Yigal Cohen

β-Aminobutyric Acid-Induced Resistance Against Plant Pathogens

The potential of a plant to resist attack by a pathogen is enhanced after an initial inoculation with a necrotrophic pathogen. Not only the initially inoculated tissue, but also the remote organs such as leaves and roots, become resistant. Such immunization, named systemic acquired resistance (SAR), was first observed by Chester (4) with Tobacco mosaic virus (TMV) in tobacco and was further demonstrated by the pioneering work of Ross (50) and Kuc (28,29). Efforts devoted to discovering the signal translocating during SAR revealed (26,35,40) the central role of salicylic acid (SA) in activating the defense mechanisms leading to SAR. Functional analogs of SA, such as 2,6-dichloroisonicotinic acid (INA) or benzo[1,2,3] thiadiazole-7-carbothionic acid S-methyl ester (BTH or acibenzolar-Smethyl), were developed which activate the resistance mechanisms downstream of SA (16,26,35,40,57). Since the observations of Chester, little attention was given to the role of induced resistance in general, and of amino acids in particular, in plant defense (29). Kuc et al. were the first to notice in 1957 (31) and 1959 (30) that Dphenylalanine, D-alanine, and DL-tryptophan injected into apple leaves increased resistance against scab without affecting the causal pathogen in vitro. As early as 1958, Van Andel (60) examined 50 amino acids for inducing resistance against Cladosporium cucumerinum in cucumber and found only D-serine, D-threonine, and L-threo-β-phenylserine highly active in vivo but not in vitro. Interestingly, she found no activity with DL-α-aminobutyric (AABA), DL-β-aminobutyric (BABA), γaminobutyric (GABA), or their iso isomers (60). In 1960, Oort and Van Andel first noted induced resistance to tomato late blight following BABA treatment (39). In 1963, two groups reported on the activity of aminobutrates. MacLennan et al. (34) showed that D-AABA and AIB (2-aminoisobutyric acid) were active in apple leaves against scab (but not L-AABA, BABA, or isoBABA). Papavizas and Davey (45) reported on the partial activity of AABA and high activity of BABA against Aphanomyces euteiches causing root rot in peas. They also showed high SAR activity with longer, straight-chain aminobutyrates such as DL-α-aminovaleric (DL-norvaline, five carbons) and DL-α-aminocaprylic acid (eight carbons). Harnack and Schwarz (19) showed that N-substituted glycine derivatives (especially ethyl and butyl) were systemically effective against Phytophthora infestans in tomato and Septoria apii in celery without being active in vitro. The interest in amino acid–mediated induced resistance was renewed about 30 years later when we discovered a strong activity of BABA against disease in potato (10), tomato (6), and tobacco (5), and revealed some of the defense mechanisms it activates in tomato (13) and tobacco (5). In recent years, substantial evidence has been accumulated, especially in Arabidopsis mutants, showing that BABA possesses a large spectrum of activity (7,22,41), as well as multiple forms of plant activation against disease (41,55,66,67).

Plant eR Genes That Encode Photorespiratory Enzymes Confer Resistance against Disease

Downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating foliar disease of cucurbits worldwide. We previously demonstrated that the wild melon line PI 124111F (PI) is highly resistant to all pathotypes of P. cubensis. That resistance was controlled genetically by two partially dominant, complementary loci. Here, we show that unlike other plant disease resistance genes, which confer an ability to resist infection by pathogens expressing corresponding avirulence genes, the resistance of PI to P. cubensis is controlled by enhanced expression of the enzymatic resistance (eR) genes At1 and At2. These constitutively expressed genes encode the photorespiratory peroxisomal enzyme proteins glyoxylate aminotransferases. The low expression of At1 and At2 in susceptible melon lines is regulated mainly at the transcriptional level. This regulation is independent of infection with the pathogen. Transgenic melon plants overexpressing either of these eR genes displayed enhanced activity of glyoxylate aminotransferases and remarkable resistance against P. cubensis. The cloned eR genes provide a new resource for developing downy mildew–resistant melon varieties.

Systemic resistance of potato plants against Phytophthora infestans induced by unsaturated fatty acids

Five unsaturated fatty acids were tested for their ability to induce systemic resistance in potato plants (cv. Bintje) to the late blight fungus Phytophthora infestans . Arachidonic acid and eicosapentaenoic acid applied to leaves 1–3 of potato plants at a dose of about 1 mg per plant induced 94% to 97% protection in leaves 4 to 11, respectively. Linoleic acid, linolenic acid and oleic acid provided 82%, 39% and 42% protection, respectively. Protection was evident as a reduction in lesion number and lesion diameter. It was maximal in plants challenged 5 days after induction and lasted for at least 12 days. A prior infection of lower leaves with P. infestans also induced up to 88% protection in the upper leaves. The mechanism involved in the induction of systemic resistance of potato plants by fatty acids is not understood.

