Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Changes in proteins and isoenzymes in leaves of wheat when infected by stem rust

Summary Biuret assay, gel electrophoresis and immunochemistry were used to study concentrations, forms and activities of proteins of uredospores of Puccinia graminis Pers. f. sp. tritici, in healthy wheat leaves, wheat leaves that had been inoculated with incompatible races of stem rust and leaves which had become rusted.The soluble proteins of primary leaves increased by 25–117% following infection by compatible races of stem rust. There was a corresponding decrease of proteins in uninfected younger leaves. Infection by an incompatible strain of rust led to a temporary 29% increase in soluble proteins.Immunoelectrophoresis and gel electrophoresis of infected leaves showed the presence in them of the forms of malate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and -amylase characteristic of the rust fungus. In the infected leaves, the activity of certain bands of host glucose-6-phosphate dehydrogenase and catalase changed with the development of the pathogen; the malate dehydrogenase and -amylase of the host were unaffected. In leaves inoculated with an incompatible race there were no obvious changes of any of these enzymes.     

Biological Control of Groundnut Late Leaf Spot and Rust by Seed and Foliar Applications of a Powder Formulation of Pseudomonas fluorescens

Glasshouse and field trials were conducted to determine the efficacy of a talc-based powder formulation of Pseudomonas fluorescens strain Pf1 in controlling groundnut leaf spot ( Cercosporidium personatum ) and rust ( Puccinia arachidis ). Seed treatment with the talc-based powder formulation of the bacterium alone effectively reduced the severity of leaf spot and rust. When the treated seeds were sown in soil, the antagonist moved to the rhizosphere and multiplied well in it. Foliar application with the powder formulation effectively controlled the groundnut leaf spot and rust. P. fluorescens multiplied well in the phyllosphere after foliar application of powder formulation. Combined application of the P. fluorescens formulation to seed and foliage effectively controlled leaf spot and rust, and increased the pod yield in greenhouse and field tests.     

Isoenzymes of P-diphenol oxidase in the stem rust-wheat interacting system

Summary Electrophoretic, histochemical and serological techniques were used to study the forms and activities of p-diphenol oxidase in healthy wheat leaves, wheat leaves that had been inoculated with incompatible races of stem rust, leaves that were rusted and in uredospores of stem rust. Three isoenzymes were detected, one in uredospores and two in infected, susceptible wheat leaves. However, these two forms were not detected in control leaves nor in actively growing, inoculated, resistant leaves. Histochemical observations suggest that the two isoenzymes found in the host tissue were secreted by the pathogen. An increase in the activity of one of these coincided with the appearance of flecks in the susceptible leaves.     

Effect of temperature, light and host on prepenetration development of Puccinia graminis avenae and Puccinia coronata avenae

The influence of temperature and light on prepenetration development of single and mixed isolates of Puccinia graminis avenae and Puccinia coronata avenae was studied on 0–2% water agar and on leaves of three oat cultivars and on three non-cultivated species of Avena. Germination of uredospores of P. graminis avenae and P. coronata avenae occurred best at 10–30^oC and at 20^oC respectively. The optimum temperature for germ-tube growth and for appressorial formation was 20^oC for both rusts. An inverse relationship was observed between light intensity and prepenetration development with maximal germination of uredospores, germ-tube growth and appressorial formation occurring in darkness. Under optimum conditions maximum percentage germination and appressorium formation of both rusts was attained within 4 and 12 h after inoculation respectively. The proportion of germinated uredospores of crown rust which gave rise to appressoria was about twice that observed for stem rust. No significant differences were observed in prepenetration development between the single and mixed race inocula of the two rusts. Although germination of uredospores was significantly greater on water agar than on oat leaves, there were no significant differences in prepenetration development of the rusts on the various oat cultivars and species examined. Consequently, the data failed to indicate the presence of resistance mechanisms operating during the prepenetration phase of the infection process on the cultivars and species examined.     

Germination of Puccinia striiformis uredospores on leaves of adult winter wheat plants

In experiments with intact adult winter wheat plants and detached leaves, higher percentages of Puccinia striiformis uredospores germinated on the adaxial leaf surface, particularly the distal parts, than on the abaxial surface. This is consistent with the observation that the distal parts of the adaxial leaf surface are those which are most susceptible to yellow rust. Higher percentages of uredospores germinated on leaves of some of the winter wheat varieties examined than on those of others. These varietal differences depended on the part of leaf and the race of P. Striiformis used for the comparisons. This suggests that the differences between varieties are partly race specific.     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.     

