The experimental work described in this master thesis was carried out at the School of Biological and Conservation Sciences, University of KwaZulu-Natal, Durban, from May 2009 to September 2010, under the supervision of Professor Patricia Berjak and Professor Norman Pamenter. Where the work of others has been used, this is duly acknowledged in the text.
PLAGIARISM
To study the effects of the culture filtrates on the proliferation of contaminants from the embryonic axes of T. These issues are especially relevant when the goal is cryopreservation of the embryonic axes of recalcitrant-seeded species (Berjak et al., 1999a,b).
INTRODUCTION
Significance of seed infection and its control
The health and vigor of seedlings and their subsequent growth depend on the quality of the seeds (Vozzo, 2002), making it important to control seed-related contaminants to maintain the quality and abundance of food, feed and fiber provided by growers. are produced all over the world to sustain. world (Chet et al., 1997).
Origin of seed infection
As examples, embryonic axes of barley seeds infected with Ustilago segetum were found to produce a seedling population with symptoms of loose smut (Rennie and Cockerell, 2006) and systemic transmission of Aspergillus flavus var. These examples illustrate why it is important to prevent infections at an early stage with treatments that do not adversely affect the embryos/embryonic axes of the seeds.
Seed categorization, seed storage and problems with stored seeds
It is only during long-term storage that fungal proliferation becomes apparent (Mycock and Berjak, 1990; Calistru et al., 2000) adversely affecting the vigor and viability of stored rebel seeds. Once the seeds are obtained, they should be surface decontaminated and stored in pre-sterilized containers (Sutherland et al., 2002).
Methods of storing recalcitrant germplasm
Although defense mechanisms have been shown to be present in resistant seeds (Calistru, 2004), they are not able to oppose the spread of the fungus during storage. While fungal propagation may initially be superficial, the embryonic axis, which may constitute only a small fraction of the seed mass and volume, is ultimately adversely affected (Calistru et al., 2000).
Fungal contamination of recalcitrant seeds
Control of seed-associated micro-organisms
- Chemical control and its hazards
- Fungicides
- Categories of fungicides
- Disinfection based on electrochemistry
- Preservatives used as fungicides
- Sodium dichloro-isocyanurate (Medi-Chlor ® / NaDCC)
- Benlate
- Biological control of plant diseases
- Trichoderma as a biocontrol agent
- Morphology and distribution
- Biocontrol characteristics of Trichoderma
- Mechanisms of biological control
- Trichoderma harzianum relative to other Trichoderma spp. as a biocontrol
- Commercial products of Trichoderma harzianum
- Strains of Trichoderma harzianum
- Cultivation of Trichoderma harzianum for the production of metabolites
Electrolyzed oxidation water is reported to be a powerful decontaminant used in the agricultural and food industries (Huang et al., 2008). This is initially achieved by recognition and growth towards the contaminant (Chet et al., 1981).
Encapsulated (minimal-growth) storage of axes of Trichilia dregeana
- Alginate gel encapsulation
Among the plant materials studied, shoot tips are the best samples for artificial seed production because of the mitotic potential of the meristems (Ballester et al., 1997). In addition to the nutrients, antibiotics (Bekheet, 2006), fungicides, pesticides (Bapat and Rao, 1990) and biocontrol agents (Russo et al., 2001; Haverson and Kimbrough, 2002) can be used as supplements to avoid bacterial or fungal contamination of the artificial seeds.
Trichilia dregeana
Trichilia dregeana seeds were originally categorized as resistant because of their requirement for high relative humidity (RH) to survive after germination and their high sensitivity to desiccation (Han et al., 1997; Kioko et al., 1998; Drew et al., 2000). It is therefore very important to eliminate infections at an early stage to extend the storage life of seeds of T.
Aim and objectives of the current study
- Objectives of the current research
Therefore, 1% v/v NaOCl was chosen as the best treatment for the surface disinfection of the embryonic axes. The effect of culture filtrate on germination of the embryonic axes alone was studied by disinfecting them before plating.
MATERIALS AND METHODS
Seed collection and handling
The seeds (Fig. 2.1B) were extracted from the capsules and the waxy aril and seed coat (now attached) were removed with a scalpel blade. The rest of the seeds were decontaminated with 1% (v/v) sodium hypochlorite (NaOCl) for 20 min and rinsed three times with distilled water to remove all traces of the chemical, then air-dried overnight on a paper towel.
Isolation and identification of fungal strains
Fungal colonies were suspended in sterile distilled water and a portion of the suspension was spread on a PDA plate. A little of the mycelium was removed using an inoculation loop and placed on the blot spot.
