Journal of
Undergraduate Research
Volume 1, Issue 8 - May 2000
An Evaluation of Fungal Isolates for the Biological Control of Waterhyacinth, Eichhornia crassipes
Alison Walker
ABSTRACT
Eichhornia crassipes causes serious aquatic weed problems in many tropical and subtropical countries. It is recognized that biological control must be an integral component of any management strategy for this weed, and that there is a need to find highly virulent pathogens to improve the chances of success in the biological control of this weed. In this study, approximately 800 fungal isolates collected from several countries were screened for their pathogenicity to waterhyacinth. Following a two-step screening, eight of the most highly pathogenic isolates were chosen for further study, identification and evaluation as biocontrol agents of waterhyacinth. These results are presented and discussed.
INTRODUCTION
Eichhornia crassipes, commonly known as waterhyacinth, is a floating aquatic plant belonging to the monocotyledonous family Ponteridaceae. It poses a serious aquatic weed problem in tropical and subtropical countries of the world. Its explosive growth rate and the ability to infest a wide range of freshwater habitats have created enormous environmental and economic problems
Figure 1. A river in Mexico completely clogged by a rapidly growing population of waterhyacinth (left). Waterhyacinth flowers (middle). A population of blooming waterhyacinth plants. The botanical curiousity of this plant led to its worldwide distribution.
It is widely recognized that biological control must be an integral component of any management strategy for waterhyacinth. Accordingly, Charudattan (unpublished survey records) undertook several surveys in Brazil, Mexico, and several other countries and collected nearly 800 fungal isolates.
The purpose of this research was to 1) screen this collection of fungal isolates obtained from natural infestations of waterhyacinth in several countries, 2) rank these isolates on the basis of their pathogenicity and damage to waterhyacinth, and 3) identify highly pathogenic fungi. A specific objective was to identify pathogens previously unreported on waterhyacinth, or previously unreported on this plant in the United States.
MATERIALS AND METHODS
Approximately 800 fungal isolates, collected over the course of several years mostly from Brazil and Mexico, but also from some other countries, were used in this study. The isolates have been stored and maintained in slants of potato dextrose agar (PDA; Difco, Detroit, MI), a growth medium, in an incubator at 27 C. The isolates were first grown out on a PDA plate, checked for purity, and multiplied as a source of inoculum. Cultures that appeared contaminated with another fungus were subcultured and purified. Eighty-three isolates were eliminated from further testing because they were either contaminated or failed to grow. Isolates that appeared to be duplicates or appeared to be a species of Cercospora, a pathogen that has been previously studied as a possible biocontrol agent on waterhyacinth, were eliminated from further study. After this initial step, 630 of the isolates were used in the following screening process.
Healthy waterhyacinth plants were collected from natural infestations in Gainesville, Florida and maintained in a quarantine greenhouse, free of insects and diseases. For the inoculation procedure, plants were kept in plastic cups (9 cm diam x 12 cm ht) filled with water, one plant per cup.
Each of the 630 isolates went through a three-step screening process in a hierarchical order to select the most highly pathogenic isolates for further study. In the first stage of screening, a 2-mm plug of each culture was grown out on a PDA plate under aseptic conditions. The plates were placed in a diurnal incubator with 12 h light/12 h dark cycles at 27C. After two weeks, mycelium and, if present, spores were harvested by adding 10 ml of distilled water to each culture plate and collecting the contents. Each suspension was then liberally applied to the surface of a waterhyacinth plant set up in the quarantine greenhouse. Each plant was individually enclosed in a plastic bag that was tied to create a dew-effect that is conducive for fungal growth. After two weeks, the plants were removed from the plastic bags and placed on benches in the quarantine greenhouse at approximately 28C. Plants were rated weekly for four weeks for disease symptoms including leaf spots, leaf lesions, and leaf death.
Forty-two isolates showing the most severe symptoms in the first stage of screening were chosen to go on to the second stage of screening. Each of these isolates was grown on three PDA plates. Mycelium and, if present, spores were harvested in the same way as in the first screening. The same inoculation technique was used as well. During the second screening, three plants were inoculated per isolate to repeat the results. In the second screening, inoculated plants were set up in the quarantine greenhouse under plastic tents (approximately 30 x 30 x 30 cm) built with PVC plastic frames to create a dew-chamber effect. The plants remained under these tents for two weeks, at which time they were removed from the tents and placed on the benches. Again, these plants were rated weekly for a period of four weeks, for the same type of disease symptoms as in the first stage of screening.
