B. Schippers

Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas SPP-mediated plant growth-stimulation

Inhibition of root cell energy metabolism is suggested to be responsible for potato yield reductions in short potato-rotation soils. Hydrogen cyanide is the microbial metabolile possibly involved in inhibition of energy metabolism. This is supported by the following observations: (1) approximately 50% of potato rhizosphere pseudomonads was shown to produce cyanide in vitro; (2) 5 μM HCN inhibited cytochrome oxidase respiration by at least 40% in intact potato roots in vitro; (3) cyanide production in vitro by Pseudomonas sp. isolate WCS361 depended on the Fe3+ concentration of the medium. Growth promoting fluorescent Pseudomonas spp isolates WCS374 and WCS358 did not produce cyanide in vitro. A hypothesis, that potato plant growth is depressed in short potato rotation soils by the microbial production of cyanide in the rhizosphere is discussed. In such soils, bacteria producing specific siderophores increase growth by competing with cyanide-producing organisms for Fe3+.

Lipopolysaccharides of plant-growth promoting Pseudomonas sp. strain WCS417r induce resistance in carnation to Fusarium wilt

The numbers of diseased plants could significantly be reduced when microconidia ofFusarium oxysporum f. sp.dianthi were inoculated into the stem and viable-, heat-killed cells or purified LPS of the bacteriumPseudomonas sp. strain WCS417r were applied to the roots. Because the competition betweenF. o. dianthi and the bacterium could be excluded, the disease suppression seems to be due to an induced resistance. Accumulation of phytoalexins was found in the stem segments. No accumulation of these compounds was found when the plants were bacterized but noninfected. It is concluded that cell surface components present in the lipopolysaccharides of the bacterium are the inducing factors.Increased peroxidase activity could be measured in root washes and root extracts after only bacterial preparations were added. No significant differences in peroxidase activity were found in stem extracts. The possible role of increased peroxidase activity in suppression of Fusarium wilt in carnation is discussed.

Induction of systemic resistance byPseudomonas fluorescens in radish cultivars differing in susceptibility to fusarium wilt, using a novel bioassay

Pseudomonas fluorescens-mediated induction of systemic resistance in radish against fusarium wilt (Fusarium oxysporum f. sp.raphani) was studied in a newly developed bioassay using a rockwool system. In this bioassay the pathogen and bacterium were confirmed to be confined to spatially separate locations on the plant root, throughout the experiment. Pathogen inoculum obtained by mixing peat with microconidia and subsequent incubation for four days at 22 °C, yielded a better percentage of diseased plants than a microconidial suspension drench, an injection of a microconidial suspension into the hypocotyl, or a talcum inoculum.Pseudomonas fluorescens strain WCS374 applied in talcum or peat, but not as a suspension drench, induced systemic resistance. A minimal initial bacterial inoculum density of ≥105 CFU WCS374 root−1 was required to significantly reduce the percentage diseased plants. At least one day was necessary between bacterization of strain WCS374 in talcum on the root tips and inoculation of the pathogen in peat on the root base, for an optimal induction of systemic resistance. Strain WCS374 induced systemic resistance in six radish cultivars differing in their susceptibility toF. oxysporum f. sp.raphani. Significant suppression of disease by bacterial treatments was generally observed when disease incidence in the control treatment, depending on pathogen inoculum density, ranged between approximately 40 to 80%. Strains WCS374 and WCS417 ofPseudomonas fluorescens induced systemic resistance against fusarium wilt, whereasP. putida WCS358 did not. This suggests that the induction of systemic resistance byPseudomonas spp. is dependent on strain-specific traits.

The role of siderophores in potato tuber yield increase by Pseudomonas putida in a short rotation of potato

