Analysis of Genetic Diversity of Fusarium tupiense, the Main Causal Agent of Mango Malformation Disease in Southern Spain.

Mango malformation disease (MMD) has become an important global disease affecting this crop. The aim of this study was to identify the main causal agents of MMD in the Axarquía region of southern Spain and determine their genetic diversity. Fusarium mangiferae was previously described in the Axarquía region but it represented only one-third of the fusaria recovered from malformed trees. In the present work, fusaria associated with MMD were analyzed by arbitrary primed polymerase chain reaction (ap-PCR), random amplified polymorphic DNA (RAPD), vegetative compatibility grouping (VCG), a PCR screen for mating type idiomorph, and phylogenetic analyses of multilocus DNA sequence data to identify and characterize the genetic diversity of the MMD pathogens. These analyses confirmed that 92 of the isolates were F. tupiense, which was previously only known from Brazil and Senegal. In addition, two isolates of a putatively novel MMD pathogen were discovered, nested within the African clade of the Fusarium fujikuroi species complex. The F. tupiense isolates all belonged to VCG I, which was first described in Brazil, and the 11 isolates tested showed pathogenicity on mango seedlings. Including the prior discovery of F. mangiferae, three exotic MMD pathogenic species have been found in southern Spain, which suggests multiple independent introductions of MMD pathogens in the Axarquía region.

First Report of Mango Malformation Disease Caused by Fusarium pseudocircinatum in Mexico.

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, causing severe economic loss due to reduction of yield. After the first report in India in 1891 (3), MMD has spread worldwide to most mango-growing regions. Several species of Fusarium cause the disease, including F. mangiferae in India, Israel, the USA (Florida), Egypt, South Africa, Oman, and elsewhere; F. sterilihyphosum in South Africa and Brazil; F. proliferatum in China; F. mexicanum in Mexico; and recently, F. tupiense in Brazil (1,2,3,4). Besides F. mexicanum, F. pseudocircinatum, not yet reported as a causal agent of MMD, was isolated in Mexico from affected inflorescences and vegetative malformed tissues (4). Symptoms of vegetative malformation caused by F. pseudocircinatum included hypertrophied, tightly bunched young shoots, with swollen apical and lateral buds producing misshapen terminals with shortened internodes and dwarfed leaves. Infected inflorescences of primary or secondary axes on affected panicles were shortened, thickened, and highly branched, while the peduncles became thick, remained green and fleshy, and branches profusely resembled a cauliflower in shape and size (3). Ten isolates of F. pseudocircinatum were recovered from cultivars Ataulfo, Criollo, Haden, and Tommy Atkins in Guerrero, Campeche, and Chiapas states and characterized. Isolates produced mostly 0-septate but occasionally 1- to 3-septate oval, obovoid, or elliptical aerial conidia (0-septate: 4 to 19 [avg. 8.7] × 1.5 to 4 [avg. 2.6] µm) in false heads in the dark and in short false chains under black light, unbranched or sympodially branched prostrate aerial conidiophores producing mono- and polyphialides, and sporodochia with straight or falcate conidia that were mostly 3- to 5-septate, but sometimes up to 7-septate (3-septate: 25 to 58 [avg. 41] × 2 to 3.3 [avg. 2.9] µm; 5-septate: 33.5 to 76.5 [avg. 56.7] × 2.5 to 6 [avg. 3.5] µm). Circinate sterile hyphae were rarely formed. Two representative isolates, NRRL 53570 and 53573, were subjected to multilocus molecular phylogenetic analyses of portions of five genes: nuclear large subunit 28S ribosomal RNA, ß-tubulin, calmodulin, histone H3, and translation elongation factor (TEF)-1α (GenBank GU737456, GU737457, GU737290, GU737291, GU737371, GU737372, GU737425, GU737426, GU737398, and GU737399). Two pathogenicity tests were conducted with NRRL 53570 and 53573 on healthy 2-year-old nucellar seedlings of polyembryonic Criollo; 20 µl conidial suspensions (5 × 106 conidia/ml) of each isolate and water controls were inoculated separately on 15 buds on 3 different trees, as described previously (1). The following conditions were used in experiment 1: 24 to 27°C with light intensity of 16.2 to 19.8 •Mol m-2s-1 in the range of 400 to 700 nm, and photoperiods of 14 h light and 10 h dark. Typical vegetative disease symptoms were discernible in plants inoculated with NRRL 53570 (20%) and 53573 (7%) after 8 months. In experiment 2, after 3 months growth under the above conditions, seedlings were transferred to an outdoor nursery in Iguala, Guerrero. Typical vegetative symptoms of MMD were observed in 86.7 and 13.3% of the buds inoculated with F. pseudocircinatum NRRL 53570 and 53573, respectively, after 9 months. Isolates from typical symptomatic vegetative buds were confirmed as F. pseudocircinatum by sequencing a portion of their TEF-1α gene, thus fulfilling Koch's postulates. This is the first report of F. pseudocircinatum as a causal agent of MMD. References: (1) S. Freeman et al. Phytopathology 89:456, 1999. (2) C. S. Lima et al. Mycologia 104:1408, 2012. (3) W. F. O. Marasas et al. Phytopathology 96:667, 2006. (4) G. Otero-Colina et al. Phytopathology 100:1176, 2010.

