Electrophysiological Responses to Fusaric Acid of Root Hairs from Seedlings of Date Palm-susceptible and -resistant to Fusarium oxysporum f. sp. albedinis

Bayoud, a vascular wilt of date palm caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most devastating disease in palm groves of north Africa. Although Foa is able to induce resistance mechanisms in its host plant, no data are available on the early responses of the root cells. Fusaric acid (FA) is the main toxin found in culture filtrates from Foa aggressive strains. This phytotoxin induces modifications of membrane permeability or membrane potential in various cell types and could thus be involved in the early steps of signal exchange between the pathogen and the plant. We showed an early differential behaviour of the disease‐resistant and ‐susceptible cultivars from date palm when challenged by FA. This response could be due to a differential sensitivity of H⁺‐ATPases to FA.     

Yellow Death: A Disease of Date Palm in Iran Caused by Fusarium solani

In December 2003, a severe general yellowing and death of the fronds of date palm (Phoenix dactylifera) occurred in a grove in the vicinity of Kazeron district, west of Fars province, Iran. Fusarium solani was isolated from the crown, and xylem rays sampled from the trunk 1.5 m above soil level. In pathogenicity tests using artificially infested soil and 1‐year‐old date palm seedlings, an isolate from the trunk (FST) induced general chlorosis and death of seedlings 25–28 days after inoculation. Similar results were obtained when seedlings were planted in naturally infested soil. In both procedures, distal portions of the roots and crown were affected. The fungus was re‐isolated from the crown and leaf bases of the inoculated seedlings. This is the first report of a serious disease of date palm, which we call yellow death, incited by F. solani in Iran.     

A Sinapic Derivative as an Induced Defence Compound of Date Palm Against Fusarium oxysporum f.sp. albedinis, the Agent Causing Bayoud Disease

A non-constitutive phenolic compound was isolated from date palm ( Phoenix dactylifera L.) roots and calli derived from offshoots. It inhibits greatly conidial germination as well as infectious germ tube growth and must be considered as a phytoalexin. Its accumulation occurred when calli turned brown or when roots were in contact with Fusarium oxysporum f.sp. albedinis , the agent causing the date palm vascular fusariosis'bayoud'. Tentatively, this compound has been identified as sinapoyl-amide by acid and alkaline hydrolysis, dansylation of amines and polyamines and by ninhydrin treatment. The relationship between this methoxy-substituted hydroxycinnamic acidamide and biotic and abiotic stress leading to tissue browning or vascular disease resistance is discussed.     

Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici

Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M _r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K _m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate.     

Mechanisms of Enhancing Photosynthetic Gas Exchange in Date Palm Seedlings (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) under Salinity Stress by a 5-Aminolevulinic Acid-based Fertilizer

Photosynthetic gas exchange characteristics, salt uptake, pigment contents, and electrolyte leakage were examined in date palm seedlings (Phoenix dactylifera L.) subject to seawater treatments at 1-, 15-, and 30-mS cm⁻¹ salinity levels in the presence or absence of 0.08% ALA-based (5-aminolevulinic acid-based) functional fertilizer commercially known as Pentakeep-v. Date palm seedlings accumulated significant amounts of Na⁺ in the foliage with increasing salinity, about a threefold increase in the accumulated Na⁺ between the control and 30-mS cm⁻¹salinity treatment. Electrolyte leakage indicated a significant reduction in membrane integrity as salinity increased. A strong linear correlation was observed between the chlorophyll (chl) a/b ratio and assimilation rate throughout salinity treatments. The slope (b) and the correlation coefficient between the chl a/b ratio and assimilation suggested that salinity reduced assimilation predominantly via the reduction in chlorophyll a contents (r ² = 0.885 and b = 1.77, P < 0.05). Plants treated with Pentakeep-v showed a similar response with increasing salinity but at higher levels of both chl a/b ratios and assimilation rates. Mechanistic analysis of A:Ci response curves showed that photosynthetic gas exchange in seedlings of the date palm was significantly reduced with increasing salinity due to gas phase limitation (S _L) as evident by stomatal conductance (g _s) values. Salinity did not induce any change in the carboxylation efficiency of the rubisco enzyme (Vc,max), or in the rate of electrons supplied by the electron transport system for ribulose 1,5-bisphosphate (RuBP) regeneration (Jmax). Accelerated carbon loss through respiration has significantly contributed to the described reduction in assimilation and increased CO₂ compensation point (Γ). Only at the 30-mS cm⁻¹ salinity level did treatment with Pentakeep-v reduce Na⁺ accumulation in the leaves, and caused a reduction in K⁺ selective uptake, leading to a concomitant reduction in K⁺/Na⁺ ratios. Pentakeep-v significantly improved chl a contents in all treatments, which was subsequently reflected in total chlorophyll and chl a/b ratios. The non-gas-phase components of the photosynthetic process (biochemical factors limiting gas exchange) were significantly improved by Pentakeep-v applications. Specifically, Pentakeep-v enhanced the biochemical efficiency of carbon fixation (Vc,max) and the rate of electron transport required for RuBP regeneration (Jmax) by 37.4% and 17.8%, respectively, over untreated plants at a salinity level of 15 mS cm^–1. In addition, Pentakeep-v reduced S _L to values similar to those of control plants (9.07%) and lowered CO₂ compensation points by reducing respiratory CO₂ loss, with increasing salinity to 30 mS cm⁻¹. We, therefore, conclude that the ALA-based fertilizer Pentakeep-v improves salt tolerance in date palm seedlings by increasing photosynthetic assimilation. The latter is mediated via boosting light-harvesting capabilities of the treated plants by increasing chl a content and by reducing stomatal limitation to photosynthetic gas exchange.     

