Flax Rust Research Articles

The effects of heat shock and inoculum density on growth and sporulation in axenic cultures of the flax rust fungus

The number of mycelial colonies of Melampsora lini (Ehrenb.) Lév. was increased by a 2-h heat shock at 31 °C delivered 3 h after uredospores were seeded on a liquid medium at 17 ± 0.5 °C under axenic conditions. At 35–38 days from seeding, the ratio of the respective number of colonies in shock and nonshock treatments in 125-mL culture flasks was highest (7.5 to 22) at inoculum levels (1 to 4 × 103 uredospores cm−2) that yielded about 10 colonies per flask without heat shock; the ratio declined sharply at inoculum levels adjusted to yield either smaller or larger numbers of colonies per flask without heat shock. Sporulation (extinction at 458 nm) and protein per colony were positively correlated; were not directly affected by heat shock; were determined by colony number; increased to maxima as mean colony number per flask increased from 1 or less to from 10 to 100 in different experiments; and decreased at higher colony numbers per flask. The results indicate that heat shock promotes the initiation of colonies; that colony number determines the duration of logarithmic growth; and that increasing competition among individual colonies is the primary factor limiting growth and sporulation, whether or not the uredospore germlings are subjected to heat shock.

H.h. Flor: pioneer in phytopathology.

Harold Henry Flor, in plant pathology? When we were asked to write this article, it took quite some time to think of Dr. Flor as pioneer­ after all he is only twelve years older than the senior author, and he's no pioneer. Maybe pioneer has meanings other than the idea of long ago, and in Webster's Dictionary there it was: a person ... that ... opens new line of thought. In this sense, he was true in developing the gene-for­ gene concept to explain the genetic relationships in flax rust. This idea has impacted not only on plant pathology, but also on genetics, and indirectly on many areas of science that related to disease of plants, animals, and man.

Protein synthesis in flax following inoculation with flax rust

Resistance to flax rust Melampsora lini (Ehrenb.) Lév. in flax carrying the N resistance gene is determined by 24 h postinoculation, at which time hypersensitivity is observed. We have examined protein synthesis in cotyledons inoculated with both virulent and avirulent races of rust by in vivo labelling with [35S]methionine. The pattern of protein synthesis was assessed by one- and two-dimensional electrophoresis 8, 13, and 18 h after inoculation. No changes in protein synthesis were observed in the first 14 h following inoculation; however, by 18 h after inoculation the susceptible combination showed a marked decrease in protein synthesis (22%; P = 0.01). This could be largely accounted for by the reduced synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit, which was readily quantified on electrophoresis gels. In addition, a 30-kDa polypeptide also declined in the susceptible combination. Two-dimensional electrophoresis enabled changes to be detected in the synthesis of other minor polypeptides. None of these changes were observed in the resistant combination in which a small increase in the synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit and the 30-kDa polypeptide was found. These results indicate that the outcome of the host–pathogen interaction has already been determined by 18 h after inoculation.

Effect of heat shock on protein synthesis in flax rust uredosporelings

In uredosporelings of Melampsora lini (Ehrenb.) Lév. germinated for 3 h, uptake of [35S]methionine and its incorporation into protein were depressed during a 2-h heat shock induced by transfer from 17 ± 0.5 to 31 °C. Spectrophotometric scans of fluorograms, prepared after one-dimensional sodium dodecyl sulphate – polyacrylamide gel electrophoresis of aliquots of protein extracts containing equal numbers of disintegrations per minute (1 dps = 1 Bq) in protein, showed that heat shock induced statistically significant changes in the relative degrees of incorporation of [35S]methionine into 15 polypeptide bands. Increased labelling occurred in seven bands, which appear to be heat shock proteins with apparent molecular masses of 84, 71, 43.5, 30.5, 19.5, 18, and 17 kD. Decreased labelling occurred in eight bands, which appear to be heat stroke proteins with apparent molecular masses of 56, 54, 48, 46, 34, 32, 31.5, and 14 kD. When [35S]methionine was administered during heat shock at 31 °C the same pattern of polypeptide labelling was observed in extracts made immediately without return to 17 °C and in extracts made after uredosporelings were returned to 17 °C for 24 h. Thus label incorporated into each polypeptide during heat shock was retained for at least 24 h after the return to 17 °C. Administration of [35S]methionine at 17 °C after completion of the heat shock showed that the "normal" or "nonshock" pattern of labelling began to be resumed within 2 h after transfer from 31 to 17 °C. The results are discussed in relation to the effect of heat shock in promoting the initiation and growth of mycelial colonies from uredosporelings in axenic culture.