Fusarium euwallaceae sp. nov.--a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California.

The invasive Asian ambrosia beetle Euwallacea sp. (Coleoptera, Scolytinae, Xyleborini) and a novel Fusarium sp. that it farms in its galleries as a source of nutrition causes serious damage to more than 20 species of live trees and pose a serious threat to avocado production (Persea americana) in Israel and California. Adult female beetles are equipped with mandibular mycangia in which its fungal symbiont is transported within and from the natal galleries. Damage caused to the xylem is associated with disease symptoms that include sugar or gum exudates, dieback, wilt and ultimately host tree mortality. In 2012 the beetle was recorded on more than 200 and 20 different urban landscape species in southern California and Israel respectively. Euwallacea sp. and its symbiont are closely related to the tea shot-hole borer (E. fornicatus) and its obligate symbiont, F. ambrosium occurring in Sri Lanka and India. To distinguish these beetles, hereafter the unnamed xyleborine in Israel and California will be referred to as Euwallacea sp. IS/CA. Both fusaria exhibit distinctive ecologies and produce clavate macroconidia, which we think might represent an adaption to the species-specific beetle partner. Both fusaria comprise a genealogically exclusive lineage within Clade 3 of the Fusarium solani species complex (FSSC) that can be differentiated with arbitrarily primed PCR. Currently these fusaria can be distinguished only phenotypically by the abundant production of blue to brownish macroconidia in the symbiont of Euwallacea sp. IS/CA and their rarity or absence in F. ambrosium. We speculate that obligate symbiosis of Euwallacea and Fusarium, might have driven ecological speciation in these mutualists. Thus, the purpose of this paper is to describe and illustrate the novel, economically destructive avocado pathogen as Fusarium euwallaceae sp. nov. S. Freeman et al.

First Report of Mango Malformation Disease Caused by Fusarium mangiferae in Sri Lanka.

