Hormonomic Changes Driving the Negative Impact of Broomrape on Plant Host Interactions with Arbuscular Mycorrhizal Fungi.

Belowground interactions of plants with other organisms in the rhizosphere rely on extensive small-molecule communication. Chemical signals released from host plant roots ensure the development of beneficial arbuscular mycorrhizal (AM) fungi which in turn modulate host plant growth and stress tolerance. However, parasitic plants have adopted the capacity to sense the same signaling molecules and to trigger their own seed germination in the immediate vicinity of host roots. The contribution of AM fungi and parasitic plants to the regulation of phytohormone levels in host plant roots and root exudates remains largely obscure. Here, we studied the hormonome in the model system comprising tobacco as a host plant, Phelipanche spp. as a holoparasitic plant, and the AM fungus Rhizophagus irregularis. Co-cultivation of tobacco with broomrape and AM fungi alone or in combination led to characteristic changes in the levels of endogenous and exuded abscisic acid, indole-3-acetic acid, cytokinins, salicylic acid, and orobanchol-type strigolactones. The hormonal content in exudates of broomrape-infested mycorrhizal roots resembled that in exudates of infested non-mycorrhizal roots and differed from that observed in exudates of non-infested mycorrhizal roots. Moreover, we observed a significant reduction in AM colonization of infested tobacco plants, pointing to a dominant role of the holoparasite within the tripartite system.

Evidence that AGL17 is a significant downstream target of CLF in floral transition control.

Polycomb-group (PcG) proteins are evolutionarily conserved in higher organisms and play essential roles in many developmental processes by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3), a key repressive histone mark. In Arabidopsis (Arabidopsis thaliana), histone methyltransferase CURLY LEAF (CLF) is one of the major PcG catalytic components, playing critical roles in plant growth and development, especially the floral transition. We have recently profiled the genome-wide occupancy of CLF by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Interestingly, AGAMOUS-LIKE 17 (AGL17) that encodes a known flowering activator was found to be a CLF direct target. Based on this observation, we set out to examine to what extent this genetic module regulates the flowering. First, we validated the ChIP-seq results by ChIP-qPCR and show that CLF indeed targets AGL17, and the level of H3K27me3 is decreased when CLF is lost. Further, we show that the expression of AGL17 is significantly up-regulated in the clf-29 mutant compared to wild-type (WT). Finally, our clf agl17 double mutant analysis suggests that AGL17 is a significant downstream target of CLF in floral transition control.

Aspergillus alliaceus infection fatally shifts Orobanche hormones and phenolic metabolism.

In this study, the physio pathological effects of Aspergillus alliaceus (Aa, fungi, biocontrol agent) on Orobanche (parasitic plant) were investigated by hormone and phenolic substance tests. In experimental group, Orobanches were treated with the fungi, considering control group was fungus-free. Based on the hormonal tests, in the experimental group, salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and gibberellic acid (GA) levels significantly decreased, and only indole acetic acid (IAA) hormone levels were fairly higher than the control group. According to phenolic substance tests, it was found that only gallic acid, syringic acid and caffeic acid values significantly increased compared with control, and catechin and p-coumaric acid values were significantly lower. Consequently, it was determined that Aa pathogenesis (1) considerably reduces the effects of all defence hormones (JA, ABA, SA), (2) operates an inadequate defence based solely on the IAA hormone and several phenolic substances (gallic acid, syringic acid and caffeic acid), (3) and inevitably the fungi lead the Orobanche to a slow and continuous death. The results were evaluated in detail in the light of similar recent article and current literature in terms of biocontrol and pathology.

Fusarium infection causes genotoxic disorders and antioxidant-based damages in Orobanche spp.

This study aims to evaluate the toxic effects of Fusarium oxysporum on root parasitic weed, Orobanche spp. Comparative genetic and gene expression studies were conducted on uninfected and fungus-infected orobanches. In genetic studies, isolated total DNA was amplified by RAPD PCR. Fragment properties were analysed by GTS test. According to the results, the fragment properties of control and Fusarium infected (experimental) groups varied widely; and it has been observed that Fusarium has genotoxic effects on the DNA of orobanches. In gene expression studies, the expression levels of genes encoding enzymes or proteins were associated with ROS damage and toxic effects, therefore, gene expressions of Mn-superoxide dismutase (SOD), Zn-superoxide dismutase (=SOD2, mitochondrial), glutamine synthetase (GS), heat shock protein gene (HSP70), BAX, Caspase-3 and BCL2 were significantly higher in the experimental group. In the light of obtained data, it was concluded that F. oxysporum (1) caused heavy ROS damage in Orobanche (2) induced significant irrevocable genotoxic effects on the DNA of Orobanche, (3) degraded protein metabolism and synthesis, and finally (4) triggered apoptosis. The results of this study can be a ground for further research on reducing the toxic effects of Fusarium on agricultural products, so that advancements in bio-herbicide technology may provide a sustainable agricultural production.