The effects of CO2 and membranes on sporulation in axenic cultures of flax rust

Uredospore production by axenically grown flax rust (Melampsora lini (Ehrenb.) Lev.) was measured as carotenoids (extinction units at 458 nm) per milligram protein. Sporulation was not affected by raising (flushing with 1–5% (v/v) CO2 in air) or lowering (KOH well in culture flasks) the level of CO2 in the air space above the cultures. Significant (two- to four-fold) increases in sporulation occurred beneath impermeable membranes of Parafilm or Saran wrap placed on the surface of young (3 weeks from seeding) mycelial mats for 2 weeks. The stimulatory effect was confined strictly to those areas of the mycelial mats in contact with the membranes. Both Parafilm and Saran wrap were easily and cleanly peeled away from the mycelial mats. Permeable Unipore and HVHP membranes, to which the fungus adhered strongly, did not stimulate sporulation. The fungus did not adhere to Unipore or HVHP membranes treated with silicone or paraffin oil; membranes thus treated stimulated sporulation. The stimulatory effect of membranes on sporulation appears to depend on the nature of the contact between the membrane surface and the mycelium and to be unrelated to the effect of the membranes on the diffusion of gases or other volatile substances.

Further observations on the growth of flax rust fungus in axenic culture

Axenic cultures of flax rust (Melampsora lini (Ehrenb.) Lév.) grown from sterile uredospores on a defined liquid medium in 125-mL Erlenmeyer flasks exhibited an incubation period of 12 to 20 days followed by a period of rapid growth and sporulation. A heat shock (31–33 °C for 2–2.5 h) administered 3 h after seeding the medium did not induce the formation of infection structures, but doubled the number of vegetative colonies initiated and increased total growth per flask (protein/flask) in 4 weeks by 20-fold. The optimum timing of the heat shock corresponded closely with the maximum rate of germ tube emergence. Ambient CO2 was not essential for germination or perception of the heat shock, but was essential, during the first half of the incubation period, for the subsequent development of vegetative colonies. CO2 fixation occurred at all stages of growth from 1 day onward, 14CO2 being incorporated into ethanol-insoluble and ethanol-soluble fractions, including amino acids. Growth rate following the incubation period was not affected by the initial pH of the medium between 5.0 and 6.0, but the incubation period was shorter at pH 6.0.

A quantitative histological and ultrastructural analysis of interactions between the flax rust and near-isogenic host lines varying in their degree of incompatibility

Histological differences were evident in the leaves of eight near-isogenic lines of flax infected with the rust fungus Melampsora lini. Compared with the compatible L9 and M1 interactions, fungal growth was progressively more restricted in the moderately incompatible K and M4, incompatible M and P, and highly incompatible L and M3 interactions. This restriction took place in advance of appreciable necrosis of host cells in K, M4, M, and P. At 72 h the proportion of haustoria-containing cells which were necrotic was only 10–15% in K and M4. In M at 72 h necrosis was 80% or more at infection sites with small colonies but was negligible at sites with large colonies. In P, by contrast, a similar proportion of necrosis, 40% at 72 h, was present at all infection sites. However, in L and M3 host necrosis was much more rapid and the fungus was restricted to a few host cells. An early ultrastructural event was the appearance of extensive fibrillar deposits in the initially electron-lucent extrahaustorial matrices of both the incompatible M and P and moderately incompatible M4 and K interactions. A positive reaction with silver proteinate indicated that these matrical deposits contained carbohydrate, possibly a mucopolysaccharide or glycoprotein, but they were not extracted by either cellulase, pectinase, or chitinase. The extrahaustorial membrane, surrounding the haustoria in the compatible L9 and M1 interactions and the moderately incompatible K interaction was not stained by the periodic acid – phosphotungstic acid – chromic acid (PACP) procedure believed specific for the plasmalemma. In incompatible reactions clusters of electron-dense particles sometimes replaced starch in plastids of infected host cells. This event usually coincided with the appearance of extensive matrical deposits around fungal haustoria. At the same time particles were also found in plastids in uninfected host cells immediately adjacent to infection sites, particularly in the M and P interactions. These particles were extracted from thin sections by using pullulanase followed by α-amylase, indicating that they consisted of highly branched amylopectin.

