Biological control of avocado white root rot with combined applications of Trichoderma spp. and rhizobacteria

This study tested the effectiveness of single and combined applications of Trichoderma and rhizobacterial strains to control white root rot (WRR) caused by Rosellinia necatrix in avocado plants. Three Trichoderma, two T. atroviride and one T. virens monoconidal strains and four bacterial strains (Bacillus subtilis, Pseudomonas pseudoalcaligenes and two P. chlororaphis) were assayed to determine their compatibilities in vitro. In addition, the effects of the bacterial filtrates were evaluated against the Trichoderma strains and reciprocally; these filtrates were applied alone or in combination to determine their effectiveness against R. necatrix. Individual control agents or combinations of them were applied to avocado plants that were artificially inoculated with a virulent R. necatrix strain. Compatibility between the combined Trichoderma applications and the bacterial strains was observed and these combinations significantly improved the control of R. necatrix during the in vitro experiments. A relative protective effect of some Trichoderma and bacteria was observed on the control of avocado WRR when they were applied singly. The combinations of T. atroviride strains with bacterial strains P. chlororaphis and P. pseudoalcaligenes showed a better control of avocado WRR, whereas the rest of Trichoderma and bacteria combinations also reduced significantly the level of disease and induced a delay at the onset of disease with respect to avocado plants inoculated either with Trichoderma or bacteria.     

Contribution of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> toxins to avocado white root rot

By transversely cutting infected avocado plant stems and using PCR techniques on avocado leaves, two experiments were carried out to determine whether Rosellinia necatrix can invade avocado vascular tissues. We were unable to detect the pathogen in either stems or leaves in either experiment, so we concluded that R. necatrix does not invade the vascular system of the plant. Additionally, the toxins produced by the pathogen were also studied to determine whether such toxins could contribute to the wilting and death of avocado plants infected by R. necatrix, having an effect on avocado leaves, where they can hinder the photosynthetic process. First, we isolated and identified the toxins cytochalasin E and rosnecatrone from filtrates of six R. necatrix isolates. Second, we tried to detect cytochalasin E in sap and leaves from infected avocado plants, and it was not detected at the minimum level of 50 μg/kg in leaves or 25 μg/kg on sap. Finally, we observed changes in fluorescence emitted by the avocado leaf surface (to detect photosynthetic efficiency) after inoculating avocado plants with this toxin. Fluorescence was higher in the leaves of plants immersed in toxin solution after 4 and 8 days, but not after longer periods of time. In this work, we demonstrated that although R. necatrix is not a fungus that invades the vascular system, its toxins are probably involved in the wilting and death of infected avocado plants, decreasing the efficiency of photosynthesis.     

Intraspecific diversity within avocado field isolates of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from south-east Spain

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.     

Hot water treatment of Japanese pear trees is effective against white root rot caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> Prillieux

Hot water was dripped into the rhizosphere of Japanese pear trees (Pyrus serotina Rehd. grafted on P. betulifolia Bunge.) infested with the white root rot fungus Rosellinia necatrix Prillieux, to destroy the fungus. Isolates of R. necatrix from diseased roots of Japanese pear were vulnerable to water at temperatures above 35°C, and the fungus was eradicated from the colonized substrate when water at 35°C was provided for 3 days. The time required to eradicate R. necatrix decreased exponentially with increasing temperature. Japanese pear trees tolerated a temperature of 45°C without reduction in vigor. Field experiments demonstrated the practical use of hot water drip irrigation (HWD). HWD at 50°C completely destroyed white root rot mycelia on diseased roots, and many rootlets grew after the treatment. HWD at this temperature caused no injury to the trees. HWD of diseased orchard trees was assessed in Takamori and Iida in southern Nagano, Japan. The fungus recurred in two of four trees 28 months after treatment in Takamori and in two of ten trees 16 months after treatment in Iida. The new mycelia emerged on thick roots deep within the soil. Although there is a possibility of recurrence, HWD treatment is a practical control measure for white root rot.     

