apricot isolates, including the almost complete Apr 184R isolate and almond isolate Alm138, showed homology within the phylogenetic group G5. The analysis clearly demonstrates the presence of isolates of this type in Europe. The 4-year-long survey of selected pomological characteristics showed that infection with the virus is generally latent, but the infection could significantly affect the ripening of the fruit of some apricot genotypes. A significant decrease in the fruit yield was noticed for ‘Magyar Kajszi’. The potential routes of the transmission of little cherry virus 1 to apricots are discussed in the context of propagation practice.

First Report of Phytophthora cinnamomi Causing Trunk Canker of Almond in California

Almond (Prunus dulcis) is the most economically important nut crop in California, accounting for $5.9 billion value in 2016. During fall of 2015, declining 2-year-old almond trees on clonal Hansen 536 (peach × almond) rootstock were observed in a commercial orchard in Kern County, California. Affected trees expressed early defoliation with amber-colored gummosis on the trunk. Cankers developed at the bud union and extended into the scion in trees planted with their bud union placed at or below the soil surface. To isolate the causal agent, necrotic pieces of bark and subtending vascular tissue were cut from the canker margin, surface sterilized in 0.5% sodium hypochlorite solution for 3 min, rinsed twice in sterile water, and plated on corn meal agar amended with selective antibiotics (PARP) (Jeffers and Martin 1986). After several days of incubation at 25°C, Phytophthora-like colonies grew from the diseased tissues and were transferred onto acidified potato dextrose agar medium (APDA). The isolates were tentatively identified as Phytophthora cinnamomi Rands based on morphological characteristics. Sporangia were nonpapillate and ovoid to obpyriform or ellipsoidal to elongate-ellipsoid with an apical thickening, avg. 57.2 × 36.6 µm with a length-width ratio of 1.56; mycelium with abundant coralloid hyphal swellings. Globose chlamydospores formed abundantly in culture on the parent hyphae or on new hyphal branches (18 to 42 [avg. 27.65] μm in diameter) (Erwin and Ribeiro 1996). For all isolates, the internal transcribed spacer (ITS) regions of the rRNA gene and a portion of the β-tubulin (TUB) gene were amplified and sequenced with primers ITS1/ITS4 and TUBUF2/TUBUR1 (Lan et al. 2016), respectively. Two representative sequences for each locus were deposited in GenBank (ITS; KY924650 and KY924651, TUB; KY924648 and KY924649). BLASTn analysis against Phytophthora Database (www.phytophthoradb.org) showed that the ITS sequences had 99% identity and the β-tubulin sequences had 97 to 98% identity to reference sequences of P. cinnamomi (ITS: GU259227; TUB: EU079894). Two of the almond isolates of P. cinnamomi were tested for pathogenicity by wound inoculating 2-year-old potted almond trees of cv. Nonpareil on rootstocks of Hansen 536 and Nemaguard (Prunus persica × Prunus davidiana). Five-millimeter-diameter mycelial disks from PDA cultures of each isolate (and sterile PDA as a control) were used for inoculating wounds of the same size (5 mm) made in stems of: i) the almond scion on Hansen 536 rootstock, ii) the almond scion on Nemaguard rootstock, iii) Hansen 536 rootstock below the scion, and iv) Nemaguard rootstock below the scion. Five replicate trees were inoculated per combination of the three inoculants (two of P. cinnamomi, one control) and the four inoculation treatments in a randomized complete block design. After inoculation, the wounds were covered with petroleum jelly and wrapped with Parafilm. Two months after inoculation, all plants inoculated with the control remained healthy, whereas all scions and rootstocks inoculated with either isolate developed stem cankers. None of the trees inoculated on the rootstocks died during the experiment, but 40% of the trees inoculated on the scion were killed. P. cinnamomi was reisolated only from the infected plants. More than 10 other species of Phytophthora were reported to cause root and crown rots as well as aerial cankers on almond (Browne and Viveros 1999; Browne et al. 2015). To our knowledge, this is the first report of P. cinnamomi causing trunk canker disease of almond in California.

Morphological, Pathogenic, and Molecular Characterization of Colletotrichum acutatum Isolates Causing Almond Anthracnose in Spain.

