UNIVERSITY OF CALIFORNIA AGRICULTURAL EXPERIMENT STATION COLLEGE OF AGRICULTURE benj. ide wheeler, president THOMAS FORSYTH HUNT. Dean and Director BERKELEY CIRCULAR No. 85 (February, 1913) A PRELIMINARY REPORT OF A NEMATODE OBSERVED ON CITRUS ROOTS AND ITS POSSIBLE RELATION WITH THE MOTTLED APPEARANCE OF CITRUS TREES BY E. E. THOMAS UNIVERSITY OF CALIFORNIA PRESS BERKELEY INTRODUCTION The citrus growers have been much concerned in recent years over a serious trouble of a very obscure nature in their groves. It has attracted attention particularly in the older sections. One of the principal symptoms is a light mottling of the leaves — it is probable that this symptom is only one of the characteristics of the trouble. There have been many explanations offered for this disease, or, as it has generally been considered, this unhealthy condition, which is of so indefinite a nature as hardly to be called, with propriety, a single definite disease. People, competent to offer an opinion, have stated to me their belief that this trouble, generally called Mottled Leaf, is by all odds the most serious one concerning citrus trees in California. Mr. Thomas, in this paper, suggests a possible cause for this difficulty. As to whether the nematodes he mentions really constitute the sole or principal factor in mottled leaf remains to be proven in a final way. The fact which stands out from Mr. Thomas' work is that a new nematode, probably new to science and certainly new in its relation to the orange tree, has been discovered. The certainty that these nematodes penetrate deeply into the tissue of the feeding roots and that they are present in immense numbers is sufficient evidence that they must injure the trees, also their similarity to the destructive nematode of the beet is suggestive. It is probable we have before us here a very difficult problem and one which will take a long time and much original work to solve properly. The first step will be to demonstrate the precise relation between the nematodes and the unhealthy condition of the orange trees. This in itself will require several years, in my judgment. The next problem will be to study the life history and habits of these nematodes and their resistance to various agencies which can be brought against them. The preliminary investigation recorded in this paper was made by Mr. Thomas in connection with a study of soil texture and soil moisture in relation to mottled leaf. Data had accumulated at the AVhittier laboratory which suggested that these two factors had something to do with the trouble, and the work of making systematic and accurate observations had been assigned to Mr. Thomas. The following state- ment concerning nematodes and their possible relation to mottled leaf is given out so that other investigators may have their attention called to this apparently most important of all our citrus problems. Wm. T. Horne, Acting Chief Division of Plant Pathology. A PRELIMINARY REPORT OF A NEMATODE OBSERVED ON CITRUS ROOTS AND ITS POSSIBLE RELATION WITH THE MOTTLED APPEARANCE OF CITRUS TREES* By E. E. THOMAS SUMMARY 1. A certain very obscure disease of slow or retarded development of citrus trees exists in California. The most characteristic symptom of this disease is the yellowing of the leaves between tne veins, with the green color only along the mid-rib and the lateral veins. 2. A particular kind of nematode which has not before been de- scribed as occurring on citrus trees has been been found associated with this disease. Great numbers of these nematodes pierce the smal] rootlets, especially of badly diseased trees. 3. Although this is but a preliminary report of the existence of nematodes on citrus trees, yet it shows that they may be an important factor in the growth and development of these trees and justifies a thorough and comprehensive study to determine the existing relation. Mr. J. R. Hodges, Horticultural Inspector, of Covina, California, has found nematodes in the soil scraped from the roots of trees with badly mottled leaves. He examined some of the diseased trees in his district and came to the conclusion that the nematodes injured the roots and were the cause of the disease. Twice previously nematodes have been reported in connection with citrus trees. *Acknowledgments. — In presenting this article, the writei wishes to express has most sincere appreciation of the hearty cooperation of Mr. D. H. White of Whittier, the Covina Valley Farmers ' Club, and the National Orange Company, of Eiverside. Mr. A. G. Schulz, of Porterville, has also given his assistance at different times. Acknowledgment is here given of the work of J. H. Norton, formerly of the Citrus Experiment Station and W. M. Mertz, of the Citrus Experiment Station, who conducted the fertilizer experiments. Special mention is also due Howard S. Fawcett, Pathologist of the State Horticultural Commis- sion. Mr. Fawcett not only collected some of the data, but otherwise materially aided in the work. The common injurious nematode {Heterodera radicicola) pene- trates the roots of various kinds of