Effect of tannins and phenolic extracts from plant roots on the production of cellulase and polygalacturonase byDematophora necatrix

Dematophora necatrix, the causal agent of the white root rot disease in plants, produced large amounts of cellulase (Cx) and very small amounts of polygalacturonase (PG). Both tannins (100 mg/l) and phenols (200 mg/l) extracted from roots of plants showing resistance to the disease decreased Cx productionin vitro. PG production was affected only by tannin extracts. Exposure of the fungus for 2 days to the tannin (100 mg/l) but not to the phenol (200 mg/l) extracts decreased the subsequent rate of fungal growth in an agar medium free of these compounds.     

Assessment of the antifungal activity of selected biocontrol agents and their secondary metabolites against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Fusarium graminearum (teleomorph: Gibberella zeae) is the causal agent of several destructive diseases in cereal crops worldwide. In the present study we have evaluated the potential of two strains of Trichoderma sp. (T23, and T16), a strain of Paecilomyces sp. (PS1), and their secondary metabolites (SMs) in suppressing F. graminearum. Results from dual culture experiments show that in the presence of either Trichoderma sp., or Paecilomyces sp. mycelial growth of F. graminearum is considerably inhibited. Strain T23 causes the greatest inhibition (83.8%), followed by strain T16 (72.2%), and strain PS1 (61.9%). Likewise, mycelial growth of the pathogen is completely inhibited (≥ 98%) when grown under exposure to volatile metabolites excreted from Trichoderma cultures. Bioautographic analyses using culture filtrates revealed that several antifungal SMs are excreted. Among five metabolites tested, 6-pentyl-alpha-pyrone (6PAP) from strain T23, and PF3 from strain PS1 exhibit pronounced antifungal activity against F. graminearum. A new method for mass production of perithecia of F. graminearum which is simple and more effective than traditional methods was developed, which allows an increase in perithecial formation of more than 5-fold. Using this method, we found, that in the presence of SMs perithecial formation was negatively affected. Perithecial production was suppressed by 81.4% and 76.6% using 200 μg ml^?1 of either 6PAP or PF3, respectively. Moreover, ascospore discharge was significantly suppressed (67.0%) when perithecia were exposed to the metabolite F116 produced by T16. Including 6PAP or PF3 in conidial suspensions impeded germination of conidia completely. Similarly, both metabolites strongly inhibited ascospore germination (? 90%).     

A fungal elicitor enhances the resistance of tomato fruit to Fusarium oxysporum infection by activating the phenylpropanoid metabolic pathway

Fungi infection in fruits is an important factor in postharvest losses. The effect of a treatment with a fungal elicitor on the response of tomato fruit to Fusarium oxysporum infection and changes in the phenylpropanoid metabolic pathway was studied. Fungal elicitor retarded for 3 days the development of the Fusarium rot development in tomato, at concentration of 2 g?l ^?1 (B2-F treatment). This treatment also induced a 3.11- and 6.03-fold increase of caffeic and chlorogenic acids, respectively, as compared with the control. Furthermore, the flavonoids naringenin-7-O-glucoside, rutin and kaempferol-3-O-glucoside, showed a greater abundance in tomato under the B2-F treatment after 6 days at 20°C. It is concluded that the fungal elicitor reduced the development of Fusarium rot by inducing the biosynthesis of metabolites from the phenylpropanoid metabolic pathway which forms part of the defense response in tomato fruit.     

Proteomic analysis of mycelial proteins from <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

The soilborne pathogen Rosellinia necatrix causes white root rot, a serious disease of various trees, and is extremely difficult to control. In this study, using one-dimensional electrophoresis coupled with nanoliquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS), we identified 696 proteins from R. necatrix mycelium (KACC 40445) grown in liquid culture. In addition, 573 proteins were assigned to at least one gene ontology term including 26 functional groups. Most were related to catalytic activity in the molecular function category. This proteomic data set advances understanding of R. necatrix biology and will inform further investigations to manage white root rot using novel strategies.     

Species-specific PCRs differentiate <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from <Emphasis Type="Italic">R. compacta</Emphasis> as the prevalent cause of white root rot in Japan

Rosellinia compacta, described recently, resembles R. necatrix and also causes white root rot. Here a species-specific PCR was developed for R. compacta, and the two R. necatrix-specific primer sets already available were validated in terms of species specificity. PCRs using the primer sets for R. necatrix amplified specific products exclusively from R. necatrix isolates. The R. compacta-specific primer set exclusively detected R. compacta, which appears to be a rare but widely distributed species. We conclude that R. necatrix is the major cause of the disease in Japan but that the involvement of R. compacta should be studied further.     

