Growth reduction was 16 percent on the Troyer seedlings, 42 percent on the sour orange seedlings, 55 percent on the Cleopatra seedlings, 44 percent on the navel/Cleopatra combination, and 48 percent on the navel/sour combination. The final experiment consisted of seedlings of four root stock cultivars, namely sour orange, Cleopatra mandarin, Troyer citrange, and trifoliate orange, and those root stocks budded to navel orange. They were grown in an unclassified virgin soil from the nearby San Bernardino mountains. Growth reduction of the second crop varied from 5-92 percent. With the exception of Cleopatra mandarin, the addition of the navel orange scion increased the growth retarding effect of the first crop on the second. The combination of navel orange on Troyer citrange root caused the greatest reduction of the second crop and Rubidoux trifoliate orange seedlings the least. Relative growth of Rubidoux trifoliate orange seedlings and navel orange on the trifoliate orange seedlings was best, and that of the Cleopatra mandarin seedlings and the navel orange trees on Cleopatra root stock was poorest. The author will reemphasize that the citrus nematode was not present. Martin and Bitters made no attempt to extract and identify any toxic substances from these pot cultures. In South Africa, Burger and Small did some very intensive research on allelopathy in citrus orchards. Based on the fact that citrus growing in South Africa was basically a monoculture on Rough lemon root stock, they made extracts from orchard soils in which citrus trees had been grown for up to 30 years in various citrus areas of the country. The extracted samples were tested for toxicity to Rough lemon roots using a bio-assay method with very young seedlings.

Of 24 phenolic compounds which are known to occur in citrus or in decomposing organic matter, they concluded that homovanillic acid, a relatively rare phenolic acid in higher plants,vertical grow system was the major toxin present in the citrus soil extracts. However, phioretic acid, o-cournaric acid and coumarin also cause some reduced growth. A comparison of the phytotoxicity of fresh roots, and partly decomposed root and soil from a 30-year-old citrus orchard sampled at different depths, and an adjacent virgin soil, showed that only the extract from the virgin soil did not show any significant reduction of the seedlings in the bio-assay procedure. There was little effect from the fresh root extracts. However, no growth occurred with the same extract fraction of partly decomposed root residues. This observation, state Burger and Small, supports the supposition that phytotoxins are found during the decomposition of citrus roots. They also investigated the relationship between soil depth and the accumulation of the phytotoxic compounds. From an orchard at the Sundays River Research Station , they collected soil samples at four depths: 0-25, 25-50, 50-75, and 75-100 cm. Their studies showed that the extract of the 0-25 cm sample caused the greatest growth reduction and the 25-50 cm sample the least reduction, but with gradually increasing toxicity at increasing depths. They felt that phytotoxic substances might accumulate in certain soil due to poor aeration and the absorbent properties of certain soil particles. No extracts were made from other than soils of Rough lemon orchards and no phytotoxic bio-assays were run other than on Rough lemon roots. In addition to the bio-assay studies reported by Burger and Small , extensive replant studies are reported by Burger . However, in these later studies, it is not clear cut as to what effects are caused by allelopathy and those caused by nematodes, or both. Even in some of the fumigation experiments the fumigation was not adequate, or effective enough, to kill all the nematodes, so the experiments were confounded by variable nematode populations. It did appear that deep plowing to a depth of 60 cm was helpful in either case. The response of the root stock seedlings subjected to the treatments is not clearly delineated.

It is somewhat peculiar that so little attention has been directed to allelopathy in the citrus growing areas of the world. There is no reason to believe the problem exists only in California and South Africa, although the environmental conditions are very similar. The toxic substances involved are apparently biodegradable and do not last long in the soil as compared to a nematode infestation . The effect of the toxic substances is thus one of retardation rather than a permanent effect. In the author’s many trips to Japan, I occasionally visited old Satsuma orchards which had been cultivated by one family for many generations. When I would inquire about a replant problem, the owners would respond with “Do you see any?” The trees were often of variable age, but all appeared to be healthy and productive. One must remember, however, that the principle root stock in Japan is the trifoliate orange. This root stock cultivar inherently has considerable resistance to the citrus nematode. Also, the studies of Martin and Bitters showed that growth retardation following trifoliate orange was the least of the seedlings studied. Burger reports a similar effect in South Africa. Furthermore, Burger found that Rough lemon root residues caused a highly significant reduction in shoot growth and internode length. According to Monselise , a reduction of internode length in the shoot is an indication of the inhibition of gibberellic acid activity in the shoots. In Burger’s studies, neither the presence of nematode, nor trifoliate orange root residues, had any effect on shoot growth and internode length. Martin reported that the inhibition of P, Cu, and Zn absorption in citrus follows soil applications of fumigants, fungicides, heat and other treatments which kill soil organisms. This condition most commonly has occurred in greenhouses and nursery operations following heat or steam treatment of soils or the use of soil fumigants. The condition was never predictable and was often spotty. Martin further stated that when citrus seeds or young citrus seedlings were planted in the treated soil, one to several progressively poorer growth flushes occurred. The newer leaves remain smaller and turn a pale green to yellowish color as described by Martin, Baines, and Page and Martin et al. . Depending on the soil type, the plants may develop moderate to severe deficiency symptoms of Cu, Zn and necrosis of leaf edges. Root growth is severely retarded, but the root appearance is healthy. The plants are stunted.

