Methods for isolation of endophytes from plants

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Plant growth and performance

Plant fresh root weights differed significantly among experiments (P<0.0001), and a significant interaction (P=0.0006) between experiments and treatments indicated that the effects of treatments on plant fresh root weight varied across experiments. Inoculation of plants with fungal isolates resulted in variable effects on plant fresh root weights across experiments. Within each experiment, no significant differences were observed between endophyte treatments and the control treatment, except for experiment 3 (P=0.0008) (Table 9). In this experiment, plants treated with isolate V5W2 had a significantly higher fresh root weight than the control treatment and that of the treatment with isolate V2W2, but it did not differ significantly from the other fungal isolates. Plants treated with isolates V5W2 further had a higher fresh root weights for 5 out of 7 experiments.
A significant difference for fresh shoot weights was found among experiments (P<0.0001), and the interaction between treatments and experiments was not significant (P=0.0503). Within each experiment, fresh shoot weight data demonstrated a lack of significant difference between the endophyte treatments and the controls, except for experiments 3 (P=0.0156) and 8 (P=0.0170) (Table 10). In these experiments, plants treated with fungal isolates Eny7.11o and Eny1.31i, respectively, resulted in significantly higher shoot weights compared to control plants. Although differences in fresh shoot weights did not reach statistical significance in most of the experiments, a general trend was observed for plants treated with the different fungal isolates to have higher shoot weights than the control plants. For example, isolates V5W2 and Eny7.11o resulted in higher fresh shoot weights in all the experiments in which these isolates were tested, compared to the control plants.
The dry shoot weights differed significantly between experiments (P<0.0001), with a nonsignificant interaction between treatments and experiments (P=0.4090). Within each experiment, dry shoot weights did not differ between endophyte treatments and the control treatment (P=0.1305 – 0.9420), except for experiments 3 (P=0.0004) and 8 (P=0.0105). In experiment 3, plants treated with fungal isolates III4W1 and Emb2.4o had significantly higher dry shoot weights, while in experiment 8, plants treated with fungal isolate Eny1.31i had a significantly higher dry shoot weight compared to the control plants (Table 11). In most of the experiments apart from experiment 5, although not significant, a trend occurred in which plants treated with the various fungal isolates had higher dry shoot weights compared to the control was observed (Table 11). Isolates Eny7.110 and III3W3 resulted in an increase of dry shoot weight in six of the seven experiments.
Plant height of endophyte-treated and untreated plants (data not shown) differed among repeat experiments (P<0.0001). Within each experiment, however, no differences were observed in plant height between the endophyte treatments and control treatment (P=0.1525). The number of standing leaves, length and width of the youngest leaf were also not influenced by endophyte treatment (data not shown).
The total number of roots per plant in endophyte-treated and untreated plants (data not shown) also differed among repeat experiments (P<0.0001). A significant interaction between experiments and treatments was observed (P=0.0070), indicating that the effect of treatments on the total number of roots per plant varied across experiments. Within each experiment no differences were observed in the total number of roots per plant between the control and endophyte treatments, except for experiments 3 (P=0.0044) and 5 (P=0.0186). In experiment 3, plants treated with isolates III4W1, V5W2 and V4W5 had significantly more roots than all the other endophyte-treated plants and the control. In experiment 5, control plants had significantly more roots than the endophyte-treated plants, except for plants treated with isolate Eny1.31i, which had significantly less roots than control plants.

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Endophytic colonization

Percentage colonization of roots treated with the various fungal isolates ranged from 44.9 to 68.8% in experiment 2, 22.6 to 65.3% in experiment 7, and 17.5 to 22.4% in experiment 9 (Table 13). Endophytic F. oxysporum was also re-isolated from control plants. Colonization of roots of control plants by F. oxysporum was 45.2%, 36.0% and 16.6% in experiments 2, 7 and 9, respectively. Percentage colonization of plant roots of endophyte-treated plants was not statistically different from the control plants in experiments 2 (P=0.1171), 7 (P=0.4467) and 9 (P=0.8519). Other fungi were also frequently isolated from endophyte-treated and control roots. The most commonly isolated fungal genera were Penicillium spp. and other sterile fungi (data not shown). The levels of colonization of plants by fungal isolates varied across the experiments. Percentage colonization by F. oxysporum was higher in experiment 2 and 7 than in experiment 9. For example, percent colonization by isolate V5W2 was 58.4%, 34.6% and 22.4% in experiments 2, 7 and 9, respectively.

