Reliable detection of the fungal pathogenfusarium oxysporum f.sp.albedinis, causal agent of bayoud disease of date palm, using molecular techniques

Bayoud, caused by the soilborne fungusFusarium oxysporum f.sp.albedinis (FOA), is the most serious disease of date palm. Since the disease is located in the North African countries of Morocco and Algeria, and advancing steadily eastwards, the ultimate goal is to prevent spread of the pathogen to other date-growing areas in the region and farther afield. Molecular diagnostic techniques have been developed for detection of FOA. In view of the fact that the fungus does not exist in Israel, DNA of FOA was obtained to determine the reliability of these methods for diagnostic purposes. Random amplified polymorphic DNA was not reliable enough for differentiation between FOA and various pathogenic and saprophyticFusarium isolates. However, the polymerase chain reaction utilizing FOA-specific primers was accurate and enabled amplification of a unique band specific to FOA DNA alone, and not that of the other tested pathogenic and saprophyticFusaria. The availability of a rapid and reliable diagnostic tool for detection of FOA will enable the Plant Protection and Inspection Services of the Israel Ministry of Agriculture to test date palm tissue for the presence of the pathogen. Contribution no. 513/00 from the Inst. of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel.     

Effect of jasmonic acid on the induction of polyphenoloxidase and peroxidase activities in relation to date palm resistance against Fusarium oxysporum f. sp. albedinis

Bayoud, caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most damaging disease of date palm in Morocco. In the present study we have investigated the effect of jasmonic acid (JA) on two defence-related enzymes, namely peroxidases (POX) and polyphenoloxidases (PPO) in date palm seedlings root. Our data show that exogenous application of JA at a concentration of 50 μM increased the activity of both enzymes. The increase of POX activity in the presence of JA was much more important than that observed following infection with the pathogen. As compared to untreated plants, PPO activity was 2.2 and 1.3 times higher in BSTN and JHL cultivars respectively. In addition, PAGE analysis revealed increased band intensity of the major constitutive isoforms of POX and PPO in both JA-treated and Foa-treated seedlings. Close examination of symptomatic and asymptomatic plants showed that root tissues of symptomatic plants were massively colonized by Foa. Also, disease development in these plants appeared to involve a marked degradation of the host cell walls early during the process of pathogen invasion. In contrast, the presence of Foa in asymptomatic plants induced limited necrotic lesions (hypersensitive-reaction like lesions) that were probably involved in reducing the progression of the pathogen. Together, our findings indicate that JA is capable of enhancing date palm root resistance to infection by Foa via the activation of defence-related enzymes such as PPO and POX.     

AFLP as a fingerprinting tool for characterising isolates of Fusarium oxysporum f. sp. elaeidis causal organism for fusarium wilt disease of oil palm in Ghana

Journal of Plant Diseases and Protection - Fusarium wilt is a damaging disease of oil palm in Ghana. Genetic diversity of Fusarium oxysporum f. sp. elaeidis (Foe) the causal organism of the disease...     

Evaluation of world castor (Ricinus communis L.) germplasm for resistance to Fusarium wilt (Fusarium oxysporum f. sp. ricini)

Castor (Ricinus communis L.) is an important industrial oilseed crop grown worldwide. Wilt caused by Fusarium oxysporum f. sp. ricini is a devastating disease of this crop. The objective of this research was to identify stable sources of wilt resistance among the global castor germplasm collections available in India for use in cultivar improvement. The global collections comprising 1,779 Indian and 190 exotic accessions from 36 countries were screened against wilt in wilt sick plots at two sites in India in preliminary screening. None of the accessions showed high resistance to wilt, 133 accessions comprising 111 Indian and 22 exotic accessions representing 13 countries exhibited resistance. Thirteen of the 133 resistant accessions were tested further for multiple years in wilt sick plots and glasshouse under controlled artificial inoculation at two sites. All the 13 accessions consistently showed wilt resistance in both wilt sick plot and glasshouse at both sites in multiple years. Eleven of these 13 accessions were from India and two were from former USSR. Evaluation for agro-morphological traits identified high seed yielding and early maturing resistant accessions. Diversity analyses precisely revealed diversity among the resistant accessions. These 13 resistant accessions would be great value as donors of resistance.     