Standardisation of surface decontamination using sodium hypochlorite, mercuric
To obtain and maintain pure cultures of fungi isolated from embryonic axes, a small part of the medium with an established fungal colony, or a part of the infected embryonic axis, is removed using a sterile scalpel blade. Each treatment was followed by rinsing the axes three times with sterile distilled water to remove all traces of the sterilizing agent and blotting them on sterile filter paper to remove surface moisture. The efficiency of the decontamination protocol was checked by transferring samples of treated axes to the germination medium (Section 2.4).
Preparation of the growth medium
Effect of other decontamination protocols for the embryonic axes of T. dregeana
- Disinfection using electrolysed oxidising (EO) water
- Disinfection using Toxicity method
- Preparation and application of Nipastat
- Preparation and application of sodium dichloro-isocyanurate (Medi-
- Preparation and application of Benlate
The potential toxicity (antifungal activity) of various chemical preparations and biological filtrates tested for their potential to inhibit the proliferation of test isolates was evaluated in vitro after their inclusion in a PDA-based medium: the tested preparations included Nipastat® (a mixture of methyl-, butyl -, ethyl-, propyl- and isobutyl-paraben [Clariant Chemicals Ltd, India]) or Benlate 500 WP (Villa Crop Protection, South Africa; active ingredient benzimidazole [500 g kg-1]), as well as culture filtrates of Trichoderma strains harzianum from EcoT® and Eco77® (Powder formulations of Trichoderma spp. [2x109cfug-1] were obtained from Plant Health Products, KwaZulu-Natal, South Africa, and stored in sealed polyethylene bags at 4ºC). The plates were incubated at 24 ± 1 °C for 7 days, and then the area of two radial measurements. Twenty embryonic wasps were plated on this MS medium with half strength of Benlate to test the effects on contaminating fungi and bacteria.
Biocontrol of contaminants (isolated from embryonic axes of T. dregeana) by
- Cultural characteristics and growth rate of two isolates of T. harzianum (EcoT
- The effect of directly diffusible metabolites produced by T. harzianum cultures
- The effect of directly diffusible metabolites produced by T. harzianum cultures
- The effect of volatile metabolites of T. harzianum against test isolates
- The effect of non-volatile metabolites of T. harzianum against test isolates
- Extraction of non-volatile substances using sugarcane bagasse (SCB) and
- Extraction and assessment of anti-fungal effects of non-volatile
- The effect of non-volatile metabolites of T. harzianumon the embryonic axes of
- Inoculation of embryonic axes of T. dregeana with conidia of Trichoderma
- Inoculation
- Co-cultivation step
- Light microscopy
- Inhibition of external proliferation of Trichoderma
- Growth and biomass studies
- Assessment of length and dry weight of roots and seedlings
- Inoculation of the Trichoderma-treated embryonic axes of T. dregeana with
- Preparation of spore suspension of Penicillium sp. and inoculation
For controls, test isolates were inserted 10 mm from the edge of the plate but without T. To assess the effect of culture filtrate alone on germination, both control and test embryo axes were decontaminated with 1% NaOCl. for 10 minutes, rinsed three times with sterile distilled water and dried. A sample of five embryonic axes from the co-culture step of the standardized test (third) was used for microscopic studies.
Storage of the embryonic axes of T. dregeana using alginate gel encapsulation
- Alginate gel encapsulation, storage and assessment
- The effect of the use of different storage containment on alginate-encapsulated
At the end of the storage period (42 days), five embryonic axes from both encapsulated and non-encapsulated axes after NaDCC treatment were sampled to determine the water content after storage in the different containers.
Statistical analysis
On the basis of the reduction of contamination and their effect on the viability of the embryo axes This may be because the penetration of Trichoderma had negatively affected the growth potential (energy) of the embryo axes Strategies for field collection of resistant seeds and zygotic embryo axes of the tropical tree, Trichilia.
RESULTS
Isolation of microflora from T. dregeana
When fungi were isolated from the surfaces of embryonic axes excised from newly harvested seeds of T. Bacterial contamination was also evident on the surface of embryonic axes, but neither bacteria nor fungi were isolated from internal tissues from excised embryonic axes from newly harvested seeds. However, embryonic wasps excised from seeds stored for more than three months were internally infected with Penicillium spp., which emerged as the dominant surface contaminant of fresh seed wasps.
Effect of surface decontaminants on T. dregeana
Proliferation of the test isolates was increasingly inhibited with increasing concentrations of Nipastat in the medium. It was observed that proliferation of the contaminants started on the surfaces of the embryonic axes that were not in contact with the medium. The effect of the culture filtrates of Trichoderma on growth inhibition of other fungi could have been due to the presence of antibiotics.
