Thursday, February 6, 2014

UST GS: A Comparative Study on the Susceptibility of Two Species and a Variety of Erythrina to Erythrina Gall Wasp

Josephine Espinoza Tondo
Thesis for Master of Science in Biological Sciences
Graduate School,University of Santo Tomas
Adviser: Dr Abe V Rotor
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Erythrina is perhaps the most lovable ornamental tree in summer in parks, along highways, and woodlands.  It has also many medical uses. The bark is used by Malay Peninsular people for relieving toothache. In China, it serves as expectorant. Its wood is rasped in water and applied as medicament for hameturia. The leaves are made into poultice and applied to sores. Barks and leaves are used to cure boil, asthma and abscesses (Budowski, 1987). Erythrina has been used in folk medicine for the treatment of insomnia, malaria fever and venereal disease. It is also used as a narcotic and anti-helminthic. This plant is effective in curing infection due to its antimicrobial substance known as Erythrina lectins (Payne, 1991).
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 Erythrina in acrylic painting by Dr Abe V Rotor 


 Flower cluster and tree structure of Erythrina crista-galli Linn. (From Internet)

INTRODUCTION
Plants are attacked in many ways resulting in injury or damage on the leaves, stems, roots and other parts. These include curling of leaves, deformation of shoots and swelling of certain parts of the plant while severe conditions may lead to defoliation and subsequently death of plants (Manner, 1982).

In parasitism, also known as antagonistic symbiosis, one organism receives no benefits and is often injured while supplying nutrients or shelter for the other organism. This relationship can be observed on various living organisms. One of the effects of parasitism is the formation of galls on leaves and stems such as the case of Erythrina trees infested with a gall wasp. Gall infested Erythrina trees were first reported by the Department of Environment and Natural Resources in 2005 (Rivera, 2005). In the same year, the report included Metro Manila, among the regions where the infestation was observed. Earlier, there were reported infestations of Erythrina in neighboring countries such as Taiwan, Singapore, Samoa and Hawaii (Kim et al., 2004).

A gall or cesidium is the swelling or overgrowth of any part of the plant which is a result of infestation by certain pests. Generally, the growth and development of gall may later coalesce into a larger mass around the point of attack by the parasite (Agrios, 1997). But tumors may develop on any part of the plant attacked by the pest.

A gall forming bacterium in plants known us Agrobacterium tumefaciens, causes crown galls on a number of plants, however the damage is usually slight (Boothroyd, 1972). There are also fungus-induced galls which are related to Ascomycota and Basidiomycota. Other causes of galls are traced to Nematoda and mites (Arachnida) (Manner,1982). Among the plants that commonly exhibit galls are lilies, evening primrose, chestnut, apple, oak, spruce, pineapple, cypress, ficus, grape, hickory, pecan, cottonwood and ash flower (Hutchkins, 1969).

In the Philippines, gall formation was observed in different species such as in santol (Sandoricum koetjape Merr.), acacia (Samanea saman (Jacq.), potato (Solanum tuberosum L.) and dita (Alstonia scholaris L.)

A number of hypotheses were drawn out of various observations. One hypothesis which is the nutrition hypothesis attributes the cause of galls to nutrition imbalance as a result of feeding by a pest. The second hypothesis maintains that gall tissues protect the gall forming agent from unfavorable abiotic conditions most especially desiccation (Stone & Schronreogge, 2003)

The third hypothesis (Enemy Hypothesis) supports the idea that plants protect the gallers from attack by natural enemies (Enemy Hypothesis) (Stone, Schonrogge, 2003). The fourth hypothesis is the transmission of a gall-forming organism, such as, the Erythrina gall wasp from exotic plant species to native plant species (Transmission Hypothesis). Guided by these hypotheses, this study aimed at detecting the most plausible cause or causes of gall formation in Erythrina trees.

THE PROBLEM AND ITS OBJECTIVES
Gall infestation of Erythrina trees has been observed in the last three years, decimating many trees along highways and in woodlands. Severely infested trees often succumb to the pest. There have been reports of similar cases that galls production in Erythrina is caused by a species of wasp known as Quadrastichus erythrinae K. or Erythrina gall wasp as identified by Il Kwon Kim (Kim et al., 2004).

 Photo shows extent of devastation by the gall wasp resulting in the death of standing trees in Australia. 

Some of the Erythrina species which are affected by gall wasps in neighboring countries are E. variegata L., E. corallodendron L. and E. abyssinica Lam. (Heu et al., 2004). It is in the context of these reports, both foreign and local, that the researcher became interested in finding out whether or not; the responsible insect pest is of the same species that attack local Erythrina trees.

If so, how are the Erythrina species and variety compared according to their susceptibility to the pest?

The specific objectives of the study are as follows:

1. To validate the cause of gall formation and the nature of host-pest relationship of the gall insect and the Erythrina specimens.

2. To compare the resistance and susceptibility among the three specimens of Erythrina to Q. erythrinae K. through gall production and the extent of this growth.