A stimulus for sporulation of axenically grown flax rust

Flax rust ( Melampsora lini) grown axenically on a chemically defined medium sporulates only sparsely. The presence of an impermeable film on top of the mycelium stimulates sporulation where it touches the mycelium. The density of spores (as measured by the concentration of carotenoids) can increase tenfold and more. The increase in strictly localized to the area in contact with the film. If the film is applied after a certain critical age the effect drops off rapidly. The nature of the film seems to be unimportant as long as it is relatively impermeable to volatile substances. The effect is also persistent: if the film is removed after a number of days, the tissue at that location will still produce more spores.

Changes in Two Ribonuclease Isozymes During Rust Infection of Flax Cotyledons

A ribonuclease fraction previously purified from flax by gel filtration was further resolved into two components by hydroxyl apatite chromatography. These were homogeneous with respect to electrophoresis and isoelectric focusing. Both enzymes are of RNase I type but differ in substrate specificity, kinetic properties, pH response, and isoelectric point.The two RNase isozymes show consistent properties when extracted from variety Bison (susceptible) or variety Bombay (resistant) with or without infection with race 3 of flax rust. The relative amounts of these isozymes change markedly during infection. These observations provide an explanation for the apparent qualitative changes in RNase noted previously. Differences between susceptible and resistant reactions in the early stages of disease are discussed.

CO2 is essential for colony initiation by flax rust fungus grown in vitro

Cultures were grown under a constant stream of gas of known composition to eliminate accumulation of any volatiles produced by the fungus. CO2-free air completely inhibits colony initiation. Air containing 1% CO2 allows colony initiation to the same extent as occurs in plugged, nonaerated controls, while "normal air" (ca. 0.03% CO2) causes initiation to be delayed to a variable extent.

Fine structure of genes controlling pathogenicity in flax rust, Melampsora lini

The fine-structure of four genes, A-P, A-P1, A-P2 and A-P3 in the flax rust organism, Melampsora lini, that control pathogenicity on the host, Linum usitatissimum, has been investigated by using a mass-screening procedure to search for rare recombinant progeny. Three presumptive recombinant individuals were recovered from amongst 3160 dikaryotic test-cross progeny and seven from a second test in which haploid basidiospores were screened for recombinants. From the pathogenicity phenotypes of the presumptive recombinants, and their frequency of occurrence, it is concluded that genes A-P, A-P1, A-P2 and A-P3 are sufficiently closely linked to belong to the same cistron. However, the possibility that they belong to separate, closely-linked cistrons can not be excluded: if they do belong to separate cistrons, then the data suggest that these are contiguous. A tentative gene order A-P A-P2 A-P1 A-P3 is proposed.

Interactions Between Genes Controlling Pathogenicity in the Flax Rust Fungus

Interactions Between Genes Controlling Pathogenicity in the Flax Rust Fungus

Polyol Dehydrogenases in the Rust Fungus, Melampsora lini (Ehrenb.) Lev.

The activity of enzymes involved in polyol metabolism was investigated in uredospores and mycelial cultures of the flax rust fungus, Melampsora lini. Mycelial cultures displayed polyol dehydrogenase activity with a range of sugars using either NADH or NADPH as a cofactor. Highest activity was found with fructose and NADPH. The relative pattern with different substrates was unaffected by the carbohydrate used in the culture medium. Highest absolute activity was detected with culture media containing d-glucitol or d-mannitol. Mannitol and d-arabitol were the polyols oxidized by mycelial extracts. Flax rust uredospores could use aldohexoses, ketohexose and aldopentoses as substrates for polyol synthesis. Uredospore extracts were also capable of oxidizing d-arabitol with NAD+ as cofactor.

Multiple Mating-Type Specificities in the Flax Rust Melampsora lini

The results of selfing and intercrossing two strains of the flax rust fungus Melampsora lini indicate that this species does not possess a simple(+) and(-) mating system. Instead, the data are consistent with the assumption that the genetic control of mating type in Melampsora lini is similar to that in the mushroom Schizophyllum commune, in which mating type is determined by two factors, each of which is controlled by two linked loci.

Patterns of translocation, changes in invertase activity, and polyol formation in susceptible and resistant flax infected with the rust fungus Melampsora lini

In two near-isogenic lines of flax ( Linum usitatissimum L.) displaying resistance and susceptibility, respectively, to race 1 of the flax rust, Melampsora lini (Ehrenb.) Lév., retention of photoassimilates at infection sites and increased import of assimilates towards these sites were detected in susceptible cotyledons bearing a localized rust infection. No alterations in translocation patterns were detected in infected resistant seedlings. Increases in the activity of both soluble and insoluble acid invertases occurred from 6 days after inoculation in the susceptible host, but no increases were detected in the resistant variety. Fungal polyols, both hexitols and penitols, were detected in the susceptible host from five days after inoculation. By day 11 after inoculation they represented 45% of the labelled products 30 min after exposing infected cotyledons to 14CO 2. Only trace amounts of labelled polyols were detected in the rust-infected resistant variety, representing only 9% of the labelled products at 11 days after inoculation. A chemical method failed to detect any chitin formation in the rust-infected resistant host. However histological examination showed definite increases in rust colony area in resistant tissue. Increase in fungal chitin in the susceptible host was correlated with a similar increase in activity of acid invertase.