Interaction between crown rust (Puccinia coronata) and two viruses of ryegrass

In Lemtal Italian and S.24 perennial ryegrass plants, two isolates of ryegrass mosaic virus (RMV) suppressed the amount of crown rust emerging on leaves inoculated with Puccinia coronata uredospores by up to 75% compared with the amount on virus-free plants. Severity of rust infection on barley yellow dwarf virus (BYDV) infected plants generally did not differ significantly from that on virus-free plants. When both RMV and BYDV were present, rust was restricted in Lemtal plants to a level intermediate between those occurring on plants infected by either virus alone, and in S.24 plants to a level below that obtained with either virus alone. The mean water soluble carbohydrate (WSC) content of Lemtal plants was reduced more than 20% by RMV, but was not significantly altered by BYDV. In S.24 plants the WSC content was increased by 10% by RMV and by 60% by BYDV. Rust reduced the WSC content of healthy and virus-infected plants, the reduction being positively correlated with the level of rust on the sampled leaves. In plants of Lemtal, but not of S.24, the degree of rust infection was positively correlated with the WSC content of leaves from rust-free control plants.     

Degradation of Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) by Cell-Free Culture Filtrates of the Mycoparasite Verticillium Psalliotae Treschow

Verticillium psalliotae isolates Taiw and Thai C are effective parasites of the soybean rust fungus. Cell-free culture filtrates of these fungi, prepared after growth on autoclaved uredospores, contained β-1,3-glucanase, chitinase and protease activities and caused degradations, when rust spores were treated with them for 24 or 72 h. During these lytic processes carbohydrates, amino compounds and N-acetylhexosamine were released. The carbohydrate fraction was composed of mannitol, arabitol, trehalose, glucose and unidentified substances showing low R_f-values during thin layer chromatography. The amino compounds consisted of 10 amino acids (leucine and/or isoleucine, phenylalanine, glutamic acid, valine, arginine, asparagine, glutamine, serine, aspartic acid, histidine) and 5—7 substances which could not be identified. Verticillium lecanii isolate Konz is a weak parasite of soybean rust. During growth on uredospores the fungus produced culture filtrates without chitinase activity and with low total activities of β-1,3-glucanase and protease. Compared with V. psalliotae, culture filtrates of V. lecanii exerted lower lytic activities on soybean rust uredospores. The results are consistent with the aspect that the rapid growth of V. psalliotae on soybean rust fungus is primarily based on the secretion of lytic enzymes which make nutrients available to the mycoparasite.     

Development of Infection Structures by the Direct-Penetrating Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) on Artificial Membranes

The development of infection structures by the directly infecting soybean rust fungus of different artificial membranes was followed by light and scanning electron microscopy. On water agar uredospores developed germ tubes without appressoria. On dialysis membranes more than 80% of the uredospores formed appressoria. With low frequencies (1–7%) also primary hyphae and/or penetration hyphae were present. When cellulose nitrate membrane filters with pore diameters ≤ 0.2 μm were used, uredospores germinated but showed a strongly reduced appressoria formation. Membranes with pores ≥ 0.1 μm allowed a development of infection structures similar to that on dialysis membranes. In experiments with paraffin oil incorporated into collodion membranes more than 90% of the uredospores formed appressoria, about 50% of the appressoria developed hyphae. Ungerminated spores and germ tubes always contained 2 nuclei. In fully developed appressoria 4 nuclei were present. Compared with stomata entering rust fungi appressoria formation by Phakopsora pachyrhizi occurred more frequently and seemed to be less dependent on specific stimuli. Moreover, in most cases only few of the appressoria formed penetration or primary hyphae. The induction of these structures seemed to be dependent on further unknown stimuli.     

The Association of Brome Mosaic Virus and Wheat Rusts.

Evidence is presented in support of transmission of brome mosaic virus by uredospores of wheat stem rust (Puccinia graminis f. sp. tritici). The presence of BMV associated with uredospores is shown by the use of sensitive serological assays such as, fluorescent antibody binding studies, enzyme-linked immunosorbent assay and immunosorbent electron microscopy. The virus is shown to be carried externally on the uredospores. The nature of the association of the virus with uredospores and its relation to the mode of carriage and transmission of the virus by the uredospores, as well as the importance of this association in the epidemiology of the virus is discussed.     