Characterization of <Emphasis Type="Italic">Trichoderma</Emphasis> species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan

Native Trichoderma spp. were isolated from agricultural fields in several regions of Ecuador. These isolates were characterized via morphological observation as well as molecular phylogenetic analysis based on DNA sequences of the rDNA internal transcribed spacer region, elongation factor-1α gene and RNA polymerase subunit II gene. Fifteen native Trichoderma spp. were identified as T. harzianum, T. asperellum, T. virens and T. reesei. Some of these strains showed strong antagonistic activities against several important pathogens in Ecuador, such as Fusarium oxysporum f. sp. cubense (Panama disease) and Mycosphaerella fijiensis (black Sigatoka) on banana, as well as Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease) on cacao. The isolates also showed inhibitory effects on in vitro colony growth tests against Japanese isolates of Fusarium oxysporum f. sp. lycopersici, Alternaria alternata and Rosellinia necatrix. The native Trichoderma strains characterized here are potential biocontrol agents against important pathogens of banana and cacao in Ecuador.     

White root rot disease of the lacquer tree <Emphasis Type="Italic">Toxicodendron vernicifluum</Emphasis> caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

In early August 2010, lacquer trees (Toxicodendron vernicifluum) severely damaged by a root rot disease were found on plantations in Iwate, Japan. The causal agent was a fungus identified as Rosellinia necatrix, based on morphology and the sequence of the ribosomal DNA internal transcribed spacer region. The fungus was clearly pathogenic on T. vernicifluum root plantings. This report is the first of white root rot on T. vernicifluum.     

Microbial metabolism of dinitramine

The microbial metabolism of N³,N³-diethyl 2,4-dinitro-6-trifluoromethyl-m-phenylenediamine (dinitramine), N-sec-butyl-4-tert-butyl-2,6-dinitroaniline (A-820), N-n-propyl-N-cyclopropylmethyl-4-trifluoromethyl-2,6-dinitroaniline (CGA-10832), and α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin) by Aspergillus fumigatus Fres., Fusarium oxysporum Schlecht and Paecilomyces sp. was investigated. The dinitrodiamine and dinitroanilines were most readily metabolized by Paecilomyces sp. The metabolism of dinitramine was examined in detail, and four metabolites were isolated: N³-ethyl 2,4-dinitro-6-trifluoromethyl-m-phenylenediamine; 2,4-dinitro-6-tri-fluoromethyl-m-phenylenediamine; 6-amino-1-ethyl-2-methyl-7-nitro-5-trifluoromethylbenzimidazole and 6-amino-2-methyl-7-nitro-5-trifluoromethylbenzimidazole. The presence of a dinitramine-degrading enzyme system in A. fumigatus was demonstrated. The enzyme dealkylates dinitramine and requires NADPH and Fe²⁺ as cofactors.     

Development of real-time PCR assay using TaqMan probe for detection and quantification of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> in plant and soil

We developed real-time PCR assays using TaqMan probes to detect and quantify Rosellinia necatrix, the causal agent of white root rot in many plant species. Two sets of PCR primers and TaqMan probe indicated that their detection limits could be as low as 1 fg of template DNA. Using the real-time PCR assays with the TaqMan probes, we were able to quantify R. necatrix DNA in naturally diseased roots of Japanese pear and in artificially infested soil samples. Although the new assays were inadequate for use with naturally infested soil samples, nested PCR procedures improved the detectability of the new assays.     