Lysine Synthesis in Rust Infected Flax Cotyledons

Pathways of lysine biosynthesis in rust infected flax cotyledons have been investigated. Non infected cotyledons were shown to possess enzymes of the diaminopimelic acid pathway of lysine synthesis, but these enzymes could not be detected in axenic cultures of the flax rust fungus. Feeding experiments demonstrated the incorporation of diaminopimelic acid into lysine by the host but not by the fungus. Conversely, enzymes of the aminoadipic acid pathway of lysine synthesis are present in the rust fungus, but, with the exception of aminoadipate transaminase, not in non-infected cotyledons. Aminoadipic acid stimulated lysine production by the fungus but not by the host. Cotyledons infected for 7 days were shown to possess enzymes of both pathways, and seemed able to convert both diaminopimelic acid and aminoadipic acid into lysine. We consider these studies to demonstrate that infected flax cotyledons possess the ability to synthesize lysine by either pathway. The fungus may thus be capable of synthesizing at least some of the lysine it requires.

Colony initiation in flax rust in axenic culture: involvement of a volatile factor

In axenic cultures of flax rust (Melampsora lini) colonies are initiated after a lag period of 12–20 days, depending partly on incubation temperatures. Colony initiation is completely inhibited by removal of a volatile factor which is absorbed by KOH in the air space of the culture flask. The fungus remains sensitive to this inhibition for 8–10 days, i.e., until shortly before visible colonies would normally have developed. While in the presence of KOH, the fungus is not killed; cultures grow normally after removal of the KOH.Although conclusive evidence must await further work, the available data strongly suggest that carbon dioxide is responsible for this effect.

Changes in Glutamate Dehydrogenase in Flax Cotyledons Infected with the Flax Rust Fungus

Summary Following inoculation with spores of the flax rust fungus (Melampsora lini), susceptible flax (Linum usitatissimum) cotyledons exhibit a biphasic increase in glutamate dehydrogenase activity. There is a transient increase in activity over the first 24 hours after inoculation, followed by a more gradual increase in activity towards later stages of infection. Both the first phase, which also occurs in resistant cotyledons, and the second phase, which is restricted to susceptible cotyledons, represent the pres sence of enzymes with altered properties from those from non-infected cotyledons. Similar enzymes could not be detected either in senescing flax cotyledons or in axenic cultures of the flax rust fungus.

Haploid Breeding Technique for Flax1

A haploid breeding technique for flax (Linum usitatissimum L.) using a twin seedling character is described. The haploid twin can be distinguished from the diploid twin by seedling size 3 days after germination, plant height at any growth stage, and by the production of sterile flowers. The twinning frequency of 54 random entries from the USDA World Flax Collection was 1 set to 5,869 normal seed; a workable level in a flax cultivar development program. Use of a flax rust [Melampsora lint (Ehrenb.) Lev.] resistance marker gene showed the haploid twin to be of maternal origin, presumably produced by apogamety. In a breeding program this indicates haploid selection and doubling could be conducted in the F2 to allow for genetic recombination. A 16‐month breeding program to produce completely homozygous breeding lines ready for field evaluation is described. This developmental period includes screening for rust resistance and selection for oil content and quality.

Flax rust resistance involving the K gene: an ultrastructural survey

An electron microscopic study of major gene resistance involving the flax K gene and the rust fungus Melampsora lini revealed several interesting ultrastructural features. Up to 9 days after infection, most haustoria-containing resistant host cells appeared viable. During this period there was a progressive increase in a fibrillar deposit in the extrahaustorial matrix which was not detected in the susceptible host. This material was in direct continuity with the distal wall of the haustorial neck. It frequently constituted an apparently complete layer or 'encapsulation' around the haustorium and was present in 75% of the extrahaustorial matrices as early as 4 days after infection. Initially, callose-like encasements formed around only a small percentage of haustoria in the resistant host; however, by day 9, about 20% of the haustoria were encased. No encasements were found around susceptible haustoria at this stage. Hypersensitive host cell collapse occurred in a small percentage of resistant infections.