Local and Systemic Induced Resistance to the Root-Knot Nematode in Tomato by DL-β-Amino-n-Butyric Acid

ABSTRACT Chemical inducers of pathogenesis-related proteins and plant resistance were applied to tomato plants, with the aim of inducing resistance to the root-knot nematode Meloidogyne javanica. Relative to control plants, foliar spray and soil-drenching with dl-beta-amino-n-butyric acid (BABA) reduced root-galling 7 days after inoculation, as well as the number of eggs 30 days after inoculation. Other chemicals (alpha- and gamma-amino-n-butyric acid, jasmonic acid, methyl jasmonate, and salicylic acid) were either phytotoxic to tomato plants or did not improve control of root-knot nematodes. Fewer second-stage juveniles invaded BABA-treated tomato roots, and root-galling indices were lower than in control tomato plants. Resistance phenomena in seedlings lasted at least 5 days after spraying with BABA. Nematodes invading the roots of BABA-treated seedlings induced small, vacuolate giant cells. Postinfection treatment of tomato plants with BABA inhibited nematode development. It is speculated that after BABA application tomato roots become less attractive to root-knot nematodes, physically harder to invade, or some substance(s) inhibiting nematode or nematode feeding-site development is produced in roots.

Systemic translocation of 14C-dl-3-aminobutyric acid in tomato plants in relation to induced resistance against Phytophthora infestans

This paper provides evidence that the systemic resistance induced by dl -3-amino-n-butanoic acid, β-aminobutyric acid (BABA), against late blight in tomato is associated with the systemic acropetal translocation of BABA in the plant. Thus, when 14 C-BABA was applied to the bottom leaves, the upper, but not the adjacent leaves, accumulated the compound and the upper leaves only were protected against disease. Similarly, when BABA was applied to the root system it was preferentially translocated to the uppermost leaves and these leaves showed the greatest protection. These data support the hypothesis that resistance induced by BABA is dependent on the actual presence of the compound in the leaf. Almost all 14 C-BABA supplied to the tomato plants was recovered unchanged with only a small proportion of the 14 C-label being retained by cell wall fractions, probably in the form of covalently-bound proteins. We suggest that apart from enhancing pathogenesis-related protein accumulation in tomato, BABA alters cell wall structure or metabolism so making the tissues more resistant to fungal enzyme attack.

Local and Systemic Activity of BABA (DL-3-aminobutyric Acid) Against Plasmopara viticola in Grapevines

The non-protein amino acid BABA (DL-3-amino-n-butanoic acid, β-aminobutyric acid) is reported here to induce local and systemic resistance against downy mildew in grape leaves. Leaf discs of susceptible cultivars placed on BABA solutions and inoculated with Plasmopara viticola on the counter surface produced brownish restricted lesions below the inoculation site (Hypersensitive-like response, HR) which failed to support fungal sporulation. Histochemical analyses of such HR lesions revealed the accumulation of lignin-like deposits in the host cells. In contrast, water-treated inoculated discs produced expanded chlorotic lesions with profuse sporulation in which no lignin accumulation was observed. Mock-inoculated BABA-treated leaf discs showed no HR or lignin accumulation. Concentrations as low as 25 µg/ml (0.25 mM) of BABA sufficed to prevent tissue colonization with the fungus. Five other isomers of aminobutyric acid, namely L-2 aminobutyric acid, 2-amino isobutyric acid, DL-2-aminobutyric acid (AABA), DL-3-amino isobutyric acid, and 4-aminobutyric acid (GABA) gave no protection against the downy mildew fungus. Of the two (R and S) enantiomers of BABA only the R form was active in producing HR, suggesting a specific stereostructure requirement for activity. BABA could stop fungal colonization even when applied post-infectionally to leaf discs. Resistance of BABA-pulse-loaded leaf discs persisted for more than 14 days. BABA provided systemic protection against the disease when applied via the root system or via the lower leaves of grape plants. Application of 14C-BABA to a single leaf of intact plants showed the accumulation of the 14C label in upper leaves (and root tips), suggesting sink-oriented transport.

Cucurbit downy mildew (Pseudoperonospora cubensis)—biology, ecology, epidemiology, host-pathogen interaction and control

Cucurbit downy mildew, caused by the oomycete Pseudoperonospora cubensis, is a devastating, worldwide-distributed disease of cucurbit crops in the open field and under cover. This review provides recent data on the taxonomy, biology, ecology, host range, geographic distribution and epidemiology of P. cubensis. Special attention is given to host-pathogen interactions between P. cubensis and its economically-important cucurbit hosts (Cucumis sativus, C. melo, Cucurbita pepo, C. maxima, and Citrullus lanatus); pathogenic variability in P. cubensis at the species, genus, and population levels; and, differentiation of pathotypes and races. Genetics and variability of host resistance and cellular and molecular aspects of such resistance are considered. A focus is given to methods of crop protection, including prevention and agrotechnical aspects, breeding for resistance—classical and transgenic approaches, chemical control and fungicide resistance. Novel technologies in biological and integrated control are also discussed. This review also summarizes the most important topics for future research and international collaboration.