3. To find out through phytochemical analysis of plant tissues the basis of resistance/susceptibility among the Erythrina specimens.


SIGNIFICANCE OF THE STUDY
This study of the susceptibility of the three species of Erythrina on Quadrastichus erythrinae K. is significant for the following reasons:

1.3.1. By knowing the resistance/susceptibility of the different species and varieties of Erythrina, agriculturists, botanists as well as the public will be guided in instituting measures on how to save them.

1.3.2. The results of the study may be used as a guide in the planting and care of Erythrina plants on the part of DENR and DA as well as the private sector.

1..3.3. The results of this study may provide an effective solution to prevent further infestation of EGW on Erythrina in other places.

1.3.4. The results of this study could serve in developing control measures against insect pest itself.


CONCEPTUAL FRAMEWORK OF THE STUDY
The study is divided into three parts, namely:

1. Validation of the host species of and parasite and their relationship.
2. Composition of resistance/susceptibility of the three species of Erythrina.
3. Testing of chemical properties through tissue analysis among the three species of Erythrina.

Two species and a variety of Erythrina were studied. Collections of the healthy young plants were made and cultured in an improvised plant nursery. Inside the nursery the healthy plants were exposed to the Erythrina wasp to determine their susceptibility. The plant specimens and the gall wasp were brought to the Philippine National Museum for validation.

Observation of plant and insect species were done in the field, while analysis of phytochemical properties was done at DOST and TARC. These properties include alkaloid, flavonoid, glycoside, saponin, sterol, triterpene and tannin.


SCOPE AND LIMITATION OF THE STUDY
The study was conducted for period of six months, from September of 2006 to February of 2007, the flowering season. It is also during this season that the trees, being deciduous completely lose their leaves in favor of the formation of numerous flowers in clusters.

Susceptibility test was conducted in the plant nursery on the two species and one variety and the result was compared with observations in the field. Chemical analysis was done at the Industrial Technology Development Institute, Standards and Testing Division of Department of Science and Technology (DOST), and Research Center for the Natural Sciences of Thomas Aquinas Research Complex (TARC).


REVIEW OF RELATED LITERATURE

ETHNOBOTANICAL DESCRIPTION OF THE PLANT
For the Japanese and Chinese, the blooming of Erythrina portends luck. For coastal peoples of Kavarawan, Pinpu tribe and Yami/Tao tribe, this is the start of the ceremony for catching flying fish. For Puyuma people, it is the time for planting sweet potatoes. If the Erythrina trees do not bloom; folk belief is that there is likely a disaster that will occur within a year (Yang et al., 2004). In Southern Mindanao and among the Ilongot tribe, the blooming of Erythrina signals the beginning of seasonal celebration, according to Dr. Remilio C. Atabay, Regional Technical Director from DENR, Region III. The tree blooms, to usher the start of an ornamental ceremony among Ilongot tribes wherein a Christian is beheaded to complete a wedding ceremony, and to bring luck to the wedded Ilongot couple.

DESCRIPTION OF Erythrina
The name Erythrina came from the Latin word erythros meaning “red” and was obviously applied due to the plant’s red flowers (Yang et al., 2004).

Most species are trees and shrubs which grow in climates with pronounced dry and cool seasons (Neill, 1988). Erythrina trees originally were classified under Family Leguminosae (Merrill, 1912) but have been lately classified under Family Fabaceae (Madulid, 2001). It was mentioned in the rules and recommendations found in the International Code of Botanical Nomenclature that the usage of the former families is treated as validly published and the use of alternatives of the family names is authorized such as Compositae as Asteraceae, Graminaneae as Poaceae and Leguminosae as Fabaceae as its alternative family name (Doyle, 2000). The trees exhibit a wide diversity in floral structure, inflorescence orientation, fruit morphology and seed coat. Their flowers are mostly red; some are salmon, pink, orange or yellow, solitary, paired or fascicled in erect, terminal racemes leafy at the base or in axillary racemes (Allen and Allen, 1981).

In the Philippines, Erythrina is known in Tagalog as karapdap, kasindak and dapdap. In the Bicol region, it is andoragat. It is sulbang in Pampanga, bagbag in Ilocos, and vuvuk in Cagayan (Quisumbing, 1978).

THREE SPECIES OF THE GENUS Erythrina
There are a number of species and varieties of Genus Erythrina. Among them are Erythrina variegata L. and Erythrina crista-galli L. and a variety named Erythrina variegata var. orientalis (L.) Merr. Erythrina crista-galli L. or cockspur coral tree is a medium-sized tree. Leaves are with 3 leaflets, smooth, alternate and to 6 cm long. The leaflets are dark green, elliptical, acute and with entire margins. Its red flower is arranged in inflorescence of raceme, pentameric, complete, bilateral symmetry, papillionate, light to dark red and leafy with standard petals of 5 cm long. The stamens are separate and about 1 cm long. The fruits are woody, elongate, cylindrical and to 3 cm long or longer (Madulid, 2000). 