Almond anthracnose is a serious and emerging disease in several countries. All isolates causing almond anthracnose have been assigned to the Colletotrichum acutatum species complex, of which only C. fioriniae and C. godetiae have been associated with the disease to date. Here, we characterized Colletotrichum isolates from almond fruit affected by anthracnose in the Andalusia region. Two Colletotrichum isolates causing olive anthracnose were included for comparison. Morphological characteristics were useful for separating the isolates into groups based on colony morphology. Pathogenicity tests in almond, olive, and apple fruit showed differences in virulence and some degree of pathogenic specialization among isolates. Molecular characterization allowed clear identification of the Colletotrichum isolates tested. The olive isolates were identified as C. godetiae and C. nymphaeae, both previously identified in Andalusian olive orchards. Two phylogenetic species were identified among the almond isolates: C. godetiae, with gray colonies, which is well known in other countries, and C. acutatum, with pink-orange colonies. This species identification differs from those of pink-colony subpopulations described in other countries, which are C. fioriniae. Therefore, this study is also the first report of a new species of Colletotrichum causing almond anthracnose within the C. acutatum species complex.

Phenotypic and genetic characterization of Pseudomonas syringae strains associated with the recent citrus bacterial blast and bacterial black pit epidemics in Tunisia

In the spring of 2012, symptoms of a disease resembling citrus blast and citrus black pit were observed in some orchards in Tunisia. The epidemic spread rapidly in the following years. Twenty‐four commercial citrus orchards from four Tunisian regions showing characteristic symptoms of bacterial diseases were surveyed during a 3‐year study. Eighty‐eight Pseudomonas‐like bacterial isolates were successfully obtained from the northeast and west of Tunisia. No isolates were recovered from the central region. Overall, 46 isolates were identified as Pseudomonas syringae pv. syringae and most of them showed similar phenotypic and genetic profiles. The virulence of three selected isolates differed from one plant cultivar to another as well as from the type of plant organ used for the inoculation. In a bioassay test, all isolates produced syringomycin, which was confirmed by molecular detection based on the syrB and syrD genes. Only EC122 possessed syrD but not syrB. DNA fingerprints, based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) and PCR melting profile (PCR MP), were used to determine the potential genetic diversity among strains. Clustering of PCR MP fingerprinting data matched with rep‐PCR fingerprinting data. The generated distribution tree showed that Tunisian isolates were closely related to the citrus reference strain LMG5496. In contrast, EC112, isolated from citrus, and the almond isolate EC122 were distantly related to the type strain LMG1247T isolated from lilac. Such studies have not been reported until now for P. syringae from citrus.

Molecular variability of Prunus necrotic ringspot virus isolates from different Prunus hosts in Tunisia.

Prunus necrotic ringspot virus (PNRSV) was detected by PCR in Tunisian stone fruit germplasm collections. Peach had the highest infection rate reaching 50%, followed by apricot (46.8%), almond (35.5%) and plum (9.5%). The genetic variability of the CP gene of 10 PNRSV isolates was evaluated by SSCP analysis of PCR products and analysis of their sequence. In phylogenetic trees, Tunisian PNRSV isolates clustered in the three major groups, PV96, PV32 and PE5. However, an almond isolate (AlTun8) segregated as an out-group. This isolate is phylogenetically distant from other PNRSV isolates from different parts of the world and displays nucleic and amino acid identities of 83-86% and 65-74%, respectively, with PV96, PE5 and PV32. This is the first report on the molecular variability of PNRSV in different Prunus hosts in Tunisia.

Genetic variability among populations of Fusicladium species from different host trees and geographic locations in the USA

Peach and almond scab caused by Venturia carpophila and pecan scab caused by Fusicladium effusum result in yield loss, downgrading of fruit, defoliation and subsequent decline of an orchard. To understand the levels of genetic diversity and divergence of pathogens from different hosts and locations 51 isolates were genotyped and analyzed using 10 RAPD and 5 UP-PCR markers, including 18 isolates of V. carpophila from peach trees in the southeastern United States, 12 isolates of V. carpophila from almond trees in California, and 21 isolates of F. effusum (a related species) from pecan trees in the southeastern United States. The combined marker results showed a low incidence of polymorphisms among the peach isolates (4.2 % of markers), but a higher incidence of polymorphisms among the almond isolates (42.0 %) and the pecan isolates (61.0 %). The Dice coefficient of similarity ranged from 0.932 to 1.000 for the peach V. carpophila isolates, 0.214 to 0.976 for the almond V. carpophila isolates, and 0.528 to 0.920 for the pecan F. effusum isolates. UPGMA bootstrap values indicated that UP-PCR data were slightly more robust and, based on the combined data, the UPGMA bootstrap analysis (1,000 runs) gave a high node value (100 %) differentiating all the isolates of V. carpophila from F. effusum and a moderate node value differentiating the peach and almond isolates of V. carpophila (68 %). The results suggest some divergence between the V. carpophila populations of almond trees in California and peach tree populations in the southeastern United States, and different levels of genetic diversity within the two populations.

