THE PARASITOLOGY OF THE TOMATO FUSARIUM BY PAUL JONES BYRD A. B. WABASH COLLEGE 1917 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF ARTS IN BOTANY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS 1921 UNIVERSITY OF ILLINOIS THE GRADUATE SCHOOL _ 191 _ I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY P a ul J. By rd F.NTTTT ED Tl J P i t C I C "Y of th3 T ~ BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF '.tar of Art* Head of Department Recommendation concurred in* Committee on Final Examination* *Required for doctor’s degree but not for master’s / \ jr.iC 'TO . . * TABLE OF CONTENTS Page I. Introduction 1 A. The tomato wilt disease 1 1. Names i 2. Nature of the disease. 1 3. Diseases confused with tomato wilt.... 2 B. Distribution and importance 3 C. History 3 II. Methods 7 A. Inoculations 7 B. Preparation of diseased material for study. 8 III. Symptoms 10 A. External symptoms 10 B. Internal symptoms 11 IV. Correlation of symptoms with histology 12 A. Discoloration of parts. 12 B. Distribution of fungus in the vascular bundles 13 V. Conclusion Id VI, Bibliography. Digitized by the Internet Archive in 2016 https://archive.org/details/parasitologyoftoOObyrd I. Introduction The disease with which this paper deals is Known as tomato wilt, Fusarium wile, sleepy disease and blight. The name tomato wilt, since it is more descriptive, is more generally used. The causal organism is known as Fusar i um lycopersici Sacc. It belongs to a group of fungi of very similar morphological char- acters, such as those organisms that produce wilt of melons, cotton, cowpea and cabbage. Fusarium l ycopersici lives as a saprophyte in the soil, where it attacks the young roots of the tomato plant. It has not been definitely determined just where and how the fungus penetrates the roots, but discolored areas on the young roots ere easily seen and may perhaps be points at which the fungus enters. .After entering the young roots it finds its way to the large vessels of the f ibrovascular bundles and follows these into the stem where it grows rapidly upward. The fungus produces two types of conidia, the small single-celled, hyaline microspores and the large falcate, hyaline, septate macrcspores. In the latter there are usually three septa though there is often only one and sometimes as many as five. There are several other diseases of the tomato which . - 3 - are often mistaken for the tomato wilt, such as bacterial blight, early blight, Sclerotium wilt and mosaic. Bacterial blight is quite common in the tomato -growing areas of the South and is often found in the same field with the fusarial wilt with which it is quite frequently confused. However, with bacterial blight the leaves do not show the slow yellowing and wilting, but rather wilt quickly, and in a short time the whole plant is dead. In the case of the fusarial wilt it is the f ibrovascular bundles which become blackened, the pith and cortex remaining uninjured, while the bacterial blight organism attacks the pith and cortex as well as the vascular system. Early blight is a leaf disease causing definite black spots and is easy to distinguish. It becomes severe enough at times to cause defoliation. The interior of the stem is not blackened. Sclerotium wilt is a disease of the stem of the tomato plant at the surface of the ground. The cortex of the stem and the cambium layer are often rotted. The plants may wilt rapidly but there is no blackening of the internal tissues. An abun- dance of mycelium is usually produced in the affected area of the stem ana small sclerotia are also often developed. Mosaic is a disease of the leaves, causing a mottling or dwarfing. The plants are seldom killed, but appear unhealthy. The stem is unaffected. The fusarial wilt, according to Edgerton (b), occurs -o- &11 over the world though it is more abundant in the warmer re- gions. In the United States the disease has been reported by workers generally in the southern states and less frequently in the northern tier of states. It has been reported in the Plant Disease Survey Bulletin of the U. S. Department of Agriculture as occurring with more or less severity, depending on the weather conditions, in all of the northern states east of the Mississippi River. For instance, it ha 3 been reported as general in Indiana for 1916 and 191?, and as being severe in 1913. In Illinois it has been reported as local for 1911 and 1912 and as general and severe in 1917 . It is said to cause much damage in Pennsylvania every year. The greatest damage, however, occurs in the Gulf States. In 1919 Georgia lost 40 $ of its entire crop; a loss of 30$ is reported from Texas; Mississippi lost 15$; Louisiana and Alabama each lost 25$ of their entire crops; the same year Ohio had 10$ of its crop destroyed, Illinois 4$ and Indiana 3$. The loss for a total of 23 states for the year 1919 was 4£$ of the crop. In the Southwest the tomato crop is injured to some extent by the wilt disease. Kansas lost 8$ of its crop, Missouri 5$, Oklahoma 3$ and California 3$ in 1919. The importance of the tomato wilt is clearly shown by the above figures. The literature upon tomato wilt is rather scattering. Although the disease has been known for some time, prior to 1916 little was published