plants, causing galls or swellings to form where it enters, and reduces the vitality of the plant attacked. In 1889 these knots were reported on citrus roots by J. C. Neal,* who states that the orange seedlings are affected and "when the tender shoots first appear many wither and die at once, others grow vigorously till the end of the first season when they are usually budded with known and valuable varieties of fruit. The next spring these buds put out tardily and make a weak growth, the leaves become spotted or yellow, then drop, the bud dies, feeble, straggling shoots sprout around the stem which maintain a sickly vitality till the first drought, when the tree dies, and on examination discloses the cause in the knotty, decaying roots, without rootlets or fibrillae." Nematodes have also been reported on citrus trees by Gaston Lavergne.f They were found on lemon trees which were very badly injured by an excessive amount of water. These trees were planted very close together and the ground was never touched by the sunshine. This made it impossible for the soil to dry out or become aerated. The nematodes were Anguilulae similar to Heterodera radicicola but some- thing like ten times larger. Small knots were found on the roots and in these the young nematodes developed. Observing that the mottled trees were found on heavy soil as well as on sandy soil, and that experiments with irrigation and fertilizers did not in all cases have beneficial effects upon the trees, the writer has carried on an investigation in different districts affected with the disease to see if the nematodes are always found in connection with it. An examination has been made in the following counties : San Diego, Orange, San Bernardino, Riverside, Los Angeles, Tulare, Fresno and Butte. These represent the most southern citrus district of California, the large citrus belt around Los Angeles, the early orange section in Tulare County, and one of the northern districts. Groves affected with the disease were found on varying types of soil. A few of the fibrous roots of citrus trees were taken and examined under the microscope for nematodes. These samples were taken not only from badly diseased trees, but also from trees slightly affected and from good trees as well. * United States Department of Agriculture, Bulletin 20, Division of Ento- mology, page 12. fPublicationes de la Estacion de Patolojia Vejetal de Chile, Num. 9 (Febrero de 1901). A tabulation of the results follows : No. of samples examined 50 Trees Valencia Root Stock Not known Type of Soil Sandy Condition of Tree Badly mottled Nematode of Roots Numerous 5 Valencia Sour Sandy Badly mottled Numerous 10 Valencia Sweet Sandy Badly mottled Numerous 15 Valencia Not known Heavy loam Badly mottled Numerous 5 Pomelo Not known Heavy loam Badly mottled Numerous 5 Navel Trifoliata Sandy loam Badly mottled Numerous 160 Navel Not known Sandy loam Badly mottled Numerous 15 Navel Sour Sandy loam Badly mottled Numerous 5 Navel Sweet Sandy loam Badly mottled Numeroas 22 Navel Not known Heavy loam Badly mottled Numerous 2 Navel Not known Sandy loam Badly mottled Not found 4 Lemon Not known Sandy loam Badly mottled Found numerous 2 Lemon Sweet Heavy loam Badly mottled Found numerous 55 Valencia Not known Heavy loam Good Not found 30 Valencia Not known Sandy Good Not found 1 Valencia Sweet Heavy loam Good Found in large numbers 4 Navel Not known Sandy loam Good Few found 75 Navel Not known Sandy loam Good Not found 55 Navel Not known Heavy loam Good Not found 15 Navel Sour Heavy loam Good Not found 17 Lemon Not known Sandy loam Good Not found 15 Lemon Not known Heavy loam Good Not found We note that nematodes are not confined to one locality, but exist around mottled trees in all of the districts, and that out of the 300 samples examined but two exceptions occur. In all the samples where the nematodes were not found, a much closer observation was made than where they were found, and with the result above tabulated. In a few cases nematodes were observed around good trees, but with one or two exceptions they were in small numbers in comparison with those around the diseased trees in the same grove. If they are dis- covered around a few of the trees in a grove it is to be expected that they may occur on the surrounding trees, as they could easily be spread with the irrigating water, rains, the cultivator, etc., while they may not yet have become numerous enough, or have been present long enough for the trees to show the effect of their attack. In a great many cases badly diseased groves with abundant nematodes were seem adjacent to healthy groves in which ho nematodes could be detected. Our observations show that in the few instances where nematodes are found in apparently healthy groves, the worms are not at all prevalent as compared with the diseased groves. A preliminary study was made to learn the exact relation which the worm bears to the root tissue. One point stands out very clearly, and that is the fact that the roots do not develop galls or knots, so far as observed by us. (Figs. 1, 2, 3, 4, 5, 