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.     

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.     

Sampling Citrus Fibrous Roots and Tylenchulus semipenetrans

Sampling precision was investigated for Tylenchulus semipenetrans juveniles and males in soil and females from roots and for citrus fibrous root mass density. For the case of two composite samples of 15 cores each, counts of juvenile and male nematodes were estimated to be within 40% of μ, at P < 0.06 (α) in orchards where $\bar{x}$ > 1,500 nematodes/100 cm³ soil. A similar level of α was estimated for measurements of fibrous root mass density, but at a precision level of 25% of μ. Densities of female nematodes were estimated with less precision than juveniles and males. Precision estimates from a general sample plan derived from Taylor''s Power Law were in good agreement with estimates from individual orchards. Two aspects involved in deriving sampling plans for management advisory purposes were investigated. A minimum of five to six preliminary samples were required to appreciably reduce bias toward underestimation of σ. The use of a Student''s t value rather than a standard normal deviate in formulae to estimate sample size increased the estimates by an average of three units. Cases in which the use of z rather than Student''s t is appropriate for these formulae are discussed.     

Parasitism of Helicotylenchus lobus by Pasteuria penetrans in Naturally Infested Soil

The population density of Helicotylenchus lobus and the percentage of the population with spores of Pasteuria penetrans were determined for 10 monthly intervals in naturally infested turf grass soil at Riverside, California. The percentage of nematodes with attached spores ranged from 40% to 67%. No relationship was found between nematode density and the percentage of nematodes with spores. The mean and maximum numbers of spores adhering per nematode with at least one spore ranged from 2 to 8 and 7 to 66, respectively. The mean number of spores per nematode (based on total number of H. lobus) was correlated with the percentage of nematodes with spores. Spores adhered to both adult and juvenile H. lobus. Between 9% and 32% of the nematodes with spores had been penetrated and infected by the bacterium. Many infected nematodes were dead, but mature spores were also observed within living adult and juvenile H. lobus that exhibited no apparent reduction in viability and motility. Spore and central endospore diameters of this P. penetrans isolate were larger than those reported for the type isolate from Meloidogyne incognita, but transmission and scanning electron microscopy did not reveal significant morphological differences between the two isolates. Spores of the isolate associated with H. lobus did not adhere to juveniles of M. incognita.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

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.     

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.     

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.     

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.     

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.     

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.     

Biological Control of Meloidogyne incognita by Paecilomyces lilacinus and Pasteuria penetrans

The root-knot nematode Meloidogyne incognita was controlled more effectively and yields of host plants were greater when Paecilomyces lilacinus and Pasteuria penetrans were applied together in field microplots than when either was applied alone. Yields of winter vetch from microplots inoculated with the nematode and with both organisms were not statistically different from yields from uninoculated control plots.     

Suppression Mechanisms of Meloidogyne arenaria Race 1 by Pasteuria penetrans

The biological control of Meloidogyne arenaria on peanut (Arachis hypogaea) by Pasteuria penetrans was evaluated using a six x six factorial experiment in field microplots over 2 years. The main factors were six inoculum levels of second-stage juveniles (J2) of M. arenaria race 1 (0, 40, 200, 1,000, 5,000, and 25,000 J2/microplot, except that the highest level was 20,000 J2/microplot in 1995) and six infestation levels of P. penetrans as percentages of J2 with endospores attached (0, 20, 40, 60, 80, and 100%). The results were similar in 1994 and 1995. Numbers of eggs per root system, J2 per 100 cm³ soil at harvest, root galls, and pod galls increased with increasing nematode inoculum levels and decreased with increasing P. penetrans infestation levels (P ≤ 0.05), except that there was no effect of P. penetrans infestation levels on J2 per 100 cm³ soil in 1994 (P> 0.05). There were no statistical interaction effects between the inoculum levels of J2 and the infestation levels of P. penetrans (P > 0.05). When the infestation level was increased by 10%, the number of eggs per root system, root galls, and pod galls decreased 7.8% to 9.4%, 7.0% to 8.5%, and 8.0% to 8.7% in 1994 and 1995, respectively, whereas J2 per 100 cm³ soil decreased 8.8% in 1995 (P ≤ 0.05). The initial infestation level of P. penetrans contributed 81% to 95% of the total suppression of pod galls, whereas the infection of J2 of the subsequent generations contributed only 5% to 19% suppression of pod galls. The major suppressive mechanism of M. arenaria race 1 by P. penetrans on peanut is the initial endospore infestation of J2 at planting.     

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.     

Population Development of Pasteuria penetrans on Meloidogyne arenaria

A microplot study on the influence of cropping sequences with peanut in summer and bare fallowed or cover crops of rye or vetch in winter on the population development of Pasteuria penetrans was initiated in the spring of 1987. The number of spores of P. penetrans attached per second-stage juvenile of Meloidogyne arenaria race 1 increased from 0.11 in the fall of 1987 to 7.6, 8.6, and 3.6 in the fall of 1989 in the rye, vetch, and fallowed plots, respectively. Higher (P ≤ 0.05) levels of P. penetrans occurred in the rye and vetch plots than in fallowed plots. No influence of P. penetrans on peanut, rye, or vetch yield was observed in 1987 and 1988, but in 1989 peanut yield was 64% higher (P ≤ 0.05) in plots infested with P. penetrans than in plots without P. penetrans. Numbers of M. arenaria in plots without P. penetrans were influenced by the cropping sequences in the spring of 1988 and 1989 but not in the fall following the peanut crop. In the spring the plots with rye had the lowest nematode numbers in either year (P ≤ 0.05). Nematode numbers were lower (P ≤ 0.05) in plots with P. penetrans than in plots without P. penetrans in the spring of 1989 (vetch) and the fall of 1989 (rye, vetch, and fallowed).     

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.     

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.     

Temperature-Dependent Development of Pasteuria penetrans in Meloidogyne arenaria

Pasteuria penetrans is a promising biological control agent of plant-parasitic nematodes. This study was conducted to determine effects of temperature on the bacterium''s development in Meloidogyne arenaria. Developmental stages of P. penetrans were viewed with a compound microscope and verified with scanning electron microscopy within each nematode at 100 accumulated degree-day intervals by tracking accumulated degree-days at three temperatures (21, 28, and 35 °C). Five predominant developmental stages of P. penetrans were identified with light microscopy: endospore germination, vegetative growth, differentiation, sporulation, and maturation. Mature endospores were detected at 28, 35, and >90 calendar days at 35, 28, and 21 °C, respectively. The number of accumulated degree-days required for P. penetrans to reach a specific developmental stage was different for each temperature. Differences were observed in the development of P. penetrans at 21, 28, and 35 °C based on