Of the isolates screened during the second stage, eight of the most highly pathogenic ones were chosen for the third screening. During this stage, rice grain was used for inoculum production. Commercial long-grain white rice was used. Five hundred g of rice was placed in a polypropylene autoclave bag (Baxter Scientific Products, Deerfield, IL), autoclaved, and then autoclaved for a second time with 75 ml of sterile water added. Then one culture plate of each isolate was added to each bag. The inoculated bags were placed in a diurnal incubator at 27C to grow for approximately 2 weeks. To obtain the inoculum, 500 ml of sterile water was added to each bag. The contents were gently kneaded, and then the suspension was filtered out through four layers of cheesecloth. Each suspension was applied to three waterhyacinth plants, one plant per cup, in the quarantine greenhouse using a hand-sprayer. This time, plants were placed in a Percival dew-chamber (Percival Corp., Ames, IA) for the first 18 h after inoculation. After being removed from the dew-chamber, the plants were placed under plastic tents supported by PVC frames for an additional five days. Again, plants were rated weekly for 4 weeks.
The rating scale that was used was two-fold. Initially, plants were rated by the number of leaves infected per total number of leaves present (disease incidence). Subsequently, a letter designation was used to quantify the type of damage, ie., mycelial growth, leaf spots, leaf lesions, or death (disease severity). At the end of each 4 week screening, isolates were placed in one of five categories. Isolates that developed no significant damage or infection were given a rating of "NS". Plants with less than 25% of the leaf area damaged were rated as "Mild". Plants with 26-50% if the leaf area damaged were rated as "Low Moderate", and those with 51-75% of the leaf area damaged were rated as "High Moderate". Those plants with greater than 75% of the leaf area damaged were rated as "Severe".
RESULTS AND DISCUSSION
In the first stage of screening, of the 531 isolates screened, 53% showed no significant symptoms, 7% showed "Mild" symptoms, 17% had "Low Moderate" symptoms, 13% showed "High Moderate" symptoms, and 10% had damage rated as "Severe" (Table 1).
| Table 1 Results of Isolates Tested in the First and Second Stages of Screening |
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| Screening number | Ranking of isolates | No. of isolates | |||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| First screen | Total tested | 531 | |||||||||||||||||||||||||||||||||||||||||||
| NS | 283 | ||||||||||||||||||||||||||||||||||||||||||||
| Mild | 36 | ||||||||||||||||||||||||||||||||||||||||||||
| Low Moderate | 90 | ||||||||||||||||||||||||||||||||||||||||||||
| High Moderate | 69 | ||||||||||||||||||||||||||||||||||||||||||||
| Severe | 53 | ||||||||||||||||||||||||||||||||||||||||||||
| Second screen | Total tested | ||||||||||||||||||||||||||||||||||||||||||||
| NS | 0 | ||||||||||||||||||||||||||||||||||||||||||||
| Mild | 0 | ||||||||||||||||||||||||||||||||||||||||||||
| Low Moderate | 24 | ||||||||||||||||||||||||||||||||||||||||||||
| High Moderate | 15 | ||||||||||||||||||||||||||||||||||||||||||||
| Severe | 3
Not all of the damage observed in this stage of screening was solely due to the effects of the pathogen. Some of the damage can be attributed to the adverse environmental conditions created by the prolonged incubation under the plastic bags, and this simplified rating scale does not account for those effects. The first-stage screening was designed to test the isolates under conditions that are favorable for fungal growth and infection by even the weakly pathogenic fungi. The objective was to select even the most mildly pathogenic isolates. However, some non-pathogenic, physiological damage was incurred due to this bagging system, ie., leaves that touched the sides of the plastic bag often became water-soaked and developed some lesions. However, it is important to note that the second and third stages of screening were designed to minimize such negative environmental effects and further distinguish truly pathogenic effects. In the second stage of screening, of 42 isolates tested, none fell into the category of either "NS" or "Mild". There were 57% that rated as "Low Moderate", 36% rated as "High Moderate", and 7% rated as "Severe" (Table 1). The three isolates that showed severe symptoms, plus five of the most active isolates from the "High Moderate" group were chosen for the third screening. One isolate, 3447, produced significant signs of disease infection. Koch's Postulates have been filled for the two most active of the final eight isolates, 3447 and 3976. The remaining six isolates are still under evaluation. The following table lists the final eight isolates that are being evaluated and their tentative identification, based on spore morphology and mycelial growth.
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