6.) This is, therefore, different from the cases noted by J. C. Neal and Gaston Lavergne, in which they state that galls or knots were found on the roots. Fig. 1. A small orange rootlet highly magnified, showing a group of nematodes, somewhat out of focus above at the left, and a single one at the right. Nematodes sometimes damage the root of the host directly, not only by crawling inside the tissues and causing knots, but also by piercing the root with the mouth parts and feeding upon it. On close examina- tion of our specimens it was found that many of the nematodes had pierced the root and pushed their way into the tissues for quite a distance. In some cases the head of the nematode was seen to have penetrated six cells into the root (Fig. 8, also 3, 5 and 6). Upon further examination it is found that the number of nematodes which pierce a rootlet is very great. In some cases as many as fifty have been counted on a small rootlet one inch long. The females are not always attached to the root, but may be in the soil surrounding it and seem to be enveloped in a gelatinous or slimy material. A number are generally to be found together with this gelatinous material surrounding the whole group. As many as twenty Fig. 2. Two nematodes on a small orange rootlet highly magnified. These are viewed from such an angle that they appear symmetrical. Usually the tail appears as obliquely placed. females have been noticed attached to the root in one group. This bunching or grouping not only occurs with the females but is also noticeable with the males, for they are very often found in groups of from six to eight or more, piercing the root close together. Many groups were also seen in which there were > both males and females, with the anterior portion of the body imbedded in the tissues of the root. Cross sections of the roots were made and the nematodes were seen penetrating the tissue. In observations on thirty-two of the worst deteriorated orange groves in one of the oldest districts in the state, nematodes were found to be very numerous on every sample. The many samples examined from each tree showed the nematodes under the microscope immedi- ately, although these were only little pieces of the rootlet one inch long or even smaller. Since they were noted in such great numbers Fig. 3. Transverse section of a citrus rootlet highly magnified, showing a nematode piercing the surface and penetrating deeply. The head of the nematode has penetrated nearly to the point of the arrow. on the tiny rootlets, one would conclude that there must be millions of nematodes in and around the root system of one tree. These diseased groves examined are as bad as any in the entire state. In most cases they have been large producers in the past, but at the present time they are giving very little fruit that is at all marketable and this is of an inferior quality. In most cases the trees thus badly diseased have only small sickly yellow fruit which is the typical condition of badly mottled, deteriorated trees. The principal symptoms which have generally been recognized in connection with mottled leaf or "decadence," as it is popularly called, are that the tree fails to develop normally, but grows tall and slender. The leaves are small and of a sickly yellow color, or they may be mottled, the veins remaining green while the portion between is yellow (Fig. 7). Trees only slightly affected bloom normally and Fig. 4. Orange rootlet highly magnified, showing a nematode near the center of the field. 10 produce good fruit, while more seriously diseased trees often bloom profusely and produce small fruit of a pale yellow color. Such trees are unprofitable. Sometimes only a portion of a tree is affected. In a very large percent of cases the southeast side of the tree has been the most seriously affected. Mottled leaf is found mostly on sandy soil. Many instances have been noted where there is a sandy streak running through a grove, and here the diseased trees occur, while the rest of the grove which is on heavier soil may be in a healthy condition. The moisture content of sandy soil varies much more than that of heavy soil in the same Fig. 5. Transverse section or orange rootlet highly magnified, showing two females and one slender male nematode between them. grove, and too great fluctuation of the moisture content of the soil was thought possibly to cause the trouble. Experiments have been carried out increasing the frequency of irrigations, with careful de- terminations of the soil moisture. In some instances some improve- ment was noted, in others not. Sandy soil is also supposed to furnish 11 the best conditions for the growth and spread of nematodes, provided the soil is kept in good condition as regards moisture, etc. This is the case in an orange grove, or in other words, what would make the best conditions for the growth of the orange would be favorable for the nematode as well. On the other hand, the disease is not very often found on heavy black soil, but a few such cases have been noted and in each case nematodes have been found upon the