except notes on its occurrence. The name Fusariu m lycop er sic i was first given by Sac- - • -4- oardo (6) in 1882 to a fungus which he studied growing on tomato fruits. The fungus which causes tomato wilt is not usually found on decaying fruit, although Wollenweber (10) reports having suc- cessfully inoculated the fruit. Most of the investigations have been carried on within the last ten or twelve years, breeding for resistant varieties being especially considered. Norton (7) was one of the first to work with resistant varieties while Essary (4), Edgerton and Moreland (3), Humbert (5), Burst (2) and others have contributed to this phase of the work, Wollenweber (10) and Sherbakoff (9) have determined the morphological and physiological differences in the genus Fusarium. Massee (6) in 1895 was one of the first to publish anything of a technical nature on the disease, giving a short account of the life history of the fungus, and of two stages of the organism. The question of the effect of certain chemicel substances in the soil on the development of the tomato wilt fungus has beer- studied at the Louisiana experiment station (3). The results shew that corrosive sublimate when added in large quantities, about 335 pounds to the acre, will eliminate the organism. How- ever, this substance when present in the soil in such quantities greatly interferes with the germination of the tomato seed. Lime and formaldehyde treatment of the soil have been shown to give the best results for controlling the fungus. It was found ' - ’ . experimentally that there was a decrease in the amount of the wilt with an increase in the amount of lime and the fungus seemed to be delayed in its development though it was not entirely eradi- cated from the soil, even with the heaviest application of lime. Wilt has developed on plants growing in soil known to be free of the wilt organism at the time the plants were set out. / case of this kind suggested the idea that the seed could carry the spores of Fusarium lycoperaic i , and by experiment (3) it was proved that plants may become infected from seed which carry the wilt spores. Tomato seeds which were aseptic were trea.ted with a spore suspension, dried and allowed to remain in a dry place for a year, after which time they were plant ed in sterile soil. The plants coming from unese seeds showed typical wilt symptoms while the checks were free from the disease. There is not much known about the activities of the wilt organism in the soil. It is quite possible that it can live for at least two years in a soil where there are no tomatoes grown. However, because a field develops wilt after it has been planted in some other crop for several years does not mean that the Fusarium organism has remained viable in the soil all this time. There are a greet number of ways that a field might be- come infected. The organism might be brought in from other fields in surface water, or the spores ma}' be blown in from neighboring, infested fields. .Again a great many fields become infested from plants which have been grown in infested beds. . • . - 6 - One of the first cases of the disease known in the State of Indiana was traced to infested seed beds in Florida. A knowledge of the effect of varying temperatures on an organism is very essential when a study of its parasitology is being made. The fusarial wilt organism has been found to have an optimum temperature of about 28°, a maximum temperature of 3? c , and a minimum below 14° (3). It is interesting to note that the highest infection percentage results when the soil tern- perature remains close to the optimum for the growth of fungus. The present invest igat ions have been carried on with the hope of determining the correlation between the external and internal symptoms of the fusarial wilt. i II . Methods Pure cultures of Fusarium lycoperslci growing on prune juice media were received from R. E. Smith of the California Agricultural Experiment Station. Pure cultures of the organism Y*ere also received from E. E, Clayton of the Ohio Agricultural Experiment Station. These cultures were kept alive during the experiments by transferring every few weeks to fresh potato pep- tone media. Pure cultures were also grown on washed and corn- meal agar. The fungus, however, did not grow well on these media and they were finally discarded. A. large number of stems of tomato plants which had been killed by the Fusarium organism were received from the Tennessee Agricultural Experiment Station. These stems, which were per- fectly dry, were ground in a food chopper. The resulting powder and fragments were thoroughly mixed with the upper soil of the seed beds in the green- house. Tomato seed of the Ponderosa variety were then sown in this soil. Plants were also started in small flats and when they grew to be four or five inches tall they were transplanted into the inoculated soil in the seed bed. These plants showed the first symptoms of the disease at the same time as did those that were started in the diseased soil. In order to learn whether the Fusarium would grow on the surface of tomato plants, a number of as#eptic seeds were . . ' . * * . . . '■ ; . -i - 8 - sown in a sterile moist chamber on filter paper and allowed to germinate. The