The ultrastructure of the uredospore of Puccinia recondita

Dry, fresh uredospores ofPuccinia recondita that have been killed by infra-red radiation showed no striking ultrastructural differences from dry, fresh, viable uredospores. Numerous spherical and elipsoidal mitochondria with distinct and deep cristae, ribosomes, endoplasmic reticulum and convoluted plasmalemma were well shown by both.When moistened and incubated, killed uredospores lost ultrastructural organization, whereas moistened, viable, fresh uredospores imbibed moisture, became more spherical and germination commenced. A more or less centrally located nucleus was seen with the double membrane invaginated at some points. The advance of the germ tube was initially by enzymic degradation and concluded by mechanical disruption of the degraded pore plug. The mitochondria and the endoplasmic reticulum increased in number and the stored lipid bodies were gradually depleted as germination progressed.Features known as the foamy cytoplasm and folded membranes were seen in the germinating uredospores only. It was suggested that the foamy cytoplasm could be functionally similar to the glyoxisome because of the close association of the former to lipid bodies. The folded membranes may be accumulated endoplasmic reticulum being transported to the site of wall formation.Abbreviations used in electronmicrographs CW Cell wall - er Endoplasmic reticulum - f folded membranes - GP Germ pore - L Lipid bodies - M Mitochondria - P Plasmalemma - Pm foamy cytoplasm - R Ribosomes - T Germ tube     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Chitosan isolation from the chitosan-glucan complex of fungal cell wall using amylolytic enzymes

The chitosan/glucan complex isolated from the mycelia of the fungus, Gongronella butleri USDB 0201 can be cleaved with a heat-stable -amylase at 65 °C for 3 h. This results in the removal of the glucan side chain and gives a chitosan solution with 100 times lower turbidity. It is proposed that chitosan and glucan chains are bound by an (1 to 4) glucosidic bond. Both fungal chitosan and fungal glucan have been purified separately.     

The Inhibition of Rust Fungus Growth in Allopurinol-Treated Bean Plants: Indirect Evidence for the Involvement of Xanthine Oxidoreductase in the Biotrophism of Uromyces phaseoli

Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine], a specific, potent inhibitor of xanthine oxidoreductase, effective in vitro and in vivo, was applied to bean plants as soil drench at a 400 μM concentration 8–10 days before inoculation and strongly reduced the development of Uromyces phaseoli in bean leaves. Allopurinol was ineffective on uredospore germination, presumably due to the absence of any xanthine oxidoreductase activity in the extract of germinated uredospores. The concentration of allopurinol used for the treatment did not significantly influence the level of ureides in leaves mainly because low concentration of these compounds were found in leaves and also probably because allopurinol-insensitive biosynthetic route/s of these compounds are active in bean plants. This paper examines the possibility that host xanthine oxidase is in some way involved in the biotrophic nutritional process leading to the growth of bean rust fungus.     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

Defeating the Warrior: genetic architecture of triticale resistance against a novel aggressive yellow rust race

Key message

Genome-wide association mapping of resistance against the novel, aggressive ‘Warrior’ race of yellow rust in triticale revealed a genetic architecture with some medium-effect QTL and a quantitative component, which in combination confer high levels of resistance on both leaves and ears.

Abstract

Yellow rust is an important destructive fungal disease in small grain cereals and the exotic ‘Warrior’ race has recently conquered Europe. The aim of this study was to investigate the genetic architecture of yellow rust resistance in hexaploid winter triticale as the basis for a successful resistance breeding. To this end, a diverse panel of 919 genotypes was evaluated for yellow rust infection on leaves and ears in multi-location field trials and genotyped by genotyping-by-sequencing as well as for known Yr resistance loci. Genome-wide association mapping identified ten quantitative trait loci (QTL) for yellow rust resistance on the leaves and seven of these also for ear resistance. The total genotypic variance explained by the QTL amounted to 44.0% for leaf and 26.0% for ear resistance. The same three medium-effect QTL were identified for both traits on chromosomes 1B, 2B, and 7B. Interestingly, plants pyramiding the resistance allele of all three medium-effect QTL were generally most resistant, but constitute less than 5% of the investigated triticale breeding material. Nevertheless, a genome-wide prediction yielded a higher predictive ability than prediction based on these three QTL. Taken together, our results show that yellow rust resistance in winter triticale is genetically complex, including both medium-effect QTL as well as a quantitative resistance component. Resistance to the novel ‘Warrior’ race of this fungal pathogen is consequently best achieved by recurrent selection in the field based on identified resistant lines and can potentially be assisted by genomic approaches.