roots, in large quantities except in one instance. Fig. 6. Transverse section of an orange rootlet highly magnified. By stain- ing with eosin the course of the anterior part of the body was clearly shown. After entering the root the worm bent strongly toward the upper side of the picture and again strongly inward so as to be nearly in line with the arrow, and penetrated nearly to its point. The other two arrows indicate nematodes of which the posterior part of the body was lost in preparing the section. One of the trees showing mottled leaf in heavy black adobe was of especial interest. It stood in the lowest part of a ten-acre grove where the water gathered at every irrigation from the whole tract, so that the tree was in water-logged soil about half of the time. In and 12 around the roots of this tree the nematodes were found in greater numbers than in any other locality examined. In several cases the owners have done everything they could to improve the trees, trying different systems of pruning, not letting the trees dry out, using commercial fertilizers liberally, and putting on several good dressings of stable manure, but all apparently to no purpose, for the trees continue to get worse each year. The trees in these groves range in age from ten to twenty-five years. Practically none of the younger groves are bearing in the same way as these older groves did when they were at the same period of growth. The soil in Fig. 7. Mottled leaf. Healthy leaves and oranges at the left and typical mottled leaves and small fruits at the right. these groves varies from a very light sand to a sandy loam with a clay subsoil. In one instance the fertilizer was applied in maximum quantities and a large number of different substances were used and all the possible combinations of them. They were applied every week and the results carefully noted. The trees chosen for the experiment were typical of the section in which they were found, and two and one 13 half years after the experiment was begun no improvement in the mottled condition could be noted in any case by comparison with the surrounding trees. In some instances it is said lime has had a beneficial effect, but the trouble has been found in its worst form where there is an excess of lime. Temporary improvement is noted in the summer, but the writer and others have observed that the mottled leaf shows much worse in the winter months. The nematodes have not only been found on old deteriorated trees but have also been seen on the roots of nursery stock, the leaves of which are mottled. Doubtless, therefore, they have been carried from one locality to another in the soil of the balls surrounding the roots of affected nursery trees. Aside from the distribution of nematodes by infected nursery stock, there are many other ways by which they can be carried from one place to another. The irrigation water is an easy means of scat- tering them, also the cultivator, and they may be in the soil which adheres to the wheels of a wagon or the feet of animals and in these ways be spread from one part of the grove to another, and to other groves in the vicinity. Description of the Nematodes As yet only a very preliminary and incomplete study has been made of these nematodes, but the following characteristics have been observed : Fig. 8. Drawing of a section of an orange rootlet, showing a nematode penetrating to near the point of the arrow. The posterior part of the worm was destroyed in cutting the section. 14 Both the male and female nematode* have a spear which aids them in piercing the root {Fig. 8). This spear varies in length from 7 ^ to 17 fx. At the base of the spear is found the slender eosophagus. No transverse striae or hairy appendages have been noted. The male nematode (Fig. 5) has a length varying from 280 p to 420 ^ and the average length of fifty specimens examined is 340 /x. These specimens were taken from four localities. At the point of greatest diameter it measures from 10 /x to 17/x, with an average of 14 jx. The tail is long and tapers to a point. Many samples of rootlets having the anterior portion of male nematodes imbedded in their tissue were placed in water and examined under the microscope. In each case the rootlet had been carefully cleaned with a brush to remove the soil particles. This was done so that the nematodes which remained could be examined more closely. The posterior portion of the nematodes thus found was seen to move, showing that they were still alive. The male nematode while frequently found with the anterior portion of the body imbedded in the tissue of the root, more often occurs in a free state in the soil surrounding the root. The female increases in thickness until when mature the main portion of the body is irregularly elliptical and not quite symmetrical, the posterior portion narrowing abruptly into a slender obliquely placed tail. The adult female is of a brownish color. The posterior portion of the female is on the outside of the root and the eggs are evidently deposited outside the root tissues. Egg laying has not been observed.