Efficient Procedure for Extracting Tylenchulus semipenetrans from Citrus Roots

Investigations were undertaken to determine the suitability of sucrose and magnesium sulphate solutions and a silica colloidal suspension with centrifugation for extracting Tylenchulus semipenetrans from citrus roots. The efficiency of incubation, sodium hypochlorite, centrifugation, and maceration methods was also compared. Numbers of females recovered by centrifugation with colloidal silica were greater than those from sucrose or magnesium sulphate. Incubation, sodium hypochlorite, and centrifugation methods were satisfactory for extracting eggs, second-stage juveniles, and males, whereas the maceration-sieving method was less efficient. Combining the sodium hypochlorite method with a 15-second maceration followed by centrifugation in colloidal silica reduced the recovery of T. semipenetrans females from citrus roots.     

Management of the Citrus Nematode,Tylenchulus semipenetrans

Of the many nematode species that parasitize citrus, Tylenchulus semipenetrans is the most important on a worldwide basis. Management of the citrus nematode remains problematic as no one tactic gives adequate control of the nematode. An overall management strategy must include such components as site selection, use of non-infected nursery stock, use of at lease one post-plant nematode control tactic, and careful management of other elements of the environment that may stress the trees. Nematicides continue to play a key role in management of this pest. Optimum results require careful attention to application techniques.     

Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae

Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P ≤ 0.05) in plants infected by both parasites, regardless of inoculum density. Fibrous and tap root weights were 5% to 23% and 19% to 34% greater (P ≤ 0.05), respectively, in nematode-fungus combination treatments compared to the fungus alone. A second experiment was conducted, where plants were infected by the fungus, the nematode, both organisms, or neither organism. The soil mixture pH for 50% of the plants was adjusted from 4.5 to 7.0 to favor nematode infection. A higher rate of nematode infection of plants growing at pH 7.0 compared to pH 4.5 resulted in greater suppression of fungal development and greater inhibition of fungal damage to the plant. Compared to plants infected only by P. nicotianae, shoot and root weights were 37% and 33% greater (P ≤ 0.05), respectively, in plants infected by both parasites. These experiments have revealed antagonism between T. semipenetrans and P. nicotianae in citrus.     

Population Changes of Tylenchulus semipenetrans Under Localized Versus Uniform Drought in the Citrus Root Zone

Population development of Tylenchulus semipenetrans in dry soil was investigated in a greenhouse study. Citrus seedlings were grown in sandy soil in vertical tubes with upper and lower sections. Nematode population densities in the upper tubes were measured at 16, 23, and 37 days, post-treatment. Three treatments consisted of i) irrigating both tubes when soil water potential reached -1 5 kPa (non-drought), ii) irrigating only the bottom tube (local drought), and iii) no irrigation (uniform drought). Soil water potential in the upper tubes did not differ under local and uniform drought during the first 16 days post-treatment, when it approached - 125 kPa. Thereafter, the water potential of soil under uniform drought continued to decrease, while that under local drought stabilized at approximately -150 kPa. Treatments had no consistent effects on female T. semipenetrans counts from soil or roots. However, after 37 days, numbers of eggs, juvenile, and male nematodes per gram of root under local drought were more than 2.4-fold greater than those under non-drought or uniform drought. Numbers of juvenile and male nematodes in soil were 6.5 times higher under local drought than under non-drought after 37 days. Nematodes did not survive in soil under uniform drought. Most of the eggs recovered on each date, from roots under local and non-drought, hatched within 35 days. Sixteen days of uniform drought reduced cumulative egg hatch to 51%, and almost no eggs hatched after 23 and 37 days of uniform drought. Thus, the response of T. semipenetrans to dry soil is fundamentally different, depending on whether all or part of the rhizosphere experiences drought. These data and field observations suggest that hydraulic lift via the root xylem may prolong the activity of some nematodes and possibly other rhizosphere-inhabiting organisms in dry soil.     

Responses of Tylenchulus semipenetrans to Citrus Fruit Removal: Implications for Carbohydrate Competition

Sixteen mature Valencia orange trees on rough lemon rootstock were selected on the basis of approximately equal, naturally occurring populations of Tylenchulus semipenetrans in soil. In March, fruit 1 cm in diameter or less were removed from eight of the trees, which were kept free of fruit for 15 months. In July, 4 months after fruit removal, fibrous root (<2 mm d) mass density of defruited trees was 51% greater and insoluble starch in fibrous roots was 24% less than on control trees with fruit. Female T. semipenetrans per gram of root were 64% more numerous on roots of control trees than on defruited trees at this time. Numbers of female nematodes per tree and of juveniles and males in soil did not differ between treatments 4 months after fruit removal. Root mass density remained higher on defruited than control trees for the remaining 13 months that the trees were studied, while nematode density in soil beneath defruited trees rapidly increased to levels proportionate to the additional root mass density. Nine months after fruit removal (December), starch concentration was 84% higher in roots of defruited trees compared to controls and remained 28% higher than in controls 15 months (May) following fruit removal. Between months 9 and 15 following fruit removal, nematode density in soil beneath defruited trees increased at a rate five times that of nematode density beneath control trees. In May, female fecundity (eggs/female) on defruited trees was 41% greater than on control trees. The data were consistent with the hypothesis that carbohydrate competition between developing citrus fruit and T. semipenetrans influences seasonal fluctuations in nematode population densities.     