Mango malformation disease (MMD) is one of the most devastating diseases causing severe economic losses to this crop worldwide. MMD has not been reported in Sri Lanka although the disease was reported in neighboring India over a century ago. Abnormal, thick, and fleshy mango panicles (40%) and proliferating stunted shoots (<1%) showing characteristic malformation symptoms were observed in Peradeniya-Kandy area (7°17'4.15" N, 80°38'14.08" E). Malformed inflorescences and vegetative shoots were collected during January to March and September to November, in 2008 through 2012. Pieces of malformed tissues were surface sterilized in 1% sodium hypochlorite and transferred to potato dextrose agar (PDA). The plates were incubated at 26 ± 2°C for 7 days. Monoconidial cultures of 41 isolates that resembled Fusarium spp. were obtained. Colonies showed white sparse aerial mycelium and magenta-dark purple pigmentation on the underside. Growth rate of the isolates averaged 3.67 mm/day in the dark at 25°C on PDA. To stimulate conidia development, Fusarium isolates were transferred to carnation leaf agar (CLA). Sympodially branched conidiophores bearing mono- and polyphialides with 2 to 3 conidiogenus openings originated erect and prostrate on aerial mycelium. Oval to allontoid, abundant microconidia were produced in false heads on mono- and polyphialides. Dimensions of aseptate conidia were 2.5 to 12.5 (6.47) × 1.25 to 3.8 (2.29) µm. Macroconidia were long and slender, 3 to 5 celled and 27.5 to 47.5 (38.59) × 2.5 to 5 (2.94) µm. Chlamydospores were absent. These characters are consistent for F. mangiferae. DNA was extracted from 30 monoconidial Fusarium isolates (1) and amplified with species-specific PCR primers 1-3F/R (forward: 5'-TGCAGATAATGAGGGTCTGC-3'; reverse: 5'-GGAACATTGGGCAAAACTAC-3') (3). Eight isolates from malformed inflorescences (I6, I13, I15, and I16) and malformed vegetative tissues (V1, V2, V3, and V4), were identified as F. mangiferae based on a 608-bp species-specific amplified DNA fragment. Pathogenicity of F. mangiferae isolates, I15 and V2, was tested on 1-year-old seedlings cv. Willard planted in 10-liter plastic pots. Conidia suspensions (107 conidia/ml of 0.1% water agar) were obtained from 10-day-old monoconidial cultures. Each isolate was inoculated onto 15 apical buds by placing drops (20 µl) of conidia (2). Both F. mangiferae isolates, I15 and V2, on artificial inoculation produced typical floral malformation symptoms in 40% of the buds, up to 10 weeks after inoculation. The Fusarium isolates recovered were identical in colony and mycelia morphology and conidia dimensions to the original F. mangiferae isolates. No Fusarium species were recovered from control flower buds. To our knowledge, this is the first report of MMD in the inflorescence and the vegetative shoots caused by F. mangiferae in Sri Lanka. Isolation of other Fusarium spp. that were not identified as F. mangiferae in this study suggests that additional Fusarium spp. may be associated with the MMD in Sri Lanka. Further studies are needed to confirm the identity of these Fusarium isolates, their role in MMD, and the distribution over the island. Since the disease is likely to drastically reduce productivity, measures will be required to protect 12,160 ha of mango cultivation from this devastating disease. References: (1) S. Freeman et al. Exp. Mycol. 17:309, 1993. (2) S. Freeman et al. Phytopathology 89:456, 1999. (3) Q. I. Zheng and R. C. Ploetz. Plant Pathol. 51:208, 2002.

First Report of Mango Malformation Disease Caused by Fusarium mangiferae in Spain.

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, which causes severe economic losses because of the reduction of productivity. Symptoms of MMD in Spain were observed for the first time in April of 2006 in three mango orchards in the Axarquia Region (southern Spain). Symptoms included an abnormal development of vegetative shoots with shortened internodes and dwarfed leaves and hypertrophied short and thickened panicles. In the years of 2006, 2009, and 2010, isolates of Fusarium were obtained from vegetative shoots and floral tissue of symptomatic mango trees from 21 different orchards of cvs. Keitt, Kent, Osteen, Tommy Atkins, and a variety of minor commercial cultivars, all showing typical symptoms of MMD. Different Fusarium-like strains were isolated from infected tissues. Colonies from single-spored isolates possessed dark purple-to-salmon-colored mycelium when grown on potato dextrose agar medium. On fresh carnation leaf agar medium, mycelium contained aerial conidiophores possessing three- to five-celled macroconidia and abundant microconidia in false heads from mono- and polyphialides; while cream-orange-colored sporodochia were produced on the surface of the medium, typical for Fusarium mangiferae. The identification of 37 isolates was confirmed as F. mangiferae by species-specific PCR analysis with the primer pair 1-3 F/R that amplified a 608-bp DNA fragment from all Spanish isolates as well as a representative Israeli control strain, Fus 34, also designated as MRC7560 (2). Pathogenicity using four representative isolates, UMAF F02, UMAF F10, UMAF F17, and UMAF F38 of F. mangiferae from Spain as well as isolate MRC7560, was tested on 2-year-old healthy mango seedlings cv. Keitt by inoculating 15 buds from three different trees with a 20-µl conidial suspension (5 × 107 conidia per ml) per isolate (1). This experiment was conducted twice with two independent sets of plants and at different times (March and November 2010). Typical mango malformation symptoms were detected after bud break in March 2011, 5 and 12 months after inoculation. Symptoms were observed for 60% of the inoculated buds with the four F. mangiferae Spanish isolates and 75% with the MRC7560 control strain, but not with water-inoculated control plants. Recovered isolates from the infected floral and vegetative malformed buds were identical morphologically to those inoculated, and the specific 608-bp fragment described for F. mangiferae was amplified with specific-PCR, thus fulfilling Koch's postulates. To our knowledge, this is the first report of mango malformation disease caused by F. mangiferae in Spain and Europe. References: (1) S. Freeman et al. Phytopathology 89:456, 1999. (2) Q. I. Zheng and R. C. Ploetz. Plant Pathol. 51:208, 2002.