Effect of different decontaminants on the embryonic axes of T. dregeana
- Electrochemical or electrolytic disinfection by anodic water
- Chemical disinfection
- Effect of exposure to Nipastat on the embryonic axes and contaminants
- Effect of Nipastat-enriched medium on the embryonic axes and associated
- Effect of Nipastat on test isolates
- The effects of sodium dichloro-isocyanurate (Medi-Chlor/NaDCC) on the
- Effect of Benlate on test isolates
Biocontrol agents
- Cultural characteristics and growth rate of two isolates of T. harzianum (EcoT
- Plate assays for evaluation of biocontrol properties of T. harzianum (EcoT and
- Effect of directly diffusible metabolites produced by EcoT and Eco77 on
- The effect of directly diffusible metabolites produced by EcoT and Eco77
- The effect of volatile metabolites of T. harzianum against test isolates
- Trichoderma harzianum as a biocontrol agent in tissue culture methods
- Effect of Trichoderma on the biomass of the embryonic axes of T. dregeana at
- Effect of Inoculating Trichoderma-treated embryonic axes of T. dregeana with
- Effect of Trichoderma inoculation in the context of contamination of the
Although the culture filtrate reduced contamination of embryonic axes compared to the control, it had a negative effect on the vigor of the embryonic axes as the immersion time increased. The embryonic axes plated on the culture filtrate-enriched medium (Table 3.20) showed a significant reduction in contamination with Penicillium spp. However, the embryonic axes plated on the PDB culture filtrate-enriched medium appeared to have reduced root hair formation and vigor was compromised compared to the control (Table 3.27;.
Viabilty of embryonic axes of T. dregeana in storage after alginate gel
- Alginate gel encapsulation, storage and assessment under hydrated conditions
- The effect of the use of different storage containment on alginate-encapsulated
These enzymes could also have contributed to the inhibitory effects of the culture filtrates obtained from both SCB and PDB in this study. This author reported that culture filtrates of the various species of Trichoderma also had negative effects (see above). However, in this study, even after 24 h of co-cultivation with Trichoderma, delayed growth and reduced biomass of T.
DISCUSSION
The need for optimising an ideal decontamination protocol for the embryonic axes
It is necessary to make the seeds free of contamination in order to protect the internal tissues from the penetration of fungi or bacteria, which will eventually make it difficult or even impossible to eliminate the infection (Sutherland et al., 2002). If seeds are collected from the soil and decontaminated immediately at the surface, the risk of surface-settled fungi invading internal tissues should be avoided (Sutherland et al., 2002). It is also very important to choose a surface decontaminant that is non-toxic to the seed tissue and ultimately the explants.
Effect of different surface decontaminants on excised axes of T. dregeana
Optimising different methods of decontamination
- Electrolysed oxidising (EO) water
- Nipastat
- Medi-Chlor ® (sodium dichloro-isocyanurate [NaDCC])
- Benlate
- Biocontrol agents and their effects
- Effect of direct diffusible metabolites of EcoT and Eco77
- Effect of volatile metabolites
- Effect of non-volatile metabolites
- Effect of Trichoderma isolates as conidial suspensions on Trichilia
- Effect of Trichoderma on root growth and biomass
The changes in the growth and development of the seedlings were found to be due to the influence of auxin signaling as well as the ability of the Trichoderma species to produce indole compounds viz. Thus, it cannot be stated with certainty that the lack of inhibition of the test isolates was due to the ineffectiveness of volatile metabolites. However, in the present study, root growth was reduced even after the 24-h co-cultivation period with Trichoderma, although there was 100% germination of the T.
Minimal growth storage of the embryonic axes using alginate gel encapsulation
Oxygen has been shown to be essential for maintaining viability of the recalcitrant seeds of Araucaria hunsteinii (Tompsett, 1983). In addition, aluminum has been shown to be lethal to plant tissues, which may have also contributed to the death of the axes. So it appears that slow dehydration from the beginning of the storage period was the major lethal factor.
Concluding summary
In: Seeds: Biology, Development and Ecology (ed.. 2000) Production of chitinase and β1,3-glucanase by Trichoderma harzianum for control of the phytopathogenic fungus Sclerotium rolfsii. 2004) Effect of Benomyl on Chitinase and β-1,3-Glucanase. Use of demethylation inhibiting fungicides (dmis) to control garlic rot (Sclerotium cepivorum Berk.) in New Zealand, New Zealand Journal of Crop Science Effects of triadimenol and tebuconazole seed coating treatments on wheat seedling growth and development. 1997) Responses of the recalcitrant seeds of Avicennia marina to hydrated storage: events occurring at the root primordia.
University of California Division of Agriculture and Natural Sciences. in Biocontrol of fusarial wilt of tomato, Volume 44, Issue 8, pp. 1977) Surface carbohydrates of the prokaryotic cell, pp. 1987) Cone and seed diseases of North American conifers.