Use of Chitin for Controlling Heterodera avenae and Tylenchulus semipenetrans

The nematicidal effect of chitin, relative to other pesticides, was evaluated against two plant-parasitic nematodes, Heterodera avenae and Tylenchulus semipenetrans. Wheat seedlings, grown in soils artificially or naturally infested with H. avenae, were treated with 0.4% (w/w) ClandoSan (CLA) prepared from crustacean chitin, aldicarb (Temik 15G), or ethylene dibromide (EDB 90EC). The CLA treatment significantly increased wheat straw, ear, and average grain dry weights of nematode-infected plants, compared with the other two treatments. In an experiment covering two consecutive seasons, all three treatments reduced the number of cysts in the soil by 60%. In a one-season experiment, CLA reduced the number of cysts by 51% and aldicarb or EDB reduced cyst number by about 40%. A reduction of 50-90% in T. semipenetrans population densities on roots of two citrus rootstocks was recorded following an application of 0.2% (w/w) CLA to the soil.     

Osmolyte Allocation in Response to Tylenchulus semipenetrans Infection,Stem Girdling,and Root Pruning in Citrus

Previous studies indicated that Tylenchulus semipenetrans infection reduced concentrations of inorganic osmolytes, (Na⁺, Cl⁻, K⁺), in roots, along with leaf K⁺ in citrus. However, infection increased leaf Na⁺ and Cl⁻, along with carbohydrates in roots. Pruning of roots also increased carbohydrates in intact roots, whereas shoot pruning increased carbohydrates in shoots. Carbohydrates are translocated as reducing sugars, which collectively form organic osmolytes. Because changes in concentrations of osmolytes regulate osmotic potential in plant cells, we hypothesize that increasing concentrations of organic osmolytes in an organ displaces inorganic osmolytes. We measured the osmotic potentials of young citrus trees under nematode infection, stem girdling, and root pruning at two salinity levels. All treatments reduced leaf osmotic potentials at four sampling times. At harvest, 16 days after pruning and girdling treatments, organs with higher carbohydrates had lower inorganic osmolytes and vice versa, regardless of the treatment. Pruning simulated effects of nematode infection, whereas girdling reduced the effects of nematodes. Results suggested that high organic osmolytes in roots displace inorganic osmolytes, thereby avoiding very low osmotic potentials.     

Leaching Soluble Salts Increases Population Densities of Tylenchulus semipenetrans

The effect of salinity on population densities of Tylenchulus semipenetrans was measured on 3-month-old salt-tolerant Rangpur lime growing on either loamy sand, sand, or organic mix and on 4-month-old salt-sensitive Sweet lime in organic mix. Salinity treatments were initiated by watering daily with 25 mol/m³ NaCl + 3.3 mol/m³ CaCl₂ for 3 days and every other day with 50 mol/m³ NaC1 + 6.6 mol/m³ CaC1₂ for one week, with no salt (NS) treatments as controls. Salinity was discontinued in one treatment (DS) by leaching with tap water prior to inoculation with nematodes, whereas the continuous salinity (CS) treatment remained unchanged. Overall, in Rangpur lime organic soil supported the highest population densities of T. semipenetrans, followed by loamy sand and sand. The DS treatment resulted in the highest (P ≤ 0.05) mean population densities of T. semipenetrans in the three soil types. Similarly, the DS treatment in Sweet lime resulted in the highest (P ≤ 0.05) nematode populations. The DS treatment predisposed citrus to nematode infection through accumulated salt stress, whereas leaching soluble salt in soil solution offered nematodes a suitable nonosmotic habitat. Nematode females under the DS treatment also had the highest (P ≤ 0.05) fecundity.     