seedlings were inoculated by atomizing with a heavy spore suspension of the wilt fungus. These moist chambers were kept in a warm place and the seedlings studied from time to time as the fungus developed. To ascertain the distribution of the wilt organism within the host a plant about 60 c.m. tall, which showed typical wilt symptoms, w as removed from the green-house and taken to the laboratory. Here an outline diagram was made of the stem and branches, divided into numbered regions. Samples from each region were then sectioned with the hand microtome; dehydrated and decolorized in alcohol, cleared in xylol and mounted perman- ently in balsam. By arranging these mounts in sequence accord- ing to the diagram it is possible to study the distribution of the fungus within the host . Free hand sections of the diseased regions were also made and treated with the differential stain, Pianese III.B. The mounts of this material show the mycelium of the organism in the large vessels of the fibro vascular bundles. (Plate I.) A diseased tomato plant was taken from the green- house and placed in 95 $ alcohol and allowed to remain for three weeks, after which time it was placed in 100$ alcohol for two weeks. This process dehydrated and decolorized the tissue thoroughly. The specimen was then cleared by placing it in xylol. /fter a few days the discolored vascular bundles could be traced from . . . y* • . The darkened bundles were _c _ the bottom of the stem to the rop. clearly visible in the petioles of the leaves. Plate II. shows a portion of the plant after it was subjected to the above treat ment . - 10 - III. Symptoms The wilt disease may occur on tomato plants in any stage of their development. In the green-house it becomes most noticeable about blossoming time. The disease first shows externally in the lower leaves. The tips of these leaves first turn yellow and this yellowing gradually spreads over all the leaf. The leaves then soon wilt and die. fls the disease progresses upward through the main stem more of the leaves turn yellow and dry up and finally the 7/hole plant may die. The time intervening between the first appearance of the disease and the death of the plant varies, due perhaps to resistance in the plant or to other factors not known. Under green-house conditions but few fruits set on diseased plants. In cases where fruits do set they remain small and ripen prematurely. Some plants become 30 badly in- fected that they do net grow to be very large, succumbing rapid- ly. On the other hand some plants do not show external signs of infection until they are from four to six weeks old and suc- cumb slowly. The root 8, as well as the other parts of the plant, show external signs of the disease. They appear yellow and in the case of the smaller ones often turn black and rot. If a diseased plant is palled up the ends of the primary roots and « - 11 - many of the secondary roots break. The internal appearance of an infected plant is very characteristic. The best method of diagnosis of this disease is to cut the stem of the diseased plant and observe the dis- coloration of the tissue. In the stem the fungus confines it- self to the f ibrovascular bundles which are turned a brownish color. In the case of the roots all of the tissues are dis- colored. The leaves which have turned yellow show discolored veins. This discoloration is continuous from the vascular bundles of the stem out into the petiole and blade of the leaf. In a young plant which is infected the darkened vascular system may be seen clearly by holding the plant up to the light. - 12 - IV. Correlation of Symptoms with Histology The roots of seedlings grown in a moist chamber on filter paper and inoculated with the wilt organism shewed a general browning five days after inoculation. By microscopic study the internal tissues of these roots were also found to show this general browning. The mycelium of the causal organ- ism was found in abundance in the cells of the cortical tissue (Plate III.). This general browning of the internal tissues and cortex was also found to be characteristic of the roots of diseased plants grown in soil in the green-house. - .As soon as the fungus reaches the region of the stem just above the ground it confines itself to the f ibrovascular bundles. The leaves of a diseased plant do not wilt in the order of their height, as the fungus progresses up the stem. The lowest leaves may first show signs of wilting while the next to wilt may be a leaf near the top of the stem (Plate IV.). About two or three weeks after the plant becomes in- fected, or after the lower leaves are all dead, the stem near the ground loses its green color and has a brownish tinge. This color, which gradually becomes darker a3 the plant becomes older, advances up the stem as the fungus inside advances. The whole plant, however, does not show this general discoloration until some time after the last leaf has wilted and died. (Plate V.) * - -13- Fibrovascular bundles in which the mycelial 6trands of Fusarium lycopersici grow are turned a dark brown color. This characteristic discoloration is not only seen in the vessels in which the mycelium is growing but can be seen in the bundles just ahead of the advancing fungus. This discoloration is