Differential response to combined prochloraz and thyme oil drench treatment in avocados against the control of anthracnose and stem-end rot

The ability to meet consumers demand for high-quality standard fruit entails the distribution of unblemished safe fruit free of chemical residues on its edible portion. Therefore, this study was focused on investigating the influence of the combined effect of aqueous plant volatiles with half strength prochloraz solution to control anthracnose and stem-end rot in the green-skinned avocado cultivar (Fuerte). This method was applied due to its practicability on bulk fruits in packhouses and the fruits were subjected to stand-alone and combined treatments to assess the development of the disease after cold storage and observe the elicitation of the residual effect of the treatments on the defence related enzymes in ‘Fuerte’. The incidence of stem-end rot was 10% by the combination of prochloraz® (500 μg mL^?1; P50) with 0.1% v/v thyme oil compared to the 58.8% incidence exhibited by the untreated fruit during storage at 6.5 °C for 14 days followed by 3 days at retail shelf conditions (15 °C) (preventative application). Citral (0.1% v/v), P50 (500 μg mL^?1)?+?0.1% v/v citral and yucca extract alone reduced the stem-end rot incidence to about 25% during storage. More so, thyme oil (0.1% v/v) reduced both anthracnose and stem-end rot incidence to 35% after postharvest storage and P50 (500 μg mL^?1)?+?0.1% v/v thyme oil and 0.1% v/v thyme oil effectively induced the activity of phenylalanine ammonia lyase, chitinase and β-1, 3 glucanase in fruit inoculated with Lasiodiplodia theobromae and Colletotrichum gloeosporioides through the improvement of quality and firmness of the fruit after storage.     

Antagonistic activity of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. against <Emphasis Type="Italic">Scytalidium lignicola</Emphasis> CMM 1098 and antioxidant enzymatic activity in cassava

Trichoderma spp. are used as antagonists against different pathogens. Despite many possibilities of using Trichoderma as an antagonist, there are gaps in the knowledge of the interaction between Trichoderma, cassava and Scytalidium lignicola. This fungus causes cassava black root rot and is an inhabitant of the soil, so it is difficult to control. Antagonists may contribute to the possible induction of resistance of plants because, when exposed to such pathosystems, plants respond by producing antioxidative enzymes. The test for potential inhibition of growth of S. lignicola CMM 1098 in vitro was performed in potato-dextrose-agar with two Trichoderma strains T. harzianum URM3086 and T. aureoviride URM 5158. We evaluated the effect of the two selected Trichoderma to reduce the severity of cassava black root rot and shoots. Subsequently, the production of enzymes (ascorbate peroxidase, catalase, peroxidase and polyphenol oxidase) was evaluated in cassava plants. All two Trichoderma strains show an inhibition of the growth of S. lignicola CMM 1098. The most efficient was T. harzianum URM 3086, with 80.78% of mycelial growth inhibition. T. aureoviride URM 5158 was considered the best chitinase producer. All treatments were effective in reducing severity, especially treatments using Trichoderma. Cassava plants treated with T. aureoviride URM 5158 had the highest enzyme activity, especially peroxidase and ascorbate peroxidase. Trichoderma harzianum URM3086 and Trichoderma aureoviride URM 5158 were effective in reducing the severity of cassava black root rot caused by S. lignicola CMM 1098.     

Diversity of soil-dwelling Trichoderma in Colombia and their potential as biocontrol agents against the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary

Twenty-one isolates of Trichoderma spp. were collected from eight states in Colombia and characterized based on the 5′ end of the translation elongation factor-1α (EF1-α1) gene and RNA polymerase II gene encoding the second largest protein subunit (RPB2) by using mixed primers. Seven species of soil-dwelling Trichoderma were found: T. atroviride, T. koningiopsis, T. asperellum, T. spirale, T. harzianum, T. brevicompactum and T. longibrachiatum. Species identifications based on the EF1-α1 gene were consistent with those obtained from the RPB2 gene. Phylogenetic analyses with high bootstrap values supported the validity of the identification of all isolates. These results suggest that using the combination of the genes EF1-α1 and RPB2 is highly reliable for molecular characterization of Trichoderma species. Trichoderma asperellum Th034, T. atroviride Th002 and T. harzianum Th203 prevented germination of more than 70 % of sclerotia of Sclerotinia sclerotiorum in bioassay tests and are promising biological control agents. No relationship between mycelium growth rate and parasitism level was found.     