The deficiency symptoms may last from a few weeks to a year and eventually the plants recover and resume normal healthy growth. Phosphorus levels were low in affected plants. About 1970, the California State Department of Agriculture adopted a requirement that nursery trees and other plants for sale or distribution be free of specified root parasites and noxious weeds. This action, plus the tristeza quarantine,plant pot with drainage necessitated fumigation of citrus seed bed and nursery sites. Rather high dosages of fumigants were used to kill nematodes, parasitic fungi, and in some cases, noxious weeds. Hindered by these restrictions, California nurserymen could no longer afford to be nomads—the practice of planting one nursery site and then moving onto new ground after the trees were grown. Most of the nurserymen thus selected and developed a permanent site. After the first nursery, fumigation treatments were necessary. In a number of citrus seed bed and nursery sites, phytotoxic problems were encountered. Initially, Martin, Baines, and Page postulated that the apparent toxicity might be caused by an organic compound synthesized by a dominant soil organism, and that beneficial organisms capable of destroying the compound were killed by the soil fumigation treatment. Wilde was one of the first researchers to point out that in view of the close relationship of certain mycorrhizal fungi to the phosphorus nutrition of certain tree seedlings, that soil fumigants, heat, or steaming kills the beneficial fungi as well as the harmful one and therefore interferes with the ability of those seedlings to absorb phosphorus. The first reported incidence of the post-fumigation problem in a field nursery probably occurred on the sandy soils of central Florida reported by Tucker and Anderson . They were perhaps the first to apply the term “seedling stunting” to the problem. Seedling root stocks affected included sour orange, Carrizo citrange, Cleopatra mandarin, and to a lesser extent, Rough lemon. The latter observation does not agree with other observations. The problem was easily corrected by phosphate application. Kleinschmidt and Gerdemann , in field data from Illinois, indicated that Rough lemon seedlings were also most mycorrhizal dependent, and Cleopatra mandarin, Troyer citrange, and sour orange were progressively less dependent.

Greenhouse data by Schenck and Tucker on sandy soil further suggested that although the mycorrhizal dependency was similar for the citrus root stocks they tested, Rough lemon was the most mycorrhizal dependent and sour orange and Cleopatra mandarin were less mycorrhizal dependent. The problem first appeared in the early 1970’s in a large citrus nursery on a sandy soil near Thermal, California . The seed bed following fumigation was a disaster. The seeds germinated and grew normally for a short time , but when the seedlings reached a height of 7-10 cm, they were variably stunted in clusters within very short distances in the seed bed, between seedling varieties. Specialists from the Citrus Research Center, Riverside, were called in and preliminary guesses ranged from fumigant residues in the soil, poor seed lots, excess fertilizer, etc.—although there was no symptom evidence of any such suggestion. Leaf samples from the stunted seedlings when analyzed revealed a phosphorus deficiency as well as a few other micro-nutrient deficiencies, especially copper. An application of 1200 pounds of phosphorus per acre improved growth considerably. However, Rough lemon and Troyer citrange seedlings only attained 80-90 percent of normal growth while seedlings of sweet orange and sour orange only attained 40-50 percent of normal size in spite of phosphorus levels being adequate. Root samples from affected plants and healthy plants were sent to J. W. Gerdemann of the University of Illinois at someone’s suggestion. Dr. Gerdemann determined that the stunted seedlings had non-mycorrhizal roots and the healthy seedlings had roots with mycorrhizae. The first attempt at field inoculation was done at the Thermal nursery on Citrus amblycarpa seedlings by Harold Lembright of Dow Chemical Company with inoculum supplied by Dr. Gerdemann. The plants responded almost instantly.Work by Dr. Gerdemann and J. A. Menge with inoculated seed of sour orange was successful. More about this later. Newcomb was also able to improve growth essentially to normal by bringing soil from adjacent healthy nursery trees and spreading it on the retarded seedling areas. Menge, Johnson, and Platt grew six citrus seedling cultivars with and without the mycorrhizal fungus Glomus fasciculatus under three fertilizer regimes, all of which were without phosphorus. Of the six citrus cultivars, the mycorrhizal dependency of Rough lemon and Brazil sour orange were greater than Alemow , Troyer citrange, Bessie sweet orange, and trifoliate orange. Most importantly, the order of mycorrhizal dependency was different for all three fertilizer regimes. The greatest mycorrhizal dependency occurred with the least fertilizer. This may explain why field problems were greatest on sandy soils—and why sandy soils were used in the greenhouse experiments. Incidentally, Dr. Menge was not associated with the Citrus Research Center until the summer of 1974, which explains his delay in getting involved in mycorrhizal problems. Kleinschmidt and Gerdemann emphasize that heavy fertilization of a citrus nursery in California did not produce citrus seedling growth that was entirely satisfactory. The 1972 fertilizer practice of the W-N nursery on a sandy soil was to supply two metric tons of chicken manure, 2.5% and 3.5 , per acre 6-8 weeks prior to planting. This application did improve the growth of the citrus seedlings, but the more susceptible varieties produced only about 25 percent normal growth. The W-N nursery did obtain an excellent growth response following inoculation with Endogone mosseae, a VAN . Seed and seedlings of citrus cultivars were supplied to these researchers by the W-N nursery. Nemec grew seedlings of six citrus root stock cultivars in a sandy soil in a greenhouse. The root stock seedlings were Rough lemon, Rangpur lime, sweet orange, sour orange, Cleopatra mandarin, and Carrizo citrange, all of which were inoculated with the VAN fungi Glomus etunicatum, Glomus mosseae and Glomus fasciculatum. Some treatments were fertilized with phosphorus and others were without phosphorus.