Four Biotypes of Tylenchulus semipenetrans in California Identified,and Their Importance in the Development of Resistant Citrus Rootstocks

Four biotypes (pathotypes) of the citrus nematode, Tylenchulus semipenetrans, occurring in California, U.S.A. were differentiated on the basis of differences of infectivity on ''Homosassa'' sweet orange, ''Troyer'' citrange, ''Pomeroy'' and ''Rubidoux'' Poncirus trifoliata, ''Thompson Seedless'' grape, and ''Manzanillo'' olive. A method for differentiating biotypes of T. semipenetrans is described. Field observations indicate that biotypes of this nematode are very stable. The importance of using highly infective biotypes in the development and selection of satisfactory citrus-nematode-resistant rootstocks is emphasized.     

Control of Tylenchulus semipenetrans on Citrus With Aldicarb,Oxamyl, and DBCP

Soil application of DBCP (l,2-dibromo-3-chloropropane) and foliar applications of oxamyl (methyl N'',N''-dimethyl-N-[(methylcarbamoyl)oxy]-l-thiooxamimidate) were compared for control of Tylenchulus semipenetrans in a grapefruit (Citrus paradisi) orchard, DBCP reduced nematode populations and increased fruit growth rate, fruit size at harvest, and yield compared to the untreated controls in the 2 years following treatments. Foliar applications of oxamyl reduced nematode populations and increased fruit growth rate slightly the first year, but not in the second. Foliar applications of oxamyl did not improve control attained by DBCP alone. Soil application of aldicarb [2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime] or DBCP to an orange (C. sinensis) orchard reduced T. semipenetrans populations in the 3 years tested and increased yield in 1 of 3 years. Aldicarb treatment reduced fruit damage caused by the citrus rust mite, Phyllocoptruta oleivora. Aldicarb, applied at 5.7 or 11.4 kg/ha, by disk incorporation or chisel injection, was equally effective in controlling nematodes, improving yields, fruit size, and external quality. In a grapefruit orchard, chisel-applied aldicarb reduced nematode populations and rust mite damage and increased yields in both years and increased fruit size in one year. The 11.4-kg/ha rate was slightly more effective than the 5.7-kg/ha rate. Aldicarb appears to be an adequate substitute for DBCP for nematode control in Texas citrus orchards and well-suited to an overall pest management system for Texas citrus.     

Tylenchulus palustris Parasitizing Peach Trees in the of nited States

Most morphological characteristics of three populations of a Tylenchulus sp. from peach roots in Alabama, Arkansas, and Georgia did not differ from those of T. palustris paratypes. However, some mature females differed slightly from those of T. palustris paratypes from Florida. These mature females were more swollen in the posterior portion of their bodies, and they possessed digitate postvulval body sections with round rather than conoid termini. These morphological variants had a wide postvulval section core (PVSC), as do T. palustris paratypes; they did not differ from the paratypes in other characteristics. Second-stage juveniles and males were less morphologically variable and were not different from the paratypes. No males were found in populations from Alabama and Georgia. The Tylenchulus sp. from three peach sites was determined to be T. palustris. This is the first report of T. palustris on an economically important crop.     

Tylenchulus graminis n. sp. and T. palustris n. sp. (Tylenchulidae), from Native Flora of Florida,with Notes on T. semipenetrans and T. furcus

Tylenchulus graminis n. sp. and T. palustris n. sp. are described and illustrated from broomsedge (Andropogon virginicus L.) and pop ash (Fraxinus caroliniana Mill.), respectively. T. graminis resembles T. furcus in having a distinct anus, but T. graminis second-stage juveniles (J2) do not have a bifid tail. T. semipenetrans does not have a perceptible anus. The mature female of T. graminis has a mucronate pointed terminus while T. semipenetrans has a smooth and round terminus. T. graminis males have wider stylet knobs and basal bulb and a longer tail than T. semipenetrans males. T. graminis J2 have a longer posterior body portion (without large fat globules) than T. semipenetrans J2. T. palustris resembles T. semipenetrans in having an undetectable anus but differs by the short and conoid mature female postvulval section. The male of T. palustris has larger stylet knobs and basal bulb than those of T. semipenetrans and a bluntly rounded tail terminus, which is tapered in T. semipenetrans. T. palustris differs from T. furcus and T. graminis in having an undetectable anus, by the conoid postvulval section of mature females, by the shorter and rounded tail of males, and the shorter J2 posterior body section without large fat globules. T. graminis and T. palustris are parasites of indigenous flora of Florida.     

A Classification of Tylenchulus semipenetrans Biotypes

The presence of two biotypes of the citrus nematode (Tylenchulus semipenetrans) in Italian citrus and olive orchards has been confirmed by comparing host specificity. Host reaction to California biotypes C1 and C3 and to three populations from Arizona, Texas, and Florida indicates that of these five United States biotypes, all except C3 consistently fit biotype C1. These findings, and the results of host-range studies in other countries, show that four biotypes of T. semipenetrans are distributed worldwide: the Poncirus biotype, the Citrus biotype, the Mediterranean biotype, and the Grass biotype.     