It grows to a height of five to eight meters and up to ten meters. This species characteristically grows wild in the tropical forest ecosystems and also in swamps and wetlands. It is popularly found in parks. The trees usually flower beginning in October through the whole of summer. April is the peak flowering month in its native home, South America, and from April to October in the northern Hemisphere (Teketay, 1990). In tropical countries, E. crista-galli bears flowers starting in the cold months of December to the summer months.

Erythrina variegata L. or dapdap in local name is a deciduous tree 10 to 15 m tall. The branches are armed with stout, short prickles. The leaves are trifoliate, with broad-ovate leaflets, up to 18 cm long. The flowers are large, with bright red petals 9 cm long. The pods are constricted between the seeds and 25 cm long. The distribution of this species is widespread in the Philippines. It is common along seashores but also found in inland. It is propagated by seeds.

Erythrina variegata var orientalis or Indian coral tree is a medium sized tree reaching 15 m tall, with prickly, stout trunk and branches. Leaves are deciduous, alternate, pinnately trifoliate. Leaflets are broad, ovate, and dark green. Flowers are bright red, to 7 cm long. Calyx is oblique, two-lipped; standard petal is free, ovate-oblong, slightly recurved, standard wings and keel nearly equal. Fruit is turgid and to 30 cm long. The difference of E. variegata var orientalis to E. variegata is the characteristic whitish yellow midrib and vein in the former. This variety is indigenous to India, Malay Peninsula, and Polynesia and grows along seashores. It is planted as a shade or ornamental tree in parks and big gardens.

The following outline shows the taxonomic classification of the two Erythrina species and one variety.

Classification
Kingdom: Plantae
Division: Anthophyta (Magnoliophyta)
Class: Magnoliopsida
Order: Fabales
Family: Fabaceae
Genus: Erythrina
Species: E. variegata var orientalis (L.) Merr.
E. variegata L.
E. crista-galli L.

GEOGRAPHICAL ORIGIN OF THE PLANT
The origin of Erythrina, like its relatives under Leguminosae is obscure. No fossil record of the genus has been reported. Little is known about its distribution, patterns of pollination and dispersal mechanism. Legumes dates back to the upper Eocene to upper Oligocene (estimated 30 to 40 million years ago) surviving ocean drift or other long-distance dispersal and spreading in the tropical regions of America, Africa and Asia-Oceania. South America seems the most likely place of origin since the majority of primitive ancestral groups within the family occurs in these places. Africa is also another possible origin of Erythrina because it is also the home of a number of endemic groups (Neil, 1988).

PLANT GEOGRAPHICAL DISTRIBUTION
Erythrina is pantropical, consisting of some 112 species; 70 neotropical, 31 African and 12 Asian. It is distributed throughout the tropics and extends into warm temperature areas such as in Africa, lower Himalaya, South China, Rio de la Plata region, Argentina and Southern America. Most of the species are trees and shrubs (Neil, 1988). The following maps shows the distribution of E. crista-galli, E. variegata and E. variegata orientalis in Figure 2,3 and 4. Fig. 1. Distribution of Erythrina worldwide as indicated by dots.

USES OF THE PLANT
Erythrina has many medical uses. The bark is used by Malay Peninsular people for relieving toothache. In China, it serves as expectorant. Its wood is rasped in water and applied as medicament for hameturia. The leaves are made into poultice and applied to sores. Barks and leaves are used to cure boil, asthma and abscesses (Budowski, 1987). Erythrina has been used in folk medicine for the treatment of insomnia, malaria fever and venereal disease. It is also used as a narcotic and anti-helminthic. This plant is effective in curing infection due to its antimicrobial substance known as Erythrina lectins (Payne, 1991).

Various species of Erythrina are used as ornamental plants in California, Mexico and other parts of America, Africa, Australia and the Pacific countries. They add to aesthetic beauty along beaches as their roots help stabilize sandy soils. Other species of Erythrina are widely appreciated as natural shade for cocoa and coffee shops in US (Teketay, 1990). In Western Samoa, Erythrina species is used as living fencepost (Viquez, 1993).

Other uses of Erythrina species are for livestock forage, as source of green manure (Hernandez, 1993). Erythrina wood is described as grayish white, spongy, lightweight and strong but not durable. It is used as sieve frames, surfboards, canoes, boxes in art carvings (Allen and Allen, 1981). It is the most preferred material in the manufacture of bakya or wooden clogs in the Philippines and other parts of Asia.

THE INSECT AND ITS ORIGIN
Quadrastichus erythrinae K, also known as gall wasp belongs to Order Hymenoptera of Class Hexapoda under Phylum Arthropoda.. This small insect has an adult length of 1 mm to 1.6 mm. It is believed to have originated from Africa, and was possibly transported via ship carrying wood and other infested parts of the plants. Another theory is that hikers carried in their clothes or boots infested parts of the plants. The role of wind dispersal is also included among the theories (Tung, 2006).