perhaps due to some toxic substance or enzyme secreted by the fungus. The wilting and final death of the plant may be due to such sub- stance, and not to the cutting off of the water supply by the plugging of the vessels by the mycelium of the fungus, as is generally thought to be the case. Of the numerous sections of diseased stems that have been studied only a very few have shown the water-conducting vessels to be entirely plugged by the fungus. The mycelium in the vascular system does not make the same upward progress in each of the bundles. Plate VI. shows outlines of sections of a diseased tomato stem taken from the regions indicated on the diagram of the whole plant in Plate VII. The relative degree of blackening in each bundle is shown by the shading. In the primary root, section P, all of the fibrovas- cular bundles showed a general discoloration, which is rather to be expected since the entire cortex of the root was discolored. Bundle No. 1, Plate VI ., was found to be discolored almost the entire length of the stem. Bundles No. 2 and 3 show the characteristic blackening to within about 7 cm. of the top of the stem. The discoloration in bundle No. 4 extended to within about 9 cm. of the 'top of the stem, while bundle No. 5 was -14- dark ened only to about 9 cm. above the ground. The petioles of the leaves which showed typical wilting caused by the fungus, as well as those which remained normal, were sectioned, and a study of their vascular tissue made. Sections No. 1, 3, 5 and 8 are of petioles the leaves of which shewed ex- ternal signs of wilting, and, as shown, Plate VI., all of the vascular bundles were blackened. Sections No. 8 and 9 are of petioles the leaves of which externally appeared normal. In this case the bundles were not the least discolored. We &s,y say that there is no wilting unless the vascular bundles have the typical discoloration. It might be added that a number of stems and petioles, the leaves of which externally did not appear to be infected, as well as number which showed typical infec- tion symptoms have been sectioned during the course of these observations and that the above statement applies in every case. - 15 - ] V. Conclusion The attack of the fusarial organism upon the tomato plant is confined to certain tissues, namely the cortex and f ibrovascuier bundles of the roots and the f iforovascular bundles of the stem. The mycelium in the cortex of the root gees from cell to cell passing through the walls. A plant which shows external 3igns of wilting is always found to have discolored vascular bundles. /II the bundles may have this discoloration at the surface of the ground, while a few centimeters up the stem only one or two show the darkening. In some diseased plants a discolored bundle may run the full length of the plant . The blackening of the bundles is not confined to the portion of the bundle in which the mycelium is growing, but is evident in vessels perceptibly in advance of the mycelium. The stem of an infected plant assumes a brownish color- a few weeks after the wilting of the leaves. This external browning of the stem is always associated with the typical in- ternal infection. The tissues of the roots of a diseased plant show dis- coloration internally as well as externally. The ends of in- fected root 6 quite often show marked rotting. ' VI. Bibliography (1) Clayton, E. C. The Relation of Soil Temperature to the Development of the Tomato Fusarium Wilt. (Abs.) Phyto- path. 10: 63-64. 1820. (2) Durst, C. E. Tomato Selection for Fusarium Resistance. (Pta.) Phytopath. 8: 85. 1918. (3) Edgerton, C. W., and Moreland, C. C. Tomato Wilt. Louisi- ana Agr. Exp. St a. Bull. 174. 1820. (4) Essary, S. H. Notes on Tomato Diseases with Results of Selection for Resistance. Tennessee Agr. Exp. Sta. Bull. 95. 1812. (5) Humbert, -J. 0. Tomato Diseases in Ohio. Ohio P gr . Exp. Sta. Bull. 321. 1918. (6) Masses, 0. P Disease of Tomatoes. Gardener's Chronicle. Ser. 3, 17: 707-708. 1895. (7) Norton, J. B. S. Tomato Diseases. Maryland £g r. Exp. Sts. Bull. 180: 102-114. 1914. (8) Saccsrdo, P. P . Sylloge Fungorum Omnium Hucusque Cognitorum. 4: 705. Padua. 1886. (9) Sherb&koff, 0. D. Fusaria of Potatoes. Cornell Univ. P gr. Exp. Sta. Memoirs. 6. 1915. (10) Wollenweber, H. W. Parasitic Wilt Diseases of Cultivated Plants Due to Fungi. Ber. Deutsch. Bot. Gesells. 31: 17-34. 18±3 . - . . Explanation of Plates. Plate I ■ Fig. A. Photomicrograph of a section, at region G, of the stem represented in Plate VII., showing the mycelial 3trands of Fusarium lycopersici in the large vessels of the fibrovas- cular bundles. Fig. B. Photomicrograph of a section, at region B, of the same stem, showing the mycelial strands scattered in the large ducts of the bundles. Plate II. A section of a diseased tomato stem decolorized and dehydrated showing the discolored vascular bundles. Plate III. Drawing showing the mycelium of the wilt organism in the cortical cells of the root. Plate IV. A diseased tomato plant showing the wilted leaves on one side of the stem. Plate V. A tomato plant killed by the wilt organism. Plate VI. croso sections oi tne tomato stem made at regions shown in Plate VII., showing the relative amount of blackening in each bundle. PL4TE II PL/TE III. PLATE IV.