Detection and partial molecular characterization of Plum pox virus on almond trees in Turkey

Almond (Prunus dulcis) is one of the well known stone fruit species grown for its unripe fruits and delicious seeds in Turkey. In the Trakya region, however, some prevailing virus infections have reduced almond yields and quality. In ten districts of Trakya, 260 leaf samples were collected from affected almond trees in June 2010. DAS–ELISA assays and RT-PCR tests were employed for the identification of viruses. As a result of these detection studies, five of the 260 leaf samples gathered from symptomatic almond trees had Plum pox virus (PPV), 81 of them had Prunus necrotic ringspot virus (PNRSV), and 11 samples contained Prune dwarf virus (PDV). Only four out of 260 samples had a mixture of these viruses. Partial nucleotide sequences of five almond isolates of PPV were determined and compared with 17 other PPV isolates in databases. Computer analysis of obtained and published nucleotide sequences showed identity ranged from 75.72% to 96.87%. Of the five PPV almond isolates obtained, however, there was a close nucleotide identity of 95.82–96.61% to Turkish isolates. Phylogenetic analysis of nucleotides and amino acids showed that five PPV isolates of almond from the Trakya Region of Turkey were clustered in the same subgroup with PPV-T Turkish isolates in GenBank. Therefore we can consider almond isolates of PPV as PPV-T strain, like the two other isolates from apricot trees in Turkey.

Host specificity, pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.

Pistachio (Pistacia vera L.) is a new natural host of Hop stunt viroid

Besides hop, Hop stunt viroid (HpSVd) infects many woody species including grapevine, citrus, peach, plum, apricot, almond, pomegranate, mulberry and jujube. Here, we report the first detection of HpSVd in pistachio (Pistacia vera L.). Samples corresponding to 16 pistachio cultivars were obtained from a nearby almond collection. From these samples, low molecular weight RNAs were extracted for double polyacrylamide gel electrophoresis, northern-blot analysis and reverse transcription polymerase chain reaction assays. HpSVd was detected in 4 of the 16 pistachio cultivars in the first year and in 6 in the second, being also detected in the almond collection. Examination of the nucleotide sequences of pistachio and almond isolates revealed 13 new sequence variants. Sequences from pistachio shared 92-96% similarity with the first reported HpSVd sequence (GenBank X00009), and multiple alignment and phylogenetic analyses showed that one pistachio isolate (HpSVdPis67Jabari) clustered with the plum group, whereas all the others clustered with the hop, and the recombinants plum-citrus and plum-Hop/cit3 groups. By identifying pistachio as a new natural host, we confirm that HpSVd is an ubiquitous and genetically variable viroid that infects many different fruit trees cultivated worldwide.

Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus

The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima's D, and Fu and Li's D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

Strain origin drives virulence and persistence ofXylella fastidiosain alfalfa

Generalist pathogens frequently exist as a complex of genetically differentiated strains, which can differ in virulence and transmissibility. A description of the extent to which strain variability mediates host species competence is needed to understand disease dynamics for systems with both host and pathogen strain diversity. This study tested the hypothesis that strain‐specific variation of a generalist vector‐borne plant pathogen,Xylella fastidiosa, affects disease severity in alfalfa (Medicago sativa) and competence of this crop as a reservoir host. Alfalfa seedlings were inoculated with one of 23X. fastidiosaisolates collected from different hosts, eight identified as belonging to an almond strain, and the remainder from a grape strain. Pathogen population, symptom severity and infection incidence were compared over five successive harvests. Infected plant size, measured mainly by plant height, internode length and above ground biomass, was reduced up to 50% compared to buffer‐inoculated controls, and more severe symptoms were observed at later harvests and for higher pathogen populations. Grape isolates had higher bacterial populations within alfalfa than almond isolates. In addition, infection with grape isolates resulted in more severe alfalfa stunting than that caused by almond isolates. Moreover, there was a strong positive relationship between isolate multiplication rate and both symptom severity and infection persistence (i.e. maintenance of chronic infection within host). Isolates with low initial populations had low incidence at the final harvest, with one isolate dying out altogether. The results showed thatX. fastidiosa‐genetic diversity contributed to variation in alfalfa disease severity. The results also suggest that pathogen strain may mediate host competence via differences in bacterial population density and persistence.