CLASSIFICATION OF THE INSECT
Quadrastichus erythrinae K. or simply Erythrina gall wasp belongs to Family Eulophidae. Quadrastichus means “four lines” (quad-four; stichus-line). These lines are found in the scutellum on the dorsal side of the thorax (Tung, 2007).

Classification
Kingdom: Animalia
Phylum: Arthropoda
Subphylum: Hexapoda
Class: Insecta
Subclass: Pterygota
Order: Hymenoptera Suborder:Apocrita Family: Eulophidae
Genus: Quadrastichus
Species: erythrinae

RELATED STUDIES ON GALL WASP
Recently, Quadrastichus erythrinae was discovered to be the pest attacking Erythrina species in different countries in Asia such as Taiwan where there was an outbreak in the early 2000s (Yang et al., 2004). It was also noticed in Singapore, China and India. In Hawaii, it was reported that some Erythrina species were affected. It was reported in the Philippines in the latter part of 2004. Apparently the presence of the pest was not given immediate attention.

One of the manifestations of the pest in Erythrina is gall formation. In other types of plants, galls are also observed leading to the death of the plant. Galls are also found in oak, Quercus punctata. In Ficus carica, jumping gall wasp was similarly observed to cause galls. (Askew, 1984).
In the Philippines, galls found on acacia foliage and branches are caused by a rust fungus, Uromycladium tepperianum (Cristavao, 2003). Another gall causing insect was found in Hagonoy plant (Chromolaena odorata L.) identified as Procecidochares connexa M. Galls on the leaves of santol are caused by mites, Eriophyes sandorici N.

PHYTOCHEMICAL COMPOSITION OF Erythrina
Erythrina is known to contain alkaloids and other phytochemical properties. Recent studies identified various chemical compositions as isoflavones and alkaloid derived from the bark and seeds of Erythrina senegaliensa (Wandji, et al., 1995). Phenylated isoflavonoids is also derived from Erythrina eriotricha (Nkungfuch et a, 1995). Other phytochemical properties were found in Erythrina costarricensis such as erythroidine, a naturally derived drug used in the 1950’s as a neuromuscular blocking agent in surgery (Payne, 1991). Erythrina edulis is also known to produce alkaloids (Allens, 1980).

For phytochemical and biological investigation of Erythrina variegata, there were five isolated from the n-hexane and chloroform soluble fractions of a methanol extract of its stem bark. These are the alpinum isoflavone, epilupeol, 6-hydroxygenistein, dihydroxylean and stigamasterol.


RESEARCH METHODOLOGY
There are four aspects of the study as presented in the conceptual framework (Fig.1, page 6).
1. description and validation of the wasp parasite;
2. comparison of size and morphology of galls from among the three Erythrina species;
3. comparison of susceptibility among the three Erythrina species to the gall parasite; and
4. comparison of chemical properties of tissues of the three (3) Erythrina hosts.

I. IDENTIFICATIONS AND DESCRIPTIONS OF GALL INSECTS

A. Collection of the Gall Wasp
Gall wasps were collected from the following areas of Bulacan particularly in the North Luzon Expressway (Sta. Rita, Bocaue, Meycauayan and Marilao). Collection of the four stages of the insect’s life cycle namely egg, larva, pupa and adult was done by mechanical means such as using insect net, and light trapping. Fig. 5 shows infested and lifeless stands of Erythrina trees.

B. Validation of the Insect
The collected wasp parasites in all stages of development were preserved with ethyl alcohol in test tube. Insect specimens were also mounted in pin and properly labeled and stored in a box. The specimens were submitted for validation to the Philippine National Museum.

II. TREATMENT FOR THE Erythrina SPECIES
The study on comparative susceptibility of the three (3) Erythrina species was conducted in an improvised greenhouse arranged in Random Complete Block Design (Best, 2003). All the four test plants were acclimatized for a period of two weeks. There are four plants in each species arranged accordingly as follows as illustrated in Table 1.

III. GALL SIZE AND MORPHOLOGY
The development of the galls in each specimen was observed according to the following criteria:
a. development and growth of the galls;
b. size and shape of galls on leaves and petioles; and
c. anatomy of dissected galls vis-à-vis healthy tissues.
A vernier caliper was used to measure the size of the galls. Observations include the shape, changes of colors in galls, maturation and coalescing of the galls.


MATERIALS AND METHOD
A. Identification and Validation of the Host Trees
Fresh and dried specimens of the plants were submitted to the Philippine National Museum for identification. In preparing the specimen for drying, the specimens were placed on a single sheet or between the folds of a double sheet of paper. Newspaper and cheesecloth were used to dry the specimen. Dried specimens were placed between sheets of white paper. The presser is made of a pair of bamboo frames measuring 50 by 35 cm. Pressure is applied by tying the frames together wit the specimens in between. Drying period was one week.

B. Improvised Plant Nursery
An improvised nursery measuring 4m x 4m was constructed adjacent to the residence of the researcher in Sto. Rosario, Paombong, Bulacan (Fig 6). Materials used were nylon mosquito net and bamboo for posts and beams. the Four healthy plants two meters in height of each of the two species and one variety of Erythrina were introduced and acclimatized in the nursery for a week.