The effect of treatment of potato seed tubers withPseudomonas putida isolate WCS358 on tuber yield was studied in different crop rotations at the Experimental Farm ‘De Schreef’, near Lelystad. With untreated, tuber yield in a 1:3 (short) rotation compared to yield in a 1:6 (long) rotation of potato was decreased by 11% at 86 days (seed tuber harvest) and by 14% at 130 days (ware potato harvest) after seeding. Seed tuber treatment with the wild-type isolate WCS358 increased tuber yield with 13% in a short rotation of potato 86 days after seeding, whereas a siderophore-negative Tn5 transposon mutant of this isolate had no effect on tuber yield. Seed tuber treatment with the wild-type isolate or the siderophore-negative mutant in a long rotation of potato had no effect on tuber yield. At 130 days after seeding no effect of any of the seed tuber treatments was found in both short and long rotations of potato.Root colonization by siderophore-producing Tn5 transposon mutants of WCS358 was decreased at the end of the growing season. No difference in root colonization between siderophore-producing and siderophore-negative Tn5 transposon mutants was found at 130 days after seeding.Siderophore production seems to be a prerequisite in potato tuber yield increase by WCS358 under field conditions. This is the first time that the involvement of siderophores in growth stimulation has been demonstrated in the field.SamenvattingDe invloed van een behandeling van aardappelpootgoed metPseudomonas putida isolaat WCS358 op de knolopbrengst werd onderzocht in verschillende gewasrotaties on een proefveld van proefboerderij ‘De Schreef’, Flevopolder. In de controlebehandelingen werd in een nauwe aardappelrotatie (1:3) een reductie van 11% in opbrengst van pootaardappelen (86 dagen na het poten) geconstateerd ten opzichte van een ruime aardappelrotatie (1:6); 130 dagen na het poten werd een vermindering met 14% gevonden in de opbrengst van consumptieaardappelen.Pootgoedbehandeling met het siderofoorproducerende isolaat WCS358 verhoogde de opbrengst van pootaardappelen in de 1:3-rotatie met 13%. Een Tn5-transposonmutant van dit isolaat die het vermogen sideroforen te produceren had verloren, had geen effect op de opbrengst. In de 1:6-rotatie had behandeling van pootgoed met WCS358 geen effect op de opbrengst van pootaardappelen.Zowel in de nauwe (1:3) als in de ruimte (1:6) rotatie werd (130 dagen na het poten), geen effect van behandeling van pootgoed met WCS358 op de opbrengst van consumptieaardappelen gevonden.Wortelkolonisatie door siderofoorproducerende Tn5-transposonmutanten van WCS358 nam aan het eind van het seizoen af. Er werd, 130 dagen na het poten, geen verschil in wortelkolonisatie geconstateerd tussen siderofoorproducerende en siderofoornegatieve Tn5-transposonmutanten.Siderofoorproduktie blijkt een voorwaarde te zijn voor verhoging van de knolopbrengst door WCS358 onder veldomstandigheden. De verhoging van de knolopbrengst treedt alleen op in de nauwe aardappelrotatie. Dit is de eerste keer dat de betrokkenheid van sideroforen bij groeistimulatie onder veldomstandigheden is aangetoond.

Suppression of fusarium wilt of radish by co-inoculation of fluorescentPseudomonas spp. and root-colonizing fungi

In an earlier study, treatment of radish seed with the bacteriumPseudomonas fluorescens WCS374 suppressed fusarium wilt of radish (Fusarium oxysporum f. sp.raphani) in a commercial greenhouse [Leemanet al., 1991b, 1995a]. In this greenhouse, the areas with fusarium wilt were localized or expanded very slowly, possibly due to disease suppressiveness of the soil. To study this phenomenon, fungi were isolated from radish roots collected from the greenhouse soil. Roots grown from seed treated with WCS374 were more abundantly colonized by fungi than were roots from nonbacterized plants. Among these were several species known for their antagonistic potential. Three of these fungi,Acremonium rutilum, Fusarium oxysporum andVerticillium lecanii, were evaluated further and found to suppress fusarium wilt of radish in a pot bioassay. In an induced resistance bioassay on rockwool,F. oxysporum andV. lecanii suppressed the disease by the apparent induction of systemic disease resistance. In pot bioassays with thePseudomonas spp. strains, the pseudobactin-minus mutant 358PSB− did not suppress fusarium wilt, whereas its wild type strain (WCS358) suppressed disease presumably by siderophore-mediated competition for iron. The wild type strains of WCS374 and WCS417, as well as their pseudobactin-minus mutants 374PSB− and 417PSB− suppressed fusarium wilt. The latter is best explained by the fact that these strains are able to induce systemic resistance in radish, which operates as an additional mode of action. Co-inoculation in pot bioassays, ofA. rutilum, F. oxysporum orV. lecanii with thePseudomonas spp. WCS358, WCS374 or WCS417, or their pseudobactin-minus mutants, significantly suppressed disease (except forA. rutilum/417PSB− and all combinations with 358PSB−), compared with the control treatment, if the microorganisms were applied in inoculum densities which were ineffective in suppressing disease as separate inocula. If one or both of the microorganism(s) of each combination were applied as separate inocula in a density which suppressed disease, no additional suppression of disease was observed by the combination. The advantage of the co-inoculation is that combined populations significantly suppressed disease even when their individual population density was too low to do so. This may provide more consistent biological control. The co-inoculation effect obtained in the pot bioassays suggests that co-operation ofP. fluorescens WCS374 and indigenous antagonists could have been involved in the suppression of fusarium wilt of radish in the commercial greenhouse trials.