Strain-specific alfalfa water stress induced by Xylella fastidiosa

Differences in the virulence of a pathogen among host species can occur because hosts differ in their resistance or tolerance to infection or because of underlying genetic variation in the pathogen. The xylem-limited bacterium Xylella fastidiosa is pathogenic to dozens of plant species throughout the Americas, and is structured into genetically and biologically distinct strains. In some plants X. fastidiosa causes striking leaf scorch symptoms and in others, such as alfalfa, stunting is the primary symptom. The mechanism by which these symptoms occur has been debated. We tested the hypothesis that symptoms result from X. fastidiosa-induced water stress, and that the magnitude of water stress is strain-dependent. We mechanically inoculated alfalfa plants with one of 14 isolates (5 identified genetically as “almond” and 9 as “grape” isolates), and compared stable carbon isotope ratios among isolates. Infected plants showed significant isotopic shifts (up to 2% on average) relative to healthy plants that were consistent with water stress. Moreover, there were significant differences in water stress among isolates, with a tendency for grape isolates to cause more severe water stress than almond isolates. There was also a positive relationship between plant infection level and isotopic shift (slope ± SE = 0.273 ± 0.068), which supports the hypothesis that X. fastidiosa symptoms result from bacterial multiplication and vessel occlusion. Unexpectedly, however, water stress was not correlated with measures of alfalfa stunting. These results suggest X. fastidiosa induces strain-specific levels of water stress, but factors other than water stress alone are responsible for stunting.

Context‐dependent transmission of a generalist plant pathogen: host species and pathogen strain mediate insect vector competence

AbstractThe specificity of pathogen–vector–host interactions is an important element of disease epidemiology. For generalist pathogens, different pathogen strains, vector species, or host species may all contribute to variability in disease incidence. One such pathogen is Xylella fastidiosa Wells et al., a xylem‐limited bacterium that infects dozens of crop, ornamental, and native plants in the USA. This pathogen also has a diverse vector complex and multiple biologically distinct strains. We studied the implications of diversity in this pathogen–vector–host system, by quantifying variability in transmission efficiency of different X. fastidiosa strains (isolates from almond and grape genetic groups) for different host plants (grape, almond, and alfalfa) by two of the most important vectors in California: glassy‐winged sharpshooter [Homalodisca vitripennis (Germar)] and green sharpshooter (Draeculacephala minerva Ball) (both Hemiptera: Cicadellidae). Transmission of isolates of the almond strain by H. vitripennis did not differ significantly, whereas transmission varied significantly among isolates from the grape strain (15–90%). Host plant species did not affect H. vitripennis transmission. Conversely, D. minerva efficiency was mediated by both host plant species and pathogen strain. No acquisition of an almond isolate occurred regardless of plant type (0/122), whereas acquisition of a grape isolate from alfalfa was 10‐fold higher than from grape or almond plants. These results suggest that pathogen, vector, and host diversity impose contingencies on the transmission ecology of this complex disease system. Studies aimed at the development of management strategies for X. fastidiosa diseases should consider the complexity of these interactions as they relate to disease spread.

Molecular characterization of an almond isolate of Prune dwarf virus in Tunisia: putative recombination breakpoints in the partial sequences of the coat protein-encoding gene in isolates from different geographic origin

Recombination is an important driving force in evolution. To investigate this evolutionary process in Prune dwarf virus (PDV), 31 accessions retrieved from international databases, supplemented by one Tunisian isolate described here, were analyzed. Two recombination detection programs were used: RDP v3.31β and RECCO. While the first program did not detect any significant recombination events, RECCO detected several breakpoints in partial sequences of the coat protein gene (CP) of fifteen isolates. The Tajima neutrality test implemented in the MEGA4.1β program indicated that there were numerous deletion/insertion events in the sequences. The strongest signal was found in Portuguese isolate 3.12N.14 (accession number AY646846) which had position nt 296–523 in the CP gene. The evolutionary historical relationships between all analyzed isolates were determined by constructing a dendrogram using Neighbor joining (NJ), Minimum evolution (ME), Maximum parsimony (MP), Maximum likelihood (ML), and the Unweighted pair group method with arithmetic mean (UPGMA); all gave similar results. Two main clusters were delineated, one representing recombinant and one nonrecombinant isolates. The recombinant isolates were mostly collected from Portugal, whereas the nonrecombinant isolates originated in eastern Europe.