C. Cultivation of the Species Twelve healthy potted plants, four for each kind of Erythrina under study were procured from Karisz Garden and Landscaping and Fernando Torres Garden in Bulacan. The plants were given proper attention to keep them healthy, shielding them from the gall wasp and other pests. After acclimatization for two weeks, the plants were transplanted directly in RCBD arrangement. After two weeks the plants were then openly exposed to the gall wasp which was introduced into the nursery from infested stock.

D. Observation of the Erythrina specimens
Comparison was made between and among the three kinds of Erythrina under study principally for their reaction to the gall wasp. Morphological and anatomical observations on the galls produced were recorded and analyzed.

E. Susceptibility Tests
The test plants in the nursery were given equal treatment in cultivation, weeding and irrigation to enhance normal growth and development. Susceptibility of each plant to the introduced gall wasp was regularly observed and the incipient appearance recorded. Subsequent developments of infestation and gall formation were made weekly thereafter until the end of the experiment which lasted for six months.

These developments were the increase in size and number of galls, coalescing of galls, defoliation, die back of stems, yellowing or wilting of plant parts, failure to produce flowers, deformed structures, and the like.

V. Field Observation

The study included field observation of standing Erythrina trees along roads and highways, backyards, open fields and parks in Bulacan, Pampanga, Tarlac and Bataan in Region 3; Laguna and Batangas in Region 4. In Metro Manila, Erythrina trees along Macapagal Boulevard, Edsa, Makati City, Parañaque City, Pasig City and Quezon City were similarly studies. The trees were observed according to these following aspects:

• Healthy, infested or lifeless.
• Approximate age of the trees
• Stage/extent of infestation by species/variety of Erythrina

VI. Testing of Chemical Properties
Phytochemical analysis was conducted on each specimen accordinbg to the presence or absence of alkaloid, sterol, flavonoids, saponins, glycosides and tannin. The test was done to be able to find out and verify if there are differences in phytochemical composition between infested and non-infested specimens, and between and among the two species and one variety of Erythrina. Testing of the plant parts (leaves, petioles and stem) was done at the Industrial Technology Development Institute Standards and Testing Division of the Department of Science and Technology, and Thomas Aquinas Research Center.

Samples from each of the test plants were obtained and weighed. They were weighed, dried and pulverized. Each sample was placed in sterilized Erlenmeyer flask with methanol .
The filtrate was obtained using filter paper, and the methanol in the filtrate was removed by rotary evaporator.

Each sample was weighed. Hexane, dichloromethane and butanol were used to determine the non-polar, semi-polar and polar properties of the extracted materials.

The extracts were screened in the presence of tannins, saponins, sterols, glycosides, triterpenes, alkaloids and flavonoids. Thin layer chromatography using aluminum silicon gel (F254 Merck) plates were used (Wagner et al., 1983). For hexane, the solvent used is 100% chloroform; for dichloromethane, the solvent is 35%methanol, 30%chloroform and 35%hexane. For butanol, the solvent system is 40% methanol, and 52%chloroform.

After preparing the sample, each plate was sprayed with specific spraying reagent such as Ferric cyanide and Potassium ferric chloride for the test in tannin, Libermann for Saponin and Sterol, Vanillin-sulfuric acid for triterpene, NP PEG for flavonoids, Dragendorff’s for alkaloid and Kedde reagent for glycoside (Wagner et al., 1983). Detection of the presence of phytochemical properties used short and long wavelength with specific colors it emitted.

VII. Statistical Analyses and Data Presentation
All statistical analysis was done using SPSS Program version 13.0. Quantitative test parameters were averaged, the means for each data group computed for significant or non-significant differences at 0.05 per cent level by One-Way ANOVA. Aside from One-Way ANOVA, DUNCAN Multiple Range Test was used to compare its similarities and differences among the studied specimens. Results were condensed in tabular form and, for brevity of presentation, also in graphs. Other numerical data and statistical presentations were rendered as Appendices, and also cited in the text as needed. Photographs were included to supplement descriptions of some aspects of the methodology.


RESULTS AND DISCUSSION
The study was conducted to determine the cause of gall formation and host-pest relationship of the gall insect to the Erythrina trees as well to compare their resistance, susceptibility and chemical properties of the three Erythrina species. Results were presented in tables and graphs, as well photographs.

COMPARATIVE STUDY ON THE TWO SPECIES AND A VARIETY OF Erythrina

I. GALL SIZE ON PETIOLE AND LEAVES
Galls are mainly on the young petioles and leaves. There is no significant difference on the average size of galls of the three Erythrina specimens. E. variegata orientalis has the largest average gall size on the petioles with 10.27mm, followed by E. variegata with the average gall size on petioles with 9.87 mm, and E. crista-galli 5.72 mm.