Preplant Fumigation for Citrus Nematode Control in Florida

Preplant soil fumigation experiments were conducted to control the citrus nematode, Tylenchulus semipenetrans. Generally, D-D (1,3-dichloropropene, 1,2-dichloroptopane and related chlorinated C₃-hydrocarbons), Telone (1,3-dichloropropene and related chlorinated C₃-hydrocarbons), Telone PBC (80% 1,3-dichloropropene, 15% chloropicrin, 5% propargyl bromide), and EDB (ethylene dibromide) controlled T. semipenetrans effectively for 4 years. The trials involved four scion varieties, two rootstock varieties and three soil types. Tree growth and yield were increased with application of D-D at 374 or 561 liters/ha (40 or 60 gal/acre) or Telone at 299 or 449 liters/ha (32 or 48 gal/acre) in broadcast and strip treatments.     

Distribution of Tylenchulus semipenetrans in a Texas Grapefruit Orchard

Distribution of the citrus nematode (Tylenchulus semipenetrans) was studied over 18 months in a 6-year-old orchard of grapefruit (Citrus paradisi cv. Ruby Red) on sour orange (C. aurantium) rootstock. The 1.8-ha orchard was under chemical weed control, no tillage, and flood irrigation. Highest numbers of nematodes were found in the top 15 cm of the soil profile. The nematode population peaked in April and declined to lowest levels in August and September. Numbers of nematodes were negatively correlated (r = -0.95) with soil temperatures above 29 C. Soil populations of nematodes were not correlated with soil moisture. The distribution of the nematode in the field was highly skewed and was described by a negative binomial. In this 1.8-ha block, five soil samples of 12 cores each would provide an estimate within 20% of the true nematode population mean with 95% confidence.     

Estimate of Yield Loss from the Citrus Nematode in Texas Grapefruit

Chemical control of the citrus nematode, Tylenchulus semipenetrans Cobb, has consistently increased yield of grapefruit on sour orange rootstock in Texas. In this study, data from chemical control tests conducted from 1973 to 1980 were analyzed to determine the relationship between nematode counts and grapefruit yield and fruit size. The correlation between yield and nematode counts was negative (r = -0.47) and highly significant (P < 0.01). The data best fit the exponential decay curve: y = 160.3e^-0.0000429 where y = yield in kg/tree and x = nematodes/100 cm³ of soil. The correlation between fruit size and nematode counts was not significant because yield and fruit size were inversely related. Yield loss in an average untreated orchard was estimated to be 12.4 tons/ha. Economic loss to citrus nematode in Texas grapefruit, assuming no treatment and an average on-tree price of $60/ton, was estimated to be $13.2 million annually.     

Characterization of Citrus Rootstock Responses to Tylenchulus semipenetrans (Cobb)

Citrus rootstocks which significantly limited the reproduction of Tylenchulus semipenetrans (Cobb) "Citrus" and "Poncirus" biotypes responded to infection by producing a hypersensitive-type response in the root hypodermis, wound periderm and/or cavities in the root cortex, and/or abnormal vacuoles in nurse cell cytoplasm. Rootstocks which limited nematode reproduction also had significantly fewer nematodes in the rhizoplane within 8 d of inoculation than did rootstocks which did not limit reproduction. Germplasm sources of the cellular responses which limited citrus nematode reproduction were identified.     

Determining Consistency of Spatial Dispersion of Nematodes in Small Plots

Nematode population densities in field plots were estimated by collecting samples consisting of 12 soil cores. Plots encompassed a variety of plant hosts and sampling dates, and provided data on the population densities of seven species of plant-parasitic nematodes. Three separate samples were collected per plot on each sampling date to obtain estimates of the mean and variance of numbers for each species. For each nematode species, these estimates were used to derive the Taylor''s Power Law regression over plots having identical hosts and sampling dates. For some nematode species, comparisons of regression equations among different sampling dates on the same host revealed similarities in values of a and b from Taylor''s Power Law. Parameters of Taylor''s Power Law relationships were used to develop sampling plans and to obtain estimates of sample precision. Precision estimates from specific and general sampling plans are illustrated for Belonolaimus longicaudatus.     

Stability and Characteristics of Spatial Description Parameters for Nematode Populations

The parameters of Taylor''s Power Law (s² = am^b) relating variance (s²) to mean population level (m) were acceptably stable in different fields with similar cropping systems. Values of both a and b parameters varied with nematode species. The value of a was a function of sample size (number of cores) and was characterized for each species. The value of b was stable across sample size and reflective of the life history strategy of the species. The relationship between the economic threshold and sampling intensity required to allow management decisions, with specified levels of risk, indicated the need for improved sampling technology.