First Report of Papaya Fruit Rot Caused by Colletotrichum magna in Brazil.

Species of the genus Colletotrichum are commonly reported as pathogens of fruits in tropical regions. Papaya fruits (Carica papaya L.), cv. Golden, with typical lesions of anthracnose, chocolate spot, and/or stem-end rot were collected from 18 papaya-producing areas of northeast Brazil in 2007. One hundred and fifty-five isolates of Colletotrichum spp. were obtained from the fruit lesions and cultured on potato dextrose agar. Pathogenicity tests were conducted by placing a 20-µl drop of 105 conidia ml-1 suspension on a wounded area of two healthy fruits of cv Golden at the climacteric stage. Inoculated fruits were placed in a moist chamber at 26°C (±2) for 48 h. After this period, the plastic covers of the trays used to form the moist chamber were removed and the trays were kept at 26°C (±2) for 98 h when symptoms were assessed. The causal agents of fruit rot were recovered from inoculated fruits showing symptoms of anthracnose and chocolate spot. Conidia from fresh lesions were collected and measured. Conidia dimensions were 13.49 × 3.80 µm, length/width ratio = 3.55 µm. Conidia were predominantly cylindrical to bluntly rounded ends and slightly flattened. All isolates were morphologically similar to Colletotrichum gloeosporioides Penz (1). Molecular analyses of the isolates were carried out with taxon-specific primers for C. acutatum J.H Simmonds and C. gloeosporioides (3). Only one amplicon was detected for eight isolates with the C. gloeosporioides primer. All isolates were genotyped using inter-simple sequence repeat (ISSR) primers. Three groups of isolates were found, one containing the eight C. gloeosporioides isolates, a second group comprised of 141 isolates, and a third contained six isolates. The second and third groups were more similar to each other than to the first C. gloeosporioides group. Thirty two representative isolates of the three ISSR groups were sequenced for the internal transcribed spacer (ITS) and glutamine synthetase (GS) (GenBank Nos. HM163181 and HM015847) regions. With molecular phylogenetic analyses, two well-supported clades were formed, one with the C. gloeosporioides isolates and the other with sequences highly similar (99% similarity) to the two ITS sequences available in GenBank (DQ003310 and GU358453) and the GS region of G. magna Jenkins & Winstead (DQ792873). The latter was reported in the United States and Taiwan (2,4). Isolates of C. magna and C. gloeosporioides are morphologically similar and identification needs to be based on molecular analyses. To our knowledge, this is the first report of C. magna causing rot of papaya fruit in Brazil. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) M. Z. Du et al. Mycologia 97:641, 2005. (3) P. Talhinhas et al. Appl. Environ. Microbiol. 71:2987, 2005. (4) J. G. Tsay et al. Plant Dis. 94:787, 2010.

Infection dynamics of Fusarium mangiferae, causal agent of mango malformation disease.

Conditions affecting germination and growth of Fusarium mangiferae, causal agent of mango malformation disease, were studied in vitro. Both conidial germination and colony growth required temperatures >5 degrees C and reached a peak at 28 and 25 degrees C, respectively. A minimum 2-h wetness period was required for conidial germination, reaching a peak after 8 h of wetness. High incidence of fungal colonization in buds, predominantly the apical buds, was detected compared with inoculated leaves. The pathogen was detected in the roots of inoculated soil 19 weeks postinoculation but not in aboveground parts of the plants, and symptoms of the disease were not observed, either. Dry, malformed inflorescence debris serving as a source of inoculum caused significantly higher colonization (52 and 20%) of inoculated buds, compared with that (0%) of the untreated controls. Incidence of sampled leaf disks bearing propagules of F. mangiferae from an infected orchard peaked in June and July and decreased during the following months, whereas airborne infections on 1-month-old branches was the highest in May and June, corresponding with inoculum availability released from infected inflorescences. Colonization pattern, determined in naturally infected vegetative and woody branches, was significantly higher in node sections than in the internode sections. This study sheds light on infection dynamics, colonization patters, and the disease cycle of F. mangiferae in mango.