To validate the homogeneous characteristics, Duncan Multiple Range Test was used. DMRT showed the relationship of the three specimens E. variegata orientalis and E variegata exhibited homogeneous characteristics with the significant value of 0.772. This is in contrast with E crista-galli which has a significant value of 1.00.

For the gall size of leaves among the three specimens, E variegata and E varigata orientalis exhibited homogeneous characteristics with a significant value of 0.935. This is contrast to the Erythrina crista-galli which has a significant value of 1.00 (Table 5).

a. External characteristics of the gall
The gall wasp caused heavy infestation particularly on E variegata orientalis and E. variegata. In the greenhouse, infestation was observed two weeks after inoculation. All test plants of E. variegata orientalis succumbed to the pest after ninety days. Infestation was observed in E. variegata orientalis as early as September 2006. All test plants of E. variegata were heavily infested but managed to remain alive to the end of the experiment. For E. crista-galli, two of the four test plants were slightly infested, but succumbed to the pest nonetheless; the other two survived. Newly formed galls (green color) measured 3.01mm and increased to 20.33mm upon maturity (brownish).

Matured galls have “craters” which are actually exit holes for the emerging adult gall wasp. The holes remain open, and dry up contributing to the subsequent drying of the whole leaf. This is exacerbated by the coalescing of galls on a single leaf which may also include the petiole.

b. Deformation, Wilting, Yellowing and Defoliation of Leaves
Aside from the formation of galls, wilting and yellowing of leaves and consequent defoliation were observed. E variegata orientalis exhibited heavily defoliation followed by E. variegata, and least, E. crista-galli.

c. Bearing Flowers E. variegata orientalis and E. variegata species did not bear flowers, during the whole experimental period. E. crista-galli on the other hand produced flowers in the months of December and February.

d. Microscopic Anatomical Observation. The gall wasp attacked the young shoots which actually are made of succulent stems, leaves and petioles. The internal structures of these parts individually dissected, and studied under the compound microscope. These included cross section of the petioles, leaves, and galls at different stage of infestation. The gall wasp was also observes in its various stages of its life cycle.

The internal tissues are mainly composed of parenchyma cells. These have thin walls and are loosely arranged. The parenchyma cells serve as storage of food and water. They are primary sites of metabolic functions such as photosynthesis, respiration and protein synthesis (Weier, 1982).

The cross sections from healthy plants show well developed and uniform parenchyma tissues as shown in the photographs at around 40X magnification. The parenchyma tissue in E variegate and E variegata orientalis are thicker than that of E crista-galli.

Based on the observation gathered, parenchyma tissues found in the cross sections of leaves show basic similarity with those in the petioles. E variegata orientalis and E variegata have thicker portions of parenchyma tissues while Erythrina crista-galli has a relatively thinner layer of parenchyma tissues. There are collenchyma cell and fibrovascular tissues (dark colored cells) scattered among the parenchyma cells which apparently add to rigidity and resistance to other kinds of damage including pest attack. (Figure 24, 25 and 26).

f. Internal Characteristics of Galls
The wasp larvae which develop within the plant tissue induce the formation of galls in young leaves and petioles (Figure 25). As the infestation progresses, the leaves curl and appear deformed while petioles and shoots become swollen. After the larval stage the insect pupates within the leaf and stem tissue (Heu et al., 2006). Based on observation, upon reaching adult stage the wasp gnaws its way out, thus creating a crater or hole on the gall.

The stages of the life cycle of Q. erythrinae K. were spent inside the gall found in the Erythrina species. Upon the growth and development of the Erythrina gall wasp in the young petioles and leaves of the species, disarranged and non-uniform sizes of parenchyma and other parts of the ground tissues of the infested plants were observed. In the cross section of infested petioles of E. variegata and E. variegata orientalis, EGW occupied the area wherein parenchyma cells are of great number.

Observations showed that the Erythrina species particularly E. variegata and E. variegata var orientalis have more EGW to occupy the area with numerous parenchyma tisssues and at the same time, E. variegata and E. variegata var orientalis were exhibited dark structures found in the exterior part of the chamber where infested part were observed. In E. crista-galli, EGW developed on the peripheral part of the petiole.Based on observations, developing wasps stayed on more parenchyma tissues where in it grow and develop.

The same observation was made on the cross section of Erythrina leaves. Differentiated tissues consist of parenchyma cells that have no uniform structures. It has no uniform sizes of cells. Disarranged structures of cells were found. Clear areas of chambers were observed around the parasitic insect. All the three Erythrina species were stressed due to the presence of hard tissue inside the infested areas. Erythrina gall wasp occupies the center part of the differentiated tissue of leaves particularly in E. variegata and E. variegata var orientalis shown in Fig. 29 and 30 while in E. crista-galli, the peripheral portion was occupied by the insect pest shown in Fig. 31.

g. Death and Infestation of Three Erythrina Species Inside the Greenhouse
Death and infestation was observed in the two Erythrina species and a variety after the transfer of infested collected parts of E. variegata (which were taken from the field study at NLEX) inside the greenhouse and after the transfer of Q. erythrinae, the development and production of galls were observed.