Aspergillus alliaceus, a new potential biological control of the root parasitic weed Orobanche.

During extensive surveys in fields heavily infested by broomrape in the Trakya Region-Turkey, a different new fungus, Aspergillus alliaceus, was isolated from the infected broomrape. It is aimed to investigate whether or not it is really a pathogen for Orobanche. The fungi was exposed to a greenhouse environment in order to assess its pathogenicity and virulence against Orobanche cernua. In addition, infection tests on Orobanche seeds were also performed under laboratory conditions. The fungus was subjected using two different methods, exposure to a liquid culture with conidial solution and a sclerotial solid culture with fungal mycelia. Cytological studies were carried out at light, TEM and SEM levels. The results show that the sclerotial solid culture with fungal mycelia quickly caused necrosis and was more effective than the other type. It also greatly diminished attachments, tubercles, and caused the emergence of shoots and an increase in the total shoot number of Orobanche. In addition, both when the fungi was exposed to both soil and used to contaminate sunflower seeds, its pathogenicity was more effective. Consequently, it was determined that A. alliaceus was an effective potential biological control of broomrape throughout its life cycle from dormant seed to mature plant.

Reduced germination of Orobanche cumana seeds in the presence of Arbuscular Mycorrhizal fungi or their exudates.

Broomrapes (Orobanche and Phelipanche spp) are parasitic plants responsible for important crop losses, and efficient procedures to control these pests are scarce. Biological control is one of the possible strategies to tackle these pests. Arbuscular Mycorrhizal (AM) fungi are widespread soil microorganisms that live symbiotically with the roots of most plant species, and they have already been tested on sorghum for their ability to reduce infestation by witchweeds, another kind of parasitic plants. In this work AM fungi were evaluated as potential biocontrol agents against Orobanche cumana, a broomrape species that specifically attacks sunflower. When inoculated simultaneously with O. cumana seeds, AM fungi could offer a moderate level of protection against the broomrape. Interestingly, this protection did not only rely on a reduced production of parasitic seed germination stimulants, as was proposed in previous studies. Rather, mycorrhizal root exudates had a negative impact on the germination of O. cumana induced by germination stimulants. A similar effect could be obtained with AM spore exudates, establishing the fungal origin of at least part of the active compounds. Together, our results demonstrate that AM fungi themselves can lead to a reduced rate of parasitic seed germination, in addition to possible effects mediated by the mycorrhizal plant. Combined with the other benefits of AM symbiosis, these effects make AM fungi an attractive option for biological control of O. cumana.

Susceptibility of Phelipanche and Orobanche species to AAL-toxin.

Fusarium and Alternaria spp. are phytopathogenic fungi which are known to be virulent on broomrapes and to produce sphinganine-analog mycotoxins (SAMs). AAL-toxin is a SAM produced by Alternaria alternata which causes the inhibition of sphinganine N-acyltransferase, a key enzyme in sphingolipid biosynthesis, leading to accumulation of sphingoid bases. These long chain bases (LCBs) are determinant in the occurrence of programmed cell death (PCD) in susceptible plants. We showed that broomrapes are sensitive to AAL-toxin, which is not common plant behavior, and that AAL-toxin triggers cell death at the apex of the radicle as well as LCB accumulation and DNA laddering. We also demonstrated that three Lag1 homologs, encoding components of sphinganine N-acyltransferase in yeast, are present in the Orobanche cumana genome and two of them are mutated leading to an enhanced susceptibility to AAL-toxin. We therefore propose a model for the molecular mechanism governing broomrape susceptibility to the fungus Alternaria alternata.

Transforming a NEP1 toxin gene into two Fusarium spp. to enhance mycoherbicide activity on Orobanche--failure and success.