CHAPTER 1 
REVIEW OF THE ROLE OF ENDOPHYTES IN BIOLOGICAL CONTROL OF PLANT-PARASITIC NEMATODES WITH SPECIAL REFERENCE TO THE BANANA NEMATODE, RADOPHOLUS SIMILIS (COBB) THORNE 
1. INTRODUCTION
2. WHY ENDOPHYTES?
3. COLONIZATION OF PLANTS BY ENDOPHYTES
4. METHODS FOR ISOLATION OF ENDOPHYTES FROM PLANTS
5. ENDOPHYTIC FUNGI AND NEMATODE CONTROL
6. ENDOPHYTIC BACTERIA AND NEMATODE CONTROL
7. MECHANISMS OF ACTION OF ENDOPHYTES AGAINST NEMATODES
8. BANANA PRODUCTION IN UGANDA
9. NEMATODE PROBLEMS OF BANANA
10. BIOLOGY OF RADOPHOLUS SIMILIS
11. DAMAGE AND ECONOMIC IMPORTANCE OF RADOPHOLUS SIMILIS
13. CONCLUSIONS
14. REFERENCES
CHAPTER 2 
IN VITRO SCREENING OF ENDOPHYTIC FUSARIUM ISOLATES AGAINST THE BANANA BURROWING NEMATODE, RADOPHOLUS SIMILIS (COBB) THORNE 
-ABSTRACT
-INTRODUCTION
-MATERIALS AND METHODS
-DISCUSSION
-REFERENCES
CHAPTER 3 
IN VIVO SCREENING OF ENDOPHYTIC FUSARIUM OXYSPORUM ISOLATES FOR ACTIVITY AGAINST RADOPHOLUS SIMILIS IN TISSUE CULTURE BANANA PLANTS 
-ABSTRACT
-INTRODUCTION
-MATERIALS AND METHODS
-RADOPHOLUS SIMILIS CULTURES
-RESULTS
-DISCUSSION
-REFERENCES
CHAPTER 4 
EFFECT OF ENDOPHYTIC FUSARIUM OXYSPORUM ISOLATES ON HOST PREFERENCE, ATTRACTION, ROOT PENETRATION AND REPRODUCTION OF RADOPHOLUS SIMILIS IN TISSUE CULTURE BANANA PLANTS 
-ABSTRACT
-INTRODUCTION
-MATERIALS AND METHODS
-HOST PREFERENCE AND ATTRACTION EXPERIMENTS
-DETACHED ROOT BIOASSAY FOR HOST PREFERENCE AND ATTRACTION
-INTACT PLANT BIOASSAY FOR HOST PREFERENCE AND ATTRACTION
-ROOT PENETRATION EXPERIMENTS
-RADOPHOLUS SIMILIS REPRODUCTION EXPERIMENTS
-RESULTS
-HOST PREFERENCE AND ATTRACTION EXPERIMENTS
-ROOT PENETRATION EXPERIMENTS
-DISCUSSION
-REFERENCES
CHAPTER 5 
MECHANISMS OF ACTION OF ENDOPHYTIC FUSARIUM OXYSPORUM AGAINST RADOPHOLUS SIMILIS IN BANANA PLANTS 
-ABSTRACT
-INTRODUCTION
-MATERIAL AND METHODS
-RESULTS
-DISCUSSION
-REFERENCES
CHAPTER 6 
GENETIC DIVERSITY OF ENDOPHYTIC FUSARIUM SPP. ASSOCIATED WITH CAVENDISH BANANA IN SOUTH AFRICA
-ABSTRACT
-INTRODUCTION
-MATERIALS AND METHODS
-RESULTS
REFERENCES 
SUMMARY

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