Characterisation ofApple chlorotic leaf spot virus infecting almonds in India

Restricted field surveys were conducted to determine the presence and incidence of virus infections in almonds. Representative asymptomatic samples from six locations were tested by enzyme linked immunosorbent assay and reverse transcription and polymerase chain reaction for known stone fruit viruses. OnlyApple chlorotic leaf spot virus (ACLSV) infection was detected in one of the six almond samples from the state of Himachal Pradesh. Comparison of sequenced amplicons with two available sequences of ACLSV-almond isolates revealed 92% and 94% amino acid identity. However, the Indian ACLSV-almond isolate shared maximum (98%) homology at the amino acid level to ACLSV-peach (AM498050) and wild apricot (AM498048) isolates from India. The C-terminus is highly conserved within the Indian ACLSV isolates with the exception of a few residues which in the case of the Indian almond isolates are more typical of peach and wild apricot isolate sequences.

Characterisation ofApple chlorotic leaf spot virusinfecting almonds in India

Restricted field surveys were conducted to determine the presence and incidence of virus infections in almonds. Representative asymptomatic samples from six locations were tested by enzyme linked immunosorbent assay and reverse transcription and polymerase chain reaction for known stone fruit viruses. Only Apple chlorotic leaf spot virus (ACLSV) infection was detected in one of the six almond samples from the state of Himachal Pradesh. Comparison of sequenced amplicons with two available sequences of ACLSV-almond isolates revealed 92% and 94% amino acid identity. However, the Indian ACLSV-almond isolate shared maximum (98%) homology at the amino acid level to ACLSV-peach (AM498050) and wild apricot (AM498048) isolates from India. The C-terminus is highly conserved within the Indian ACLSV isolates with the exception of a few residues which in the case of the Indian almond isolates are more typical of peach and wild apricot isolate sequences.

Genetic characterization of Xylella fastidiosa isolated from citrus and coffee plants

The Citrus Variegated Chlorosis and the Coffee Leaf Scorch are some of the many destructive diseases caused by Xylella fastidiosa, a gram-negative bacterium limited to the xylem of affected plants. As its genetic characterization is still not well established, different isolates of X. fastidiosa from citrus and coffee were evaluated through RAPD (Random Amplified Polymorphic DNA) technique to characterize and classify these isolates based on similarity coefficients. Sixteen isolates of X. fastidiosa were used on this trial, obtained from citrus, coffee and almond. The genetic polymorphism evaluation was performed using six arbitrary 10-base primer pairs. It was possible to establish a dendogram in which the isolates were classified into five groups (A, B, C, D and E). A prevalence of citrus isolates in groups A and D was observed. In groups B and C, there was a prevalence of coffee isolates meanwhile the group D consisted of the almond isolate, solely.

Genetic and Pathogenic Variation in Phytophthora cactorum Affecting Fruit and Nut Crops in California

Isolates of Phytophthora cactorum and 15 other species of Phytophthora were characterized according to their genomic DNA, pathogenicity, and sensitivity to mefenoxam. Amplified fragment length polymorphism (AFLP) analysis was completed for 132 isolates of P. cactorum (30 from almond, 86 from strawberry, 5 from walnut, and 11 from other hosts) and 22 isolates of 15 other Phytophthora spp. from various hosts. All 16 Phytophthora spp. were distinguishable by unique AFLP banding patterns. Cluster analysis of the AFLP data revealed high coefficients of genetic similarity (>0.9) among all California isolates of P. cactorum. Analysis of molecular variance indicated that, among all 132 isolates of P. cactorum, 30.8 and 24.5% of the AFLP variation was associated with hosts and geographical sources of isolates, respectively, whereas 15.0% of the variation was associated with isolate niche (i.e., an aerial plant part, portion of the root system, or soil). Among the 86 isolates of P. cactorum from strawberry, characterization by source in the production system (i.e., fruiting field or plant nursery) did not account for a significant proportion of the variation (0.6%, P = 0.204). In pathogenicity tests on strawberry plants (cv. Diamante) in a greenhouse, isolates of P. cactorum from hosts other than strawberry and an isolate from a strawberry fruit caused only negligible amounts of disease, but isolates from strawberry root systems were highly aggressive. On excised shoot segments of almond (cv. Drake), all isolates of P. cactorum originally from almond were pathogenic, and 8 of 17 isolates of the pathogen from other hosts caused significantly less disease than the almond isolates. All 132 isolates of P. cactorum were sensitive to mefenoxam at 1 ppm. Populations of P. cactorum in California apparently are mefenoxam sensitive and exhibit host specificity with relatively minor variation in genomic DNA. The genetic variation observed in P. cactorum included significant geographical and host origin components, which has implications for disease management approaches.