Induction of Defence Responses in Roots and Mesocotyls of Sorghum Seedlings by Inoculation with Fusarium thapsinum and F. proliferatum, Wounding and Light

The defence reactions of sorghum seedlings 7 days after inoculation with Fusarium thapsinum and F. proliferatum, and interactions with wounding and exposure to light were studied to determine whether responses to these fungi differed from those to abiotic stresses. In non‐wounded plants, inoculation with both fungi increased concentrations of anthocyanins and soluble phenolics and activities of peroxidase (POX), chitinase and β‐1,3‐glucanase in the roots, and increased β‐1,3‐glucanase activity in the mesocotyls. There was no effect of inoculation on phenylalanine ammonia‐lyase (PAL) activity. Wounding by itself increased anthocyanin content of mesocotyls. Wounding also had a variety of interactions with inoculation. Exposure to light had very little effect on any defence response measured. A time course experiment showed that induction of chitinase and β‐1,3‐glucanase occurred in less than 24 h after inoculation. POX activity increased 2 days after inoculation, followed by a transient increase in PAL activity. The content of anthocyanins and soluble phenolics in roots of inoculated seedlings increased gradually compared with controls over 6 days. The responses of sorghum seedlings to inoculation with F. thapsinum and F. proliferatum were similar to those found by other workers following challenge by necrotrophic pathogens and were different from those induced by wounding and exposure to light.     

Screening of fungal antagonists against yellows of cabbage caused by Fusarium oxysporum f. sp. conglutinans

 Screening of fungal antagonists against yellows of cabbage caused by Fusarium oxysporum f. sp. conglutinans was carried out. We obtained 78 seed-borne fungal isolates from 20 kinds of vegetable roots. Fifty-five soilborne fungal isolates were obtained from the surface-sterilized roots of seven vegetables. Twelve isolates were from field soil using a baiting method. By in vitro and in vivo screening, two seedborne species of Penicillium (S-34) and P. citrinum (S-59), and four soilborne Epicoccum nigrum (TC-33), Fusarium solani (SS-6, CM02), and F. oxysporum f. sp. lactucae (F-9501) suppressed yellows of cabbage effectively. Reductions in disease incidence ranged from 28% to 63%. Received: August 8, 2001 / Accepted: August 28, 2002 Present address:Resource Development Division, Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0812, Japan Tel. +81-438-52-2384; Fax +81-438-52-2329 e-mail: paku-jyuyon@meti.go.jp Acknowledgments We are grateful to Dr. T. Aoki (National Institute of Agrobiological Sciences, MAFF) for identifying Fusarium species, and Dr. Kyung-min Kim (Kyungbuk University, Korea) for advising with the statistical analysis. Correspondence to:J.-Y. Park     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Biocontrol of Fusarium oxysporum f.sp. cubense tropical race 4 by formulated cells and cell-free extracts of Streptomyces griseus in sterile soil environment

The biocontrol activities of cells and cell-free extracts of Streptomyces griseus was tested against Fusarium oxysporum f.sp. cubense tropical race 4 (FOC race 4) in a sterile soil environment. They were first formulated in sodium alginate, kaolin clay and in alginate–kaolin combination, prior to introducing into sterile soil inoculated with 6 log₁₀ cfu FOC race 4 g^?1 soil. Results revealed that bioformulated cells of S. griseus, irrespective of the materials used, were generally more effective in inhibiting growth of FOC race 4 when compared to non-formulated cells of S. griseus. Kaolin was the most suitable inert material as formulation of S. griseus with kaolin effectively suppressed FOC race 4, with only 5.40 log₁₀ cfu g^?1 of FOC race 4 recovered after 20 days. Kaolin formulations also allowed good cell recovery post-formulation. Alginate was less desirable as poorer control was demonstrated, with 6.12 and 6.16 log₁₀ cfu g^?1 of FOC race 4 recovered from soils treated with alginate only and alginate–kaolin formulated S. griseus, respectively. Bioformulations did not benefit cell-free extracts at all. Our study suggests formulation of cells of S. griseus is more beneficial than cell-free extracts and kaolin is the preferred material for formulation.     