BACKGROUND: The NEP1 gene encoding a fungal toxin that successfully conferred hypervirulence when transformed into Colletotrichum coccodes (Wallr.) Hughes attacking Abutilon theophrasti (L.) Medic. was tested to ascertain if it would enhance pathogenicity of Fusarium species to Orobanche aegyptiaca Pers. parasitising crops. RESULTS: None of the Fusarium oxysporum (#CNCM I-1622) NEP1 transformants was hypervirulent. NEP1 transformants of a new but unnamed Fusarium sp. (#CNCM I-1621--previously identified as F. arthrosporioides) killed Orobanche more rapidly than the wild type. Transformed lines of both species were NEP1 PCR positive, as was the wild type of F. oxysporum #CNCM I-1622 and five other formae speciales of F. oxysporum. All six wild-type formae speciales of F. oxysporum tested excrete minute amounts of immunologically and bioassay-detectable NEP1-like protein. NEP1 expression of most F. oxysporum transformants was suppressed, suggesting that the native gene and the transgene silence each other. The sequence of the putative NEP1 gene in Fusarium oxysporum #CNCM I-1622 differs from the sequence in the toxin-overproducing strain of F. oxysporum f. sp. erythroxyli in four or five amino acids in the first exon. CONCLUSION: Wild-type Fusarium sp. #CNCM I-1621 does not contain a NEP1-like gene, explaining why it seemed amenable to transformation with high expression, and its virulence was probably enhanced by not cosuppressing the endogenous gene as occurred with Fusarium oxysporum #CNCM I-1622.

Methods for selecting hypervirulent biocontrol agents of weeds: why and how.

A considerable number of plant pathogens have been studied for their possible use in weed control. Some have proven virulent enough to control weed species and to compete commercially with chemical herbicides. However, most pathogens of weeds are not useful in their wild form because they are not sufficiently host-specific and/or virulent. The authors believe that these barriers can be overcome. The present research has focused on the inhibitory effects of certain amino acids on the growth and development of specific plants. Pathogens that overproduce these selected amino acids can be easily selected from a pool of spontaneous mutants. Such mutants can have increased pathogenicity to their target weed and enhanced field performance as biocontrol agents. Enhancement of biocontrol efficacy in three separate pathogen-host systems, two with Fusarium and one with Pseudomonas, has already been reported. It is proposed to use the same technology to enhance the biocontrol efficacy of the two species of Fusarium that are host-specific pathogens of the broomrape group of parasitic weeds. The stepwise approach outlined can lead to obtaining enhanced biocontrol agents capable of producing inhibitory levels of selected amino acids in situ. It is proposed that these approaches, in combination with other methods of virulence enhancement, will lead to sustainable systems of biological control of parasitic weeds.

Granular formulation of Fusarium oxysporum for biological control of faba bean and tomato Orobanche.

BACKGROUND: Orobanche spp. represent a serious threat to a wide range of crops. They are difficult targets for herbicides, and biological control could provide a possible solution. This work therefore aimed to formulate mycoherbicides of Fusarium with adequate shelf life and virulence against Orobanche but safe to faba bean and tomato. RESULTS: Only two isolates of Fusarium oxysporum Schlecht. (Foxy I and Foxy II) obtained from diseased Orobanche shoots were found to be pathogenic to Orobanche crenata Forsk. and Orobanche ramosa L. Conidial suspension of both isolates significantly decreased germination, attachments and tubercles of Orobanche. Microconidia and chlamydospores of both isolates were formulated as mycoherbicides encapsulated in a wheat flour-kaolin matrix (four different formulations). All formulations greatly diminished Orobanche emerged shoots, total shoot number, shoot height, attachment of emerged shoots, the germinated seeds that succeeded in emerging above the soil surface and dry weight. Meanwhile, disease incidence and disease severity of emerged shoots were enhanced. The shelf life was adequate, particularly for coarse, freshly prepared, low-temperature-stored, microconidia-rich formulations. The induced growth reduction of Orobanche-infected host plants seemed to be nullified by formulations, particularly at the highest dose. CONCLUSION: These formulations seemed to destroy Orobanche but appeared harmless to host plants. Hence, they could be efficiently used as mycoherbicides for biological control of Orobanche in faba bean and tomato.

Biological control of Orobanche aegyptiaca by Fusarium oxysporum F. sp. Orobanchein northwest Iran.

Broomrape (Orobanche aegyptiaca) is one of the most serious weed pathogen on many plants all over the world, especially in northwest Iran. It causes damage on some plants particularly on tomato, cucumber and other dicotyledonous crops in zanjan province. Broomrape as weed, caused reductions in crop yield, adversely affected crop quality, and resulted in loss of cultivated land due to reduced crop alternatives. Since there were no any chemical methods and other proper technique to control this plant parasite we tried to find a good mechanism for its management in the fields. Our study showed there was a specific species, Fusarium oxysporum f. sp. orobonche that had infected Broomrapes naturally in the field. In point of fact the species was isolated from naturally infected Broomrape in studied Locations. Our surveys showed F. oxysporum f. sp. orobanche caused disease on orobanche spp. in different agricultural fields. Although there were other fungal species which can nearly manage the orobanche but it can be fungal pathogen to other plants. However the best fungal isolate can be F. oxysporum f. sp. orobanche since it may not be pathogen for other plants.