Siderophore-mediated competition for iron and induced resistance in the suppression of fusarium wilt of carnation by fluorescent Pseudomonas spp

The mechanisms of suppression of fusarium wilt of carnation by two fluorescentPseudomonas strains were studied.Treatments of carnation roots withPseudomonas sp. WCS417r significantly reduced fusarium wilt caused byFusarium oxysporum f. sp.dianthi (Fod). Mutants of WCS417r defective in siderophore biosynthesis (sid−) were less effective in disease suppression compared with their wild-type. Treatments of carnation roots withPseudomonas putida WCS358r tended to reduce fusarium wilt, whereas a sid− mutant of WCS358 did not.Inhibition of conidial germination of Fod in vitro by purified siderophores (pseudobactins) of bothPseudomonas strains was based on competition for iron. The ferrated pseudobactins inhibited germination significantly less than the unferrated pseudobactins. Inhibition of mycelial growth of Fod by bothPseudomonas strains on agar plates was also based on competition for iron: with increasing iron content of the medium, inhibition of Fod by thePseudomonas strains decreased. The sid− mutant of WCS358 did not inhibit Fod on agar plates, whereas the sid− mutants of WCS417r still did. This suggests that inhibition of Fod by WCS358r in vitro was only based on siderophore-mediated competition for iron, whereas also a non-siderophore antifungal factor was involved in the inhibition of Fod by strain WCS417r.The ability of thePseudomonas strains to induce resistance against Fod in carnation grown in soil was studied by spatially separating the bacteria (on the roots) and the pathogen (in the stem). Both WCS417r and its sid− mutant reduced disease incidence significantly in the moderately resistant carnation cultivar Pallas, WCS358r did not.It is concluded that the effective and consistent suppression of fusarium wilt of carnation by strain WCS417r involves multiple mechanisms: induced resistance, siderophore-mediated competition for iron and possibly antibiosis. The less effective suppression of fusarium wilt by WCS358r only depends on siderophore-mediated competition for iron.

BENEFICIAL AND DELETERIOUS EFFECTS OF HCN-PRODUCING PSEUDOMONADS ON RHIZOSPHERE INTERACTIONS

Rhizobacteria live around roots but also inside the cortical root tissues by utilizing organic substances released from root cells into the intercellular spaces and the root environment. The effects of metabolites of these rhizosphere-inhabiting bacteria on root physiology and plant development have hardly been studied. However, recent studies indicate that, depending on environmental factors and plant species, certain strains of rhizosphere Pseudomonas spp. and some of their metabolites such as HCN may inhibit or enhance plant establishment or inhibit development of plant disease. Cultural practices such as cropping frequency, no tillage, and soilless cultivation, as well as edaphic factors seem to determine these rhizosphere interactions.

BIOASSAY FOR STUDYING THE ROLE OF SIDEROPHORES IN POTATO GROWTH STIMULATION BY PSEUDOMONAS SPP IN SHORT POTATO ROTATIONS

Summary-A bioassay is described for studying the mechanisms of growth stimulation by fluorescent pseudomonads using rooted potato stem cuttings. Root development of potato stem cuttings was inhibited in short potato-rotation soil compared to development in long potato-rotation soil. Treatment with Pseudomonas Juorescens isolate WCS374 or Pseudomonas putida isolate WCS358 increased root development in short potato-rotation soil, whereas siderophore-negative Tn5 transposon mutants of isolate WCS358 had no effect. Both siderophore-producing and siderophore-negative Tn5 mutants of isolate WCS358 could be recovered in similar numbers from the root systems. These results were obtained in a bioassay of 8 days duration. It is postulated that failure of siderophore-negative Tn5 transposon mutants of isolate WCS358 lo induce growth stimulation demonstrates that siderophore production is a prerequisite for growth stimulation in short potato-rotation soil.

Prospects for Management of Natural Suppresiveness to Control Soilborne Pathogens

Epidemics of soilborne plant diseases are rare in natural vegetations. If they occur, they seem to originate from a man-made disturbance of the natural equilibrium such as in natural Eucalyptus forests in Australia which enhanced Phytophthora dieback (Malajczuk, 1979).

Pseudomonas spp. with Mutational Changes in the O-Antigenic Side Chain of their Lipopolysaccharide are Affected in their Ability to Colonize Potato Roots

Microorganisms that efficiently colonize plant roots are ideal tools for the production of biocontrol agents, since many major and minor root pathogens are present at the root surface. In the past decade Pseudomonas spp. have received much attention as biocontrol agents (Kloepper et al 1980, Schippers et al 1986). The interest in this group of bacteria is mainly due to their antagonistic potentials in vitro towards pathogens combined with their ability to colonize the roots of many plants efficiently.