     

Estimates of the residual nitrogen benefit of groundnut to maize in Northeast Thailand

Four cultivars of groundnut were grown in upland soil in Northeast Thailand to study the residual benefit of the stover to a subsequent maize crop. An N-balance estimate of the total residual N in the maize supplied by the groundnut was made. In addition three independent estimates were made of the residual benefits to maize when the groundnut stover was returned to the land and incorporated. The first estimate (Estimate 1) was an N-balance estimate. A dual labelling approach was used where ¹⁵N-labelled stover was added to unlabelled microplots (Estimate 2) or unlabelled stover was added to ¹⁵N-labelled soil microplots (Estimate 3). The nodulating groundnut cultivars fixed between 59–64% of their nitrogen (as estimated by the ¹⁵N isotope dilution method using non-nodulating groundnut as a non-fixing reference) producing between 100 and 130 kg N ha^-1 in their stover. Although the following maize crop suffered from drought stress, maize grain N and dry weights were up to 80% and 65% greater respectively in the plots where the stover was returned as compared with the plots where the stover was removed. These benefits were comparable with applications of 75 kg N ha^-1 nitrogen in the form of urea. The total residual N estimates of the contribution of the nodulated groundnut to the maize ranged from 16.4–27.5 kg N ha^-1. Estimates of the residual N supplied by the stover and fallen leaves ranged from 11.9–21.3 kg N ha^-1 using the N-balance method (Estimate 1), from 6.3–9.6 kg N ha^-1 with the labelled stover method (Estimate 2) and from 0–11.4 kg N ha^-1 with the labelled soil method. There was closest agreement between the two ¹⁵N based estimates suggesting that apparent added nitrogen interactions in these soils may not be important and that N balance estimates can overestimate the residual N in crops following legumes, even in very poor soils. This work also indicates the considerable ability of local groundnut cultivars to fix atmospheric nitrogen and the potential benefits from returning and incorporating legume residues to the soil in the upland cropping systems of Northeast Thailand. The applicability of the ¹⁵N methodology used here and possible reasons for the discrepancies between estimates 1, 2 and 3 are discussed.     

Light- and Scanning Electron Microscopic Studies on the Development of the Mycoparasite Verticillium psalliotae Treschow on Uredospores of the Soybean Rust (Phakopsora pachyrhizi Syd.)

Abstract By light and scanning electron microscopy it is shown that a strain of Verticillium psalliotae , originally isolated from pustules of the soybean rust in Thailand, is able to infect uredospores of this rust fungus. In most cases appressoria-like structures were formed at the infection sites. However, only a part of the spores were infected. Most spores appeared heavily damaged without any visible mycelium inside. This indicates that growth of Verticillium psalliotae on uredospores of the soybean rust is probably mainly based on the production of lytic enzymes.     

Untersuchungen über Stoffwechselbeziehungen zwischen Parasit und Wirt am Beispiel von Puccinia graminis var. tritici auf Weizen

Summary Rust infected leaves of wheat plants were incubated with glucose-¹⁴C. Uredospores which were formed during the application of the tracer were analyzed. All isolated compounds were labeled with ¹⁴C. When germinating uredospores were incubated directly with ¹⁴C-glucose, the isolated glutamic acid, arginine and lysine had practically no radioactivity. These compounds did, however, contain considerable ¹⁴C-activity when they were isolated from uredospores formed on leaves that had been treated with the tracer. We therefore conclude that these amino acids were synthesized in the host and were taken up by the haustoria of the mycelium.High ¹⁴C-radioactivity was also found in all carbohydrates (chitin, glucomannan, polyols etc.). Hexoses isolated from the spore constituents chitin and glucomannan showed the same distribution of radioactivity as the applied glucose-1-¹⁴C or glucose-6-¹⁴C. It follows that the rust mycelium takes up glucose or a similar monosaccharide from the wheat plant. The C-6-skeleton is not degraded to smaller metabolites before it is taken up.