The effects of Fusarium oxysporum on broomrape (Orobanche egyptiaca) seed germination.

Broomrape (Orobanche aegyptiaca L.), one of the most important parasitic weeds in Iran, is a root parasitic plant that can attack several crops such as tobacco, sunflower, tomato and etc. Several methods were used for Orobanche control, however these methods are inefficient and very costly. Biological control is an additional recent tool for the control of parasitic weeds. In order to study of the fungus Fusarium oxysporum (biocontrol agent) effects on broomrape seed germination, two laboratory studies were conducted in Tehran University. In the first experiment, different concentration of GR60 (0, 1, 2 and 5 ppm) as stimulation factor for Orobanche seeds germination were experimented. Results showed that concentrations of GR60 had a significant effect on seed germination. The highest seed germination percent was obtained in 1 ppm. In the second experiment, the effect of Fusarium oxysporum was tested on O. aegyptiaca seeds germination. The fungus Fusarium oxysporum were isolated from infested and juvenile O. aegyptiaca ower stalks in tomato field in karaj. Fungus spores suspension in different concentrations (0 (Control), 10(5) (T1), 10(6) (T2), 10(7) (T3) and 3 x 10(7) (T4)) from potato dextrose agar (PDA) prepared and together with 1ppm of GR60 concentration were tested on O. aegyptiaca seeds. Results show that the highest inhibition of seed germination obtained in 10(5) spores/ml. With increasing of suspension concentrations, inhibition percent was reduced and mortality of seeds germ tube was increased. In this investigation, Fusarium oxysporum can be used to inhibit seed germination, stimulate the "suicidal germination" of seeds and reduce the Orobanche seed bank.

Metabolites inhibiting germination of Orobanche ramosa seeds produced by Myrothecium verrucaria and Fusarium compactum.

Myrothecium verrucaria and Fusarium compactum were isolated from diseased Orobanche ramosa plants collected in southern Italy to find potential biocontrol agents of this parasitic weed. Both fungi grown in liquid culture produced metabolites that inhibited the germination of O. ramosa seeds at 1-10 muM. Eight metabolites were isolated from M. verrucaria culture extracts. The main metabolite was identified as verrucarin E, a disubstituted pyrrole not belonging to the trichothecene group. Seven compounds were identified by spectroscopic methods as macrocyclic trichothecenes, namely, verrucarins A, B, M, and L acetate, roridin A, isotrichoverrin B, and trichoverrol B. The main metabolite produced by F. compactum was neosoloaniol monoacetate, a trichothecene. All the trichothecenes proved to be potent inhibitors of O. ramosa seed germination and possess strong zootoxic activity when assayed on Artemia salina brine shrimps. Verrucarin E is inactive on both seed germination and zootoxic assay.

Infection of tubercles of the parasitic weed Orobanche aegyptiaca by mycoherbicidal Fusarium species.

Progression of the infection by host-specific strains of Fusarium oxysporum and Fusarium arthrosporioides of Orobanche aegyptiaca (Egyptian broomrape) tubercles attached to tomato roots was tracked using light, confocal and electron microscopy. Mycelia transformed with the gene for green fluorescent protein were viewed using a confocal microscope. Fungal penetration was preceded by a rapid loss of starch, with approx. 10 % remaining at 9 h and no measurable starch at 24 h. Penetration into the Orobanche tubercles began by 12 h after inoculation. Hyphae penetrated the outer six cell layers by 24 h, reaching the centre of the tubercles by 48 h and infecting nearly all cells by 72 h. Most of the infected tubercles were dead by 96 h. Breakdown of cell walls and the disintegration of cytoplasm in and around the infected cells occurred between 48 and 96 h. Lignin-like material increased in tubercle cells of infected tissues over time, but did not appear to be effective in limiting fungal penetration or spread. Callose, suberin, constitutive toxins and phytoalexins were not detected in infected tubercles, suggesting that there are no obvious defence mechanisms to overcome. Both Fusarium spp. pathogenic on Orobanche produced fumonisin-like ceramide synthase inhibitors, while fusaric acid was produced only by F. oxysporum in liquid culture. The organisms do not have sufficient virulence for field use (based on glasshouse testing), suggesting that virulence should be transgenically enhanced or additional isolates sought.