PCR detection of Fusarium oxysporum f.sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms

Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large- and small-flowered Gladiolus cultivars. Race 2 isolates infect only small-flowered cultivars but can be present as epiphytes on large-flowered plants. When 160 arbitrary 10-mer oligonucleotide primers were tested on FOG by PCR to find RAPD markers specific for race 1, the RAPD primer G12 amplified two discriminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates only. Both fragments were cloned and sequenced. Two pairs of race 1-specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolates by PCR showed that, in almost all cases, race 1 isolates of vegetative compatibility group 0340 could be distinguished with these primers. Seven putative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latently infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28°C in a semiselective medium to induce growth of Fusarium . Cultivated mycelium was isolated and subjected to the developed multiplex PCR after standard DNA isolation or disruption by microwave treatment.     

Molecular records of micro-evolution within the Algerian population of Fusarium oxysporum f. sp. albedinis during its spread to new oases

The genetic diversity of the date palm wilt pathogen Fusarium oxysporum f. sp. albedinis in Algeria was assessed using vegetative compatibility, restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD). Ninety-eight isolates were collected from the main infested regions, Touat, Gourara and Mzab, and 6 isolates from Morocco were added for comparison. All isolates were vegetatively compatible and belonged to VCG 0170. No variation was detected in the mtDNA of a subset of 73 isolates and the RAPD analysis indicated that they were genetically very closely related. However, some geographic substructuring was apparent, suggesting that local diversification of the pathogen might have occurred. These results provide evidence that the Algerian isolates of F. oxysporum f. sp. albedinis belong to a same clonal lineage and support the hypothesis that they were probably founded by a single virulent clone that originated from the Moroccan oases where the date palm wilt (Bayoud disease) was first detected. Based on similarity of RAPD patterns occurring in different oases, and on historical records of the Bayoud disease in Algeria, spread of the pathogen in the different regions is discussed.     

Mechanisms of date palm resistance to Bayoud disease: Current state of knowledge and research prospects

The Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis (Foa), represents a major limiting factor of date palm culture in Morocco and constitutes a serious threat to the date palm plantations in Algeria and all other countries. Efficient disease prevention requires the development of resistant cultivars. In Morocco, among the cultivars listed, only six appear to be resistant to Bayoud disease, but they produce poor quality fruit. Thus, the Moroccan program of date palm genetic improvement is based on directed crossing between resistant cultivars and susceptible cultivars with good date quality traits to select resistant genotypes producing high quality fruits. In addition to the separation of the resistance to Bayoud disease and quality of the fruits characters, this breeding program is really complex due to the sex separation in the date palm, the duration of juvenile phase which is very long, and the lifespan of the date palm which requires a durable polygenic resistance. Then, the selected genotypes must be of female sex, of good date quality, and possess effective defense mechanisms against the pathogen. Moreover, the selection of the date palm resistance must necessarily take into account the mechanisms of pathogen aggressiveness. In this review, we will present and discuss studies developed on the Bayoud disease of the date palm, particularly on the disease control, the biochemical and molecular markers of resistance, the program of date palm genetic improvement of resistance, the Foa pathogenicity factors, and host defense mechanisms. It will also highlight the recent studies that showed that differential behaviour of the resistant and susceptible cultivars was not related to a difference of induction of the defense mechanisms, but to the suppression of their elicitation in the susceptible cultivars.     

Fusarium oxysporum f. sp. radicis‐vanillae is the causal agent of root and stem rot of vanilla

Root and stem rot (RSR) is a very detrimental disease of vanilla worldwide. Fusarium oxysporum is frequently associated with the disease but other Fusarium species are also reported. In this international study, 52 vanilla plots were surveyed in three of the most important vanilla producing countries (Madagascar, Reunion Island and French Polynesia) in order to determine the aetiology of RSR disease. Subsets from the 377 single‐spored Fusarium isolates recovered from rotten roots and stems in the surveys were characterized by molecular genotyping (EF1α and IGS gene sequences) and pathogenicity assays on Vanilla planifolia and V. ×tahitensis, the two commercially grown vanilla species. Fusarium oxysporum was shown to be the principal species responsible for the disease, representing 79% of the isolates recovered from the RSR tissues, 40% of which induced severe symptoms on inoculated plantlets. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Fusarium solani, found in 15% of the samples and inducing only mild symptoms on plantlets, was considered a secondary pathogen of vanilla. Three additional Fusarium species were occasionally isolated in the study (F. proliferatum, F. concentricum and F. mangiferae) but were nonpathogenic. Histopathological preparations observed in wide field and multiphoton microscopy showed that F. oxysporum penetrated the root hair region of roots, then invaded the cortical cells where it induced necrosis in both V. planifolia and V. ×tahitensis. The hyphae never invaded the root vascular system up to 9 days post‐inoculation. As a whole, the data demonstrated that RSR of vanilla is present worldwide and that its causal agent should be named F. oxysporum f. sp. radicis‐vanillae.     

Selection methods for determining resistance of carnation cultivars to Fusarium oxysporum f.sp. dianthi

The level of resistance of carnation ( Dianthus caryophyllus ) cultivars to wilt caused by Fusarium oxysporum f.sp. dianthi was compared in root-dip-inoculated plants grown in pots (filled with tuff or sandy soil) in a greenhouse and plants grown in a field where the soil was artificially infested with the fungus. In the field, wilt symptoms appeared first in susceptible and subsequently in resistant cultivars; none was immune. Variations in the level of resistance were expressed either by different percentages of wilted plants (i.e. disease incidence) or by delayed disease progress as compared to a susceptible cultivar. The range of disease severity in the field, ranked on a scale from 0 to 4, was highly and significantly correlated with the percentage of diseased plants. The greenhouse test was unreliable as a predictor of the degree of resistance observed in the field. Similar wilt levels in the greenhouse and the field were found only in susceptible cultivars.     

Pathogenic and Genetic Variation in the Japanese Strains of Fusarium oxysporum f. sp. melonis

ABSTRACT Pathogenic variation among 41 Japanese strains of Fusarium oxysporum f. sp. melonis was analyzed by pathogenicity tests with muskmelon, oriental melon, and oriental pickling melon cultivars. Based on pathogenicity to muskmelon cvs. Amus and Ohi and oriental melon cv. Ogon 9, 41 strains were divided into 3 groups that corresponded completely to Risser's races 0, 2, and 1,2y. To further characterize pathogenic variation within the forma specialis and races, strains were assayed for pathogenicity to 42 additional muskmelon, oriental melon, and oriental pickling melon cultivars. All strains of race 1,2y were pathogenic to all cultivars tested. Strains of race 0 were divided into six variants based on differences in pathogenicity to three muskmelon cultivars; strains of race 2 also were classified into six variants based on differences in pathogenicity to two muskmelon cultivars and one oriental melon cultivar. Genetic variation among strains was analyzed by DNA fingerprinting with four repetitive DNA sequences: FOLR1 to FOLR4. Thirty-six fingerprint types were detected among forty-one strains by pooling results of fingerprinting with four probes. Cluster analysis showed distinct genetic groups correlated with races: the fingerprint types detected in each of races 2 and 1,2y were grouped into a single cluster, and two distinct genetic groups were found in race 0. However, pathogenic variation detected within races 0 and 2 could not be differentiated based on the nuclear markers examined.     

Sensitivity of Fusarium oxysporum f. sp. cubense to phosphonate

Phosphonate (0.1 mM) significantly reduced growth of Fusarium oxysporum f. sp. cubense (Foc) race 4 grown at an optimal phosphate concentration of 0.3 mM in vitro. At higher phosphate concentrations, closer to physiological conditions within the plant, the sensitivity of Foc race 4 to phosphonate was greatly reduced, with 25 mM phosphonate required to reduce growth by 50% at 1 mM phosphate. Two isolates of Fusarium oxysporum f. sp. dianthi and another race of Foc, race 1, were shown to be similar to Foc race 4 in their sensitivity to phosphonate, while another species of Fusarium, F. avenaceum , was more sensitive to phosphonate in vitro.     

Induced resistance in tomato plants against Fusarium wilt invoked by Fusarium oxysporum f.sp. dianthi

Tomato plants, susceptible toFusarium oxysporum f. sp.lycopersici, were inoculated by immersing the roots in a conidial suspension ofF. oxysporum f. sp.lycopersici race 1,F. oxysporum f. sp.dianthi race 2 or a mixture of both fungi. Plants inoculated withF. oxysporum f. sp.lycopersici showed disease symptoms after 2 weeks, whereas plants inoculated withF. oxysporum f. sp.dianthi or a mixture of both fungi remained symptomless for over 7 weeks, the duration of the experiment. In another experiment root systems of plants were split and each half was separately inoculated. One half was firstly inoculated withF. oxysporum f. sp.dianthi or treated with water, followed after a week by a second inoculation of the other half withF. oxysporum f. sp.lycopersici or by a water treatment. The disease symptoms in the half firstly inoculated withF. oxysporum f. sp.dianthi were significantly delayed, compared to plants of which that half had been treated with water. BecauseF. oxysporum f. sp.dianthi reduced disease symptoms caused byF. oxysporum f. sp.lycopersici without any direct interaction with this pathogen, it is concluded thatF. oxysporum f. sp.dianthi is able to induce resistance againstF. oxysporum f. sp.lycopersici in tomato plants.     

Involvement of phenol metabolism in resistance of Dianthus caryophyllus to Fusarium oxysporum f.sp. dianthi

Three carnation cultivars were investigated for the effect of stem inoculation withFusarium oxysporum f.sp.dianthi on production of phenolic compounds and on fungistatic activity. Carnation stems were characterized by a complex mixture of cell wall-bound phenolics, several of which occurred in considerable amounts. Only very low amounts of phenolic compound were present in the vacuoles.Infection withF. oxysporum f.sp.dianthi induced the production and accumulation of a number of new compounds, both free in the cell sap and bound to the cell wall. In addition, the stem extracts acquired germination-inhibiting properties for conidia of the fungus. The accumulation of several phenolics and the fungistatic activity were roughly correlated to the degree of resistance of the three cultivars. Part of the differences in their resistance toF. oxysporum f.sp.dianthi might be due to an inhibition of the conversion of phenolic acid-type precursors into phytoalexins in the more susceptible cultivars.Samenvatting Drie anjercultivars werden onderzocht op de effecten van stengelinoculatie metFusarium oxysporum f.sp.dianthi op de produktie van fenolische verbindingen en op de fungistatische activiteit van stengelextracten. Anjerstengels bleken een complex mengsel van celwand-gebonden fenolzuren te bevatten, waarvan er verschillende in vrij grote hoeveelheden voorkwamen. Daartegenover waren de hoeveelheden fenolische verbindingen in de vacuole bij gezonde anjerstengels erg laag.Infectie metF. oxysporum f.sp.dianthi induceerde de produktie en accumulatie van een aantal nieuwe verbindingen in het celsap, alsook gebonden aan de celwand. Tevens bleek de remming van de kieming van conidiën vanF. oxysporum f.sp.dianthi onder invloed van deze stengelextracten sterk verhoogd, in vergelijking met de remming veroorzaakt door extracten van gezonde stengels. De accumulatie van een aantal fenolen en de verhoging van de fungistatische activiteit van de extracten waren in grote lijnen gecorreleerd met de uit de praktijk bekende resistentie van de drie cultivars. De verschillen in resistentie tegenF. oxysporum f.sp.dianthi zouden deels kunnen berusten op remming van de omzetting van precursors (fenolzuren) in fytoalexinen in de meer vatbare cultivars.     

A detailed evaluation method to identify sources of quantitative resistance to Fusarium oxysporum f. sp. pisi race 2 within a Pisum spp. germplasm collection

Fusarium oxysporum f. sp. pisi (Fop) is an important pathogen of field pea (Pisum sativum) worldwide. The constant evolution of the pathogen drives the necessity to broaden the genetic basis of resistance to Fop. To achieve this, it is important to have a large germplasm collection available and an accurate and efficient method for disease assessment. Here, a detailed evaluation method coupling disease incidence, disease rating over time and its related area under the disease progression curve (AUDPC) was established and used to screen a Pisum spp. germplasm collection against one isolate of Fop race 2. A large variation in the disease response of specific pea accessions ranging from highly resistant to susceptible was observed within the collection, indicating the quantitative expression of the resistance. The repetition of the inoculation experiments on a subset of 19 accessions, including two susceptible accessions, indicated that the scoring method was robust and reproducible and confirmed the highly resistant phenotypes of 11 accessions. To initiate the characterization of resistance mechanisms within these accessions, the external and internal stem symptoms were compared between these selected pea accessions, together with the extent of fungal colonization within plants. All these tests indicated that, in all resistant accessions, the resistance mechanisms efficiently stopped pathogen progression at the crown. Incorporation of these sources of resistance to breeding programmes will contribute to improved Fop resistance in pea cultivars.     