Interaction of the mite Aceria mangiferae with Fusarium mangiferae, the causal agent of mango malformation disease.

The role of the mango bud mite, Aceria mangiferae, in carrying conidia of Fusarium mangiferae, vectoring them into potential infection sites, and assisting fungal infection and dissemination was studied. Following the mite's exposure to a green fluorescent protein-marked isolate, conidia were observed clinging to the mite's body. Agar plugs bearing either bud mites or the pathogen were placed on leaves near the apical buds of potted mango plants. Conidia were found in bud bracts only when both mites and conidia were co-inoculated on the plant, demonstrating that the mite vectored the conidia into the apical bud. Potted mango plants were inoculated with conidia in the presence or absence of mites. Frequency and severity of infected buds were significantly higher in the presence of mites, revealing their significant role in the fungal infection process. Conidia and mite presence were monitored with traps in a diseased orchard over a 2-year period. No windborne bud mites bearing conidia were found; however, high numbers of windborne conidia were detected in the traps. These results suggest that A. mangiferae can carry and vector conidia between buds and assist in fungal penetration but does not play a role in the aerial dissemination of conidia between trees.

Inoculum availability and conidial dispersal patterns of Fusarium mangiferae, the causal agent of mango malformation disease.

Inoculum availability and conidial dispersal patterns of Fusarium mangiferae, causal agent of mango malformation disease, were studied during 2006 and 2007 in an experimental orchard. The spatial pattern of primary infections in a heavily infected commercial mango orchard corresponded with a typical dispersal pattern caused by airborne propagules. Malformed inflorescences were first observed in mid-March, gradually increased, reaching a peak in May, and declined to negligible levels in August. The sporulation capacity of the malformed inflorescences was evaluated during three consecutive months. Significantly higher numbers of conidia per gram of malformed inflorescence were detected in May and June than in April. Annual conidial dissemination patterns were evaluated by active and passive trapping of conidia. A peak in trapped airborne conidia was detected in May and June for both years. The daily pattern of conidial dispersal was not associated with a specifically discernable time of day, and an exponential correlation was determined between mean relative humidity (RH) and mean number of trapped conidia. Higher numbers of conidia were trapped when RH values were low (<55%). This is the first detailed report on airborne dispersal of F. mangiferae, serving as the primary means of inoculum spread.

First Report of Anthracnose Caused by Colletotrichum dematium on Statice (Goniolimon tataricum, Synonym Limonium tataricum) in Bulgaria.