The observed infestation of E. variegata var orientalis was started on the third week of September, 2006. First species of E. variegata var orientalis died on the second week of October in the same year. While the remaining species of E. variegata var orientalis completely died during the first week of February of 2007.

In E. variegata species, gall production was observed on the first week of October, 2006. Heavy infestations in all E. variegata were prominently exhibited in the month of February, 2007.

Meanwhile, in E. crista-galli, gall production was observed on the third week of January, 2007. Light infestation was observed in the month of February, 2007. Only two E. crista-galli were partially infested (refer to Table 6). Results of the treatment on the erythrina species and a variety were based on the observations of infestation and duration of the growth of the infested plants.

V. Field Study
Field observation and survey were made in selected areas of Regions 3, 4 and Metro Manila. For Region 3, some parts of Bulacan, Pampanga, Tarlac and Bataan were observed. For the Region 4, selected areas of Laguna and Batangas were studied. In Metro Manila, some places such as Macapagal Boulevard, Pasay City, Paranaque City, Edsa, Pasig City and Quezon City were observed.

Tremendous effects were observed among the Erythrina species and a variety (Table 7). Gall formations were evident particularly in the petioles and leaves of the plant. Wilting and yellowing of leaves, stunted growth of Erythrina and death trees were observed.

a. Effects of EGW on Field Observations
The collected data of healthy and infested from the survey study of healthy, infested and dead Erythrina trees in selected areas of Regions 3 and 4 and parts of Metro Manila were presented in Table 7.

b. Death and Infestation of the Three Erythrina Species in the Field
Field observation and survey were made in selected areas of Regions 3, 4 and Metro Manila. For Region 3, some parts of Bulacan, Pampanga, Tarlac and Bataan were observed. For the Region 4, selected areas of Laguna and Batangas were studied. In Metro Manila, some places such as Macapagal Boulevard, Pasay City, Paranaque City, Edsa, Pasig City and Quezon City were observed. Tremendous effects were observed among the Erythrina species. Gall formations were evident particularly in the petioles and leaves of the plant. Wilting and yellowing of leaves, stunted growth of Erythrina and death trees were observed.

c. Field Observations
There were 280 Erythrina trees died, 101 Erythrina trees were totally infested. Bearing of flowers by Erythrina variegata and Erythrina variegata var orientalis trees were not observed during flowering months. However, there were two trees of Erythrina crista-galli that produced flowers. Based on the field observations, of the selected areas being studied, there were still 8 trees of E. crista- galli which were healthy.As shown in Table 7, selected areas in Batangas had the highest percentage of dead Erythrina trees followed by Laguna and Bulacan. The highest infestation was observed in Bulacan followed by Laguna, Metro Manila and Batangas. Highest surviving Erythrina trees were found in Bulacan followed by Batangas. There were no healthy Erythrina trees in Metro Manila and Laguna.

VI. Validation of the Erythrina gall wasp
The adult female wasp lays its eggs into the parenchyma cells of differentiated tissues. The eggs hatch within 7 days and the larva starts feeding on the surrounding cells. The larva undergoes moulting ensconced in a chamber inside the gall eating the parenchyma tissues. As a result, cells multiply apparently triggered by the damage. The larva makes only one chamber where it feeds, grows and pupates. The pupal stage is spent in the same chamber. Adult wasps cement the lining of its chamber with its secretion to make it impervious to water. The adult emerges gnawing its way out into the open, leaving a gaping hole on the gall. This observation confirms the report of Heu et al., 2006 that the egg, larva and pupa stages of Q. erythrinae K. are spent inside the gall, ensconced in a chamber as the gall increases progressively in size and coalesce with adjacent galls.

VII. Phytochemical Analysis
Phytochemical tests were made on these Erythrina species on sterols, tripertenes, flavonoids, alkaloids, saponin, glycosides and tannins to be able to identify other factors that may contribute in the susceptibility among the species and a variety of Erythrina. Phytochemical analysis showed some similarities and differences on the three Erythrina species tested. Sterols are positive in E.variegata, E.variegata var orientalis and E. crista-galli. Flavonoids are present in E. variegata, E. variegata var orientalis and E. crista-galli. Alkaloid is present in E. variegata, E. variegata var orientalis and E. crista-galli. Tannin is present in E. variegata, E. variegata var orientalis and E. crista-galli. Triterpene is not present in E. variegata, E. variegata var orientalis and E. crista-galli (refer to Table 9). Saponin is present in E. variegata var orientalis and not present in E. variegata and E. crista-galli. Glycoside is present in E. variegata var orientalis and E. crista-galli, however, it is not present in E. variegata (refer to Table 9).

Phytochemical tests were done in Industrial Technology Development Institute in Department of Science and Technology. Aside from the tests done at the Department of Science and Technology, validation of tests were done in Thin Layer Chromatography was done in Research Center of the Natural Sciences in Thomas Aquinas Research Complex, University of Santo Tomas. There were two species used in the phytochemical analyses. These were the E. variegata and E. crista-galli. There was no available specimen of the E. variegata var orientalis.