Downy mildew of Cucurbits (Pseudoperonospora Cubensis): the Fungus and its hosts, distribution, epidemiology and control

Taxonomy of the genusPseudoperonospora, morphology ofPseudoperonospora cubensis (Berk, et Curt.) Rostow. and occurrence of its oospores, are described briefly. A list is presented of over 40 cucurbitaceous host species, representing about 20 genera, on whichP. cubensis has been recorded. Two or more races exist in Japan and the United States, but not in Europe or the Middle East. The distribution ofP. cubensis is widest on all continents on cucumbers (70 countries) and muskmelon (50 countries); onCucurbita and watermelons it extends to about 40 and 25 countries, respectively.P. cubensis may overwinter as oospores, though this seems rare, and on wild hosts or crops grown in the open or under cover. Airborne sporangia may also reach cooler countries from regions with mild winters.Apart from the leaf wetness essential for infection, the factors determining disease progress are: rate of foliage growth and physiological age of the host; amount of primary inoculum available, light, and the rate at which lesions necrotize. The interaction of these factors is described for early, mid-season, and late crops. Losses caused byP. cubensis depend on the growth stage at which the crop is attacked, and on the rate of foliage and pathogen development. Breeding has produced downy mildew resistant lines of cucumbers, used chiefly in the United States, and some resistant lines of melons and watermelons. The most important agricultural practices used to restrict downy mildew development are proper irrigation management and avoidance of sowing in proximity to infected crops.Success of control by protectant chemicals depends largely on proper timing of applications. Proximity of inoculum sources, hours of leaf wetness, age of crop, and irrigation practices are the principal factors that determine when to begin treatments. These factors and rate of leaf formation determine the frequency of applications. Application of systemic fungicides is much easier to time correctly.

Ultrastructure, autofluorescence, callose deposition and lignification in susceptible and resistant muskmelon leaves infected with the powdery mildew fungus Sphaerotheca fuliginea

Abstract Leaves of the susceptible cv. Ananas-Yokneam (AY) and of resistant cvs PI 124111F, PI 124112, PMR-45, PMR-6 of muskmelon ( Cucumis melo ) were inoculated with either race 1 or race 2 of Sphaerotheca fuliginea and examined microscopically after staining with Calcofluor, basic aniline blue or phloroglucinol. At 20–24 h post inoculation of the susceptible AY at 23 °C, the fungal spores (both race 1 or race 2) developed one or two germtubes which penetrated into one or two epidermal cells. The penetration zones were surrounded with callose-like material but no autofluorescence nor lignin-like materials were observed in the penetrated epidermal cells. A similar response was observed in PMR-45 inoculated with race 2 (compatible). In contrast, the fungus developed a single germtube on the resistant PI 124111F, PI 124112, and PMR-6 inoculated with either race, as well as in PMR-45 inoculated with race 1, which induced autofluorescence, callose accumulation and lignification in the penetrated epidermal cells. Electron microscopical studies revealed that the rapid collapse of epidermal cells in the resistant cultivars was accompanied by the accumulation of callose-like deposits in cell walls and around haustoria, electron-opaque deposits in the plasma membrane and electron-opaque deposits between the cell wall and the plasma membrane. Occasionally, callose also appeared in epidermal and mesophyll cells adjacent to the penetrated cells. By 96–120 h post inoculation, abundant sporulation was observed in the compatible interactions whereas only 1–3 germtubes with no sporulation were seen in the incompatible interactions. Heat shock or chemical inhibitors (cycloheximide, blasticidin-S, cordycepin, 2-deoxy-D-glucose, α-aminooxyacetic acid, 2,4-dinitrophenol and sodium azide) failed to induce susceptibility in the resistant cultivars. The results suggest a similar structural response to powdery mildew in C. melo cultivars carrying the resistance genes Pm-1 (PMR-45), Pm-2 (PMR-6), Pm-3 (PI 124111F), Pm-4 (PI 124112), Pm-5 (PI 124112) and Pm-6 (PI 124111F).

Induced Resistance to Cyst and Root-knot Nematodes in Cereals by DL-β-amino-n-butyric Acid

Foliar sprays and soil drenches with DL-β-amino-n-butyric acid (BABA) reduced the number of Heterodera avenae and H. latipons cysts on wheat and barley. Foliar sprays of wheat with 8000 mg l−1 BABA reduced the number of H. avenae cysts by 90%, whereas 2000 mg l−1 BABA was enough to reduce the number of H. latipons cysts by 79%. Multiple spray treatments with 2000 mg l−1 BABA at 10-day intervals reduced the number of H. avenae cysts on wheat and barley. A soil drench of wheat with 125 mg l−1 BABA reduced the number of H. latipons cysts by 93% and H. avenae cysts by 43%. Second-stage juveniles of these nematodes penetrated and formed syncytia in wheat roots soil-drenched with BABA. More adult males of H. avenae were produced in BABA (<250 mg 1−1)-treated wheat roots (~76%) than in untreated roots (27%). Soil drenches with higher concentrations of BABA inhibited development of adult males and females. Several chemical elicitors of induced resistance were tested for their ability to reduce the number of H. avenae cysts on wheat. Only BABA was found to be an effective resistance inducer. The number of egg masses of an unidentified Meloidogyne sp. root-knot nematode, which infects only monocots, was also reduced by 95% by a soil drench of wheat with 500 mg l−1 BABA. Development of this nematode inside the BABA-treated roots was also inhibited.