Bait twig method for soil detection of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>, causal agent of white root rot of Japanese pear and apple,at an early stage of tree infection

Mulberry twigs were inserted into the soil as bait to detect Rosellinia necatrix at an early stage of tree infection in the orchard. R. necatrix was frequently trapped on twigs near the trunk base at soil depths of 6–20 cm within 10–20 days in May–July, suggesting that the incubation period was dependent on soil temperature. Subsequently, we inserted twig in the soil around healthy-looking trees in naturally infested orchards. R. necatrix was trapped from 80.0% of Japanese pear and 75.0% of apple trees that later proved to be infected. This bait twig method facilitated quicker diagnosis of white root rot on Japanese pear and apple at early stages of infection and can be used to detect recurrence of the fungus after fungicide treatment.     

Isolation,production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases

Antibiosis has been shown to be an important mode of action by Trichoderma species used in the protection of grapevine pruning wounds from infection by trunk pathogens. The major active compound from Trichoderma isolates known to protect grapevine pruning wounds from trunk pathogen infection was isolated and identified. The compound, a 6‐pentyl‐α‐pyrone (6PP), was found to be the major secondary metabolite, by quantity, which accumulated in the culture filtrate of T. harzianum isolate T77 and the two T. atroviride isolates UST1 and UST2. Benzimidazole resistant mutants generated from these isolates also produced 6PP as their main secondary metabolite, except for a mutant of T77 that had lost its ability to produce 6PP. The isolates UST1 and UST2 were co‐cultured with the grapevine trunk pathogens Eutypa lata and Neofusicoccum parvum in a minimal defined medium and a grapevine cane‐based medium (GCBM). Co‐culturing UST1 with N. parvum induced 6PP production in the minimal defined medium and the GCBM. The production of 6PP by UST2 was induced in the GCBM, while co‐culturing with the two trunk pathogens either reduced or had no effect on 6PP production. Mycelial growth and ascospore/conidia germination of E. lata, N. australe, N. parvum and Phaeomoniella chlamydospora were inhibited by 6PP in a concentration‐dependent manner. The results show that the presence of N. parvum and grapevine wood elicits the production of 6PP, suggesting that this metabolite is involved in Trichoderma–pathogen interactions on grapevine pruning wounds.     

Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates

Metribuzin [4-amino-6-tert-butyl-3(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in soybean [Glycine max (L.) Merr. Tracy]. Pulse treatment studies with seedlings and excised mature leaves showed that [5-¹⁴C]metribuzin was absorbed rapidly and translocated acropetally. In seedlings, >97% of the root-absorbed ¹⁴C was present in foliar tissues after 24 hr. In excised leaves, 50–60% of the absorbed ¹⁴C remained as metribuzin 48 hr after pulse treatment, 12–20% was present as polar metabolites, and 20–30% was present as an insoluble residue. Metabolites were isolated by solvent partitioning, and were purified by adsorption, ion-exchange, thin-layer, and high-performance liquid chromatography. The major metabolite (I) was identified as a homoglutathione conjugate, 4-amino-6-tert-butyl-3-S-(γ-glutamyl-cysteinyl-β-alanine)-1,2,4-triazin-5(4H)-one. Metabolite identification was confirmed by qualitative analysis of amino acid hydrolysis products, fast atom bombardment (FAB), and chemical ionization (CI) mass spectrometry, and by comparison with a reference glutathione conjugate synthesized in vitro with a hepatic microsomal oxidase system from rat. Minor metabolites were identified as an intermediate N-glucoside conjugate (II), a malonyl N-glucoside conjugate (III), and 4-malonylamido-6-tert-butyl-1,2,4-triazin-3,5(2H,4H)-dione (N-malonyl DK, IV) by CI and FAB mass spectrometry. Alternative pathways of metribuzin metabolism are proposed.     

Heat shock-induced resistance in strawberry against crown rot fungus Colletotrichum gloeosporioides

This present study investigated the effects of heat shock treatments in strawberry seedlings against crown rot caused by Colletotrichum gloeosporioides. Heat shock treatment at 50 °C for 20 s reduced the disease index of strawberry crown rot and increased chitinase 2-1 gene expression as well as free salycilic acid accumulation. Heat shock treatment did not reduce mycelial growth of C. gloeosporioides. BIT(2-benzisothiazol-3(2H)-one,1,1-dioxide) treatment could not protect strawberry seedlings against crown rot disease. This finding suggests that heat shock induces strawberry resistance against Colletotrichum crown rot and assumes that mechanisms other than SAR probably mediated the protective effect of heat shock-induced resistance.