German statice (Goniolimon tataricum, synonym Limonium tataricum) is a popular ornamental species, which is frequently used in bouquet arrangements. During a field survey of statice farms in the Plovdiv Region of Bulgaria (August 2007), lesions were observed predominantly on the peduncles and rarely on wilted leaves of 2- and 3-year-old plants. Symptoms appeared on the base of peduncles as irregular, brown necrotic lesions ranging from 30 to 40 mm that coalesced, whereas lesions on leaves were initially round to elliptical with dimensions from 5 to 15 mm and developed a necrosis that subsequently spread toward the petioles. Rounded and elongated setose acervuli were observed on the lesions of peduncles. Isolations on potato dextrose agar (PDA) produced fungal colonies that initially were whitish but turned gray 4 to 5 days after incubation at 25°C. Falcate, hyaline, and aseptate conidia with mean dimensions of 22.0 × 4.5 µm, ranging from 18.3 to 25.0 × 4.2 to 5.8 µm, were observed from acervuli of both naturally infected peduncles and PDA-cultured colonies. Pathogenicity of the fungus (three single-conidium representative isolates) was tested by spray inoculating 4-month-old intact plantlets (12 to 15 fully developed leaf stage) with a conidial suspension (106 conidia/ml, 15 ml/plant) and maintaining them in a humidity chamber for 30 h. Plants sprayed with sterile water served as controls. There were three replicates per treatment per isolate and the experiment was conducted twice at room temperature (22 to 26°C). After 10 to 12 days, the spray-inoculated plants exhibited light brown lesions mainly on the older leaves that gradually expanded and caused leaf mortality. The pathogen was reisolated from all inoculated samples but not from any of the control and symptomless treatments, thus fulfilling Koch's postulates. It should be noted that symptoms caused by the pathogen in artificially inoculated plants were seen as wilting of petioles and leaves, as opposed to necrotic lesions observed on leaves under field conditions. This may be related to the method of inoculation, leaf age, and texture, as well as environmental factors affecting symptomology under natural field conditions. Sequence analysis of the rDNA internal transcribed spacer region of three representative isolates (GenBank Accession Nos. FJ236461-FJ236463) showed the fungus to be 99% similar to an isolate of Colletotrichum dematium (GenBank Accession No. AJ301954), consistent with the observed morphological characters. On the basis of observed symptoms, morphology, and molecular characterization, it can be concluded that C. dematium is the causal agent of anthracnose of German statice in Bulgaria. To our knowledge, this is the first report of this pathogen on G. tataricum in Bulgaria, although it has been reported that C. dematium (1) and C. gloeosporioides (1-3) may attack other Limonium species. References: (1) C. F. Hong et al. Plant Pathol. Bull. 15:241, 2006. (2) T. Kagiwata. J. Agric. Sci. (Jpn.) 31:101, 1986. (3) M. Maymon et al. Phytopathology 96:542, 2006.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Pepper and Tomato in Bulgaria.

In the late summer of 2005, sporadic and unusual damage was observed on pepper (Capsicum annuum cv. Kurtovska kapia and local cv. Ribka) on two farms and tomato (Lycopersicon esculentum cv. Florida 47) fruits on one farm in the Plovdiv Region of Bulgaria. Dry, round, sunken zones (10 to 20 mm) were observed on pepper fruits that preserved their natural skin color even after black acervuli containing orange masses of conidia appeared. Eventually, the lesions turned brown, coalesced, and the fruits mummified on the plants. Tomato fruits developed similar symptoms, with less prominent discoloration and fewer acervuli. The pathogen was easily isolated from both hosts on potato dextrose agar where it formed white-to-gray colonies with salmon orange pigmentation on the reverse side of the plates. Conidia that formed were hyaline, fusiform, aseptate, and measured 13.3 to 17.4 × 3.5 to 5.5 µm and 11.6 to 15.5 × 4.1 to 5.0 µm for pepper and tomato isolates, respectively. Both cultural and morphological characteristics of the isolates were similar to those described for Colletotrichum acutatum (3). Koch's postulates were performed with two representative isolates from each host by artificial inoculation of healthy, green pepper and ripe tomato fruits from the respective cultivars. Fruits were wound inoculated with a sterile scalpel, and small agar plugs (3 to 4 mm) containing 7-day-old sporulating cultures were placed on each wound (five fruits per isolate), or by pipette tip-pricking and pipetting a 5-µl droplet of a conidial suspension (5 × 106 conidia ml-1) on each wound. The same number of wounded, noninoculated fruits was used as a control. Fruits were maintained in a humidity chamber at 22 to 25°C, and 4 days later, sunken necrotic zones were observed around the wounds of inoculated fruit, whereas control fruits remained symptomless. The pathogen was subsequently reisolated from the inoculated diseased tissues but not from the control fruits. Species-specific PCR (using primer pair CaInt2/ITS4) (2,4) of genomic DNA from three representative isolates (two from pepper and one from tomato) resulted in an amplification product of 490 bp, specific for C. acutatum, further confirming the identity of the pathogen. To our knowledge, this is the second report of C. acutatum in Bulgaria (1), and the first occurrence of that agent on tomato and pepper in this country. References: (1) S. G. Bobev et al. Plant Dis. 86:1178, 2002. (2) S. Freeman et al. Phytopathology 91:586, 2001. (3) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (4) M. L. Lewis Ivey et al. Plant Dis. 88:1198, 2004.

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds.

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.

Molecular analyses of colletotrichum species from almond and other fruits.

ABSTRACT Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.