The results of the study were the following. To test the presence of sterol, Libermann- Burchard test was used as spraying reagent. Both infested and non-infested E. variegata and E. crista-galli exhibited red color, it shows the existence of sterol. For the presence of triterpene, vanillin-sulfuric acid reagent was used as spraying reagent. After spraying, there is no pink, violet or red color for the infested E. variegata and E. crista-galli. However, in non-infested E. crista-galli, there is pink or violet color after the spray. For the test of flavonoids, NP-PEG reagent was used. After spraying, there was the yellow to orange colors found in both infested and non-infested E. variegata and E. crista-galli. Dragendorff’s reagent was used for testing the presence of alkaloid. The results exhibited orange color for both infested and non-infested E. variegata and E. crista-galli. Libermann- Burchard test was used for saponin. 

As a result, there is no saponin in infested and non-infested E. variegata and E. crista-galli. Kedde reagent was used to test glycoside. For the infested and non-infested E. variegata, glycoside is not present while in E. crista-galli, there is the presence of glycoside. For tannin, Ferric cyanide and Potassium Ferric Chloride reagents were used. There is the presence of blue color in infested and non-infested E. variegata and E. crista-galli (see Table 10).


SUMMARY, CONCLUSION AND RECOMMENDATION
Erythrina species and variety exhibited variations with respect to their susceptibility. Factors that validate susceptibility were gall sizes on petioles and leaves, nature and extent of damage by EGW which includes external characteristics of the galls, deformation, wilting yellowing and defoliation of leaves, bearing of flowers, cross sections of petioles and leaves, internal characteristics of galls, and death of species and a variety of Erythrina field study and observations and phytochemical analyses.

In the gall sizes on petioles and leaves, Erythrina crista-galli exhibited the least average size of galls. For the nature of damage by EGW, thin portions of parenchyma tissues in cross sections of petioles and leaves were observed in Erythrina crista-galli while thick portion of parenchyma tissues in cross sections of petioles and leaves were found in E. variegata and E. variegata var orientalis. There is the partial infestations occurred in E. crista-galli, heavy infestations occurred in E. variegata and death of E. variegata var orientalis were observed based on the production of galls among the species and a variety of Erythrina.

In the field study exhibited that identified numbers of E.variegata died due to EGW followed by E. variegata var orientalis and the least infestation occurred in Erythrina crista-galli.
It was validated that the same wasp infested the species and a variety of Erythrina in some parts covered by the study such as in Bulacan, Laguna, Batangas and Metro Manila. Aside from the cursory study, it was also validated in the infested species and a variety of Erythrina in the improvised nursery done in the study. Erythrina gall wasp allows numbers of wasp to lay eggs and penetrate the immature and young parts of the plants for its development and infestations. The study also revealed that EGW grows and develops in parenchymatous tissues of the plants particularly in petioles and leaves of the plants.

Phytochemical analyses exhibited no differences in infested species and a variety of Erythrina, however, there is the difference showed by the non-infested species of E. crista-galli having triterpene.

Validation of insect pest and plant species and variety was done in Philippine National Museum.
From the results of the experiment, the researcher has arrived at the following conclusions:

1. It is the same gall wasp (Q. erythrinae K.) that infests and causes galls in the two Erythrina species and a variety, thus proving the hypothesis of a single wasp parasite responsible in the formation of galls in the two host species and a variety.

2. Gall size significantly varies in the three species with E. variegata of having the biggest (5.54mm) and E. crista-galli the least (3.34 mm), which proves that there is a significant difference between and among the two Erythrina species and a variety as far as the size, morphology of the galls is concerned.

3. Of the two species of Erythrina and a variety, E. crista-galli is resistant, while the other a Erythrina species and a variety are susceptible to the parasitic wasp. There is no significant difference between E. var orientalis and E. variegata on their susceptibility to wasp parasite.

4. There is a difference on the presence of triterpenes found in species and variety of Erythrina. Triterpene is present in non-infested E. crista-galli while not absent in infested and non-infested E.variegata and in infested E.crista-galli.


RECOMMENDATIONS
Considering the limited scope of this study, the researcher recommends further studies specifically in the following areas of research:

1. Geographic distribution of the parasitic wasp in different parts of the country where the host-parasite relationship can be further studied, to include possible alternative host plants.

2. Resistance and susceptibility levels among standing Erythrina trees by species, and season.

3. Natural enemies of the parasitic wasp to include predators, parasites and pathogens with the purpose of developing a practical biological control method.

4. Genetic studies on trees as a basis in establishing a propagation program for Erythrina species.

5. Phytochemical analysis may serve as a basis for further research in
answering the infestations of EGW on Erythrina species.~

1 comment:

  1. it's good to see this article, I saw this gall effect in our areas in srilanka, it gives good answer to me

    ReplyDelete