Impact of beef extract and six carbon source on antifungal metabolites production by bacterium associated with entomopathogenic nematode against Fusarium oxysporum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp., was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with beef extract on the production of antifungal substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for fructose plus beef extract (956?mg/l). The antifungal activity was significantly high in beef extract plus maltose (21?±?1.5?mm) followed by beef extract plus glucose and beef extract plus fructose. Antifungal activity was significantly reduced in beef extract plus lactose and sucrose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antifungal production was substantially reduced. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Beef extract and maltose as nitrogen and carbon sources in the fermentation medium produced maximum antifungal activity. It is concluded that Beef extract and maltose as nitrogen and carbon sources produced maximum activity which can effectively control the Fusarium oxysporum which causes vascular fusarium wilt in tomato, tobacco, legumes, cucurbits, sweet potatoes, banana, etc.     

Induction of defence-related enzymes in onion by combined application of fungal and bacterial biocontrol agents against Fusarium oxysporum f. sp. cepae

Basal rot disease of onion is a major problem in different onion growing regions of Tamil Nadu, India. Fungal and bacterial cultures were isolated and tested their efficiency against Fusarium oxysporum f. sp. cepae under in vitro conditions. Effective bacterial and fungal antagonists were tested alone and in combinations for the control of F. oxysporum f. sp. cepae in glasshouse experiments. Defence-related enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase were induced and accumulated in onion treated with fungal and bacterial antagonists. Defence-related enzymes were significantly higher in onion pretreated with consortial formulation of Pf12 + Pf27 + TH3 at 5 days after the challenge inoculation with F. oxysporum f. sp. cepae and gave resistance to onion against basal rot disease.     

Combined application of Pseudomonas fluorescens strain LRB3W1 with a low dosage of benomyl for control of cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans

An antagonistic bacterium, Pseudomonas fluorescens strain LRB3W1, inhibited the fungal growth of Fusarium oxysporum f. sp. conglutinans in vitro and suppressed cabbage yellows caused by F. oxysporum f. sp. conglutinans. Under glasshouse conditions, the bacterium survived at ca. 10⁶-10⁷ CFU g^-1 in the cabbage rhizosphere for 4 weeks after the initial application. The chemical fungicide, benomyl, did not suppress the disease severity at low concentration (1 or 10 µg mL^-1). However, the disease severity was decreased by the combined application of a low dosage of benomyl with strain LRB3W1. Combined application of a low dosage of benomyl with strain LRB3W1 was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol agent, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing cabbage yellows in sustainable agriculture because of the reduced chemical dosages needed for disease management.     

Colonization of Fusarium Wilt‐Resistant and Susceptible Watermelon Roots by a Green‐Fluorescent‐Protein‐tagged Isolate of Fusarium oxysporum f.sp. niveum

Interactions between watermelon and a green fluorescent protein (GFP)‐tagged isolate of Fusarium oxysporum f.sp. niveum race 1 (Fon‐1) were studied to determine the differences in infection and colonization of watermelon roots in cultivars resistant to and susceptible to Fusarium wilt. The roots of watermelon seedlings were inoculated with a conidial suspension of the GFP‐tagged isolate, and confocal laser scanning microscopy was used to visualize colonization, infection and disease development. The initial infection stages were similar in both the resistant and susceptible cultivars, but the resistant cultivar responded differentially after the pathogen had penetrated the root. The pathogen penetrated and colonized resistant watermelon roots, but further fungal advance appeared to be halted, and the fungus did not enter the taproot, suggesting that resistance is initiated postpenetration. However, the tertiary and secondary lateral roots of resistant watermelon also were colonized, although not as extensively as susceptible roots, and the hyphae had penetrated into the central cylinder of lateral roots forming a dense hyphal mat, which was followed by a subsequent collapse of the lateral roots. The initial infection zone for both the wilt‐susceptible and wilt‐resistant watermelon roots appeared to be the epidermal cells within the root hair zone, which the fungus penetrated directly after forming appressoria. Areas where secondary roots emerged and wounded root tissue also were penetrated preferentially.     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.     

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes. Received: 6 June 1997 / Accepted: 8 July 1997