Variation in susceptibility of lettuce cultivars to fusarium wilt caused by Fusarium oxysporum f.sp. lactucae

Lettuce cultivars adapted to Californian growing conditions were screened for resistance to fusarium wilt caused by Fusarium oxysporum f.sp. lactucae in order to determine if differences in susceptibility among currently grown cultivars might contribute to management of this disease. Based on a preliminary evaluation of 46 cultivars, eight that were among the most resistant of their horticultural type (iceberg, romaine or leaf) were selected for further testing. The relative susceptibility of these cultivars was assessed by: (i) root-dip inoculation, (ii) sowing seeds into infested potting mix and (iii) transplanting seedlings into an infested field. Evaluations of disease severity showed that both methods (i) and (ii) produced cultivar rankings that were significantly correlated with rankings from field trials [method (iii)]. Two romaine and two leaf cultivars were highly resistant to fusarium wilt (mean disease severity rating of ≤1·3 on a 1–4 scale) under all test conditions. Other romaine and leaf cultivars, however, were highly susceptible in root–dip tests, so there was no consistent association between cultivar type and susceptibility to fusarium wilt. Likewise, there was considerable variation in susceptibility to wilt among iceberg cultivars, but all were significantly more susceptible than the most resistant romaine and leaf cultivars.     

Potential role for salicylic acid in induced resistance of asparagus roots to Fusarium oxysporum f.sp. asparagi

Pre-inoculation of asparagus ( Asparagus officinalis ) roots with selected nonpathogenic isolates of Fusarium oxysporum (np Fo ) has previously been shown to induce systemic resistance against infection by F. oxysporum f.sp. asparagi ( Foa ) through activation of plant-defence mechanisms. To elucidate the putative np Fo -mediated defence pathways, the effect of salicylic acid (SA) was examined in a split-root system of asparagus where one half of the seedling root system was drenched with SA and the activation of defence responses was measured subsequently on the remaining roots. SA-treated plants exhibited enhanced systemic resistance, with a significant reduction in disease severity of the roots inoculated with Foa , compared with untreated plants. SA activated peroxidase and phenylalanine ammonia-lyase, as well as lignification, upon Foa attack, in a manner similar to that observed with np Fo pretreatment. In addition, application of diphenyleneiodonium, an SA biosynthesis inhibitor, led to failure of np Fo to induce lignin deposition and systemic resistance. Treatment of fungal spores with SA did not affect germination and growth of either np Fo or Foa in in vitro antifungal assays. Production of SA at the site of np Fo infection may be involved in the induction of Foa resistance in asparagus roots.     

Fusaric acid: phytotoxicity and in vitro production by Fusarium oxysporum f.sp. lilii,the causal agent of basal rot in lilies

The production of phytotoxins byFusarium oxysporum f.sp.lilii, a pathogen causing bulb and scale rot in lilies, was investigated. To determine the toxic activity of culture filtrate, a bioassay with in vitro grown scale bulblets or with callus was set up. The fungus produces toxic components in different culture media. The highest toxicity was observed in Czapek Dox medium. HPLC and GC/MS analyses revealed the presence of fusaric acid at toxic concentrations in this medium. The production of fusaric acid in time coincides with the increase of toxic activity in the culture filtrate. It is concluded that at least part of this toxicity is due to fusaric acid.     

Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markers

Screening of genotypes of melon ( Cucumis melo ) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth-chamber experiments. Disease incidence varied from 0 to 100% in a genotype-dependent manner. Using four combinations of light (60 and 90  µ E m⁻² s⁻¹) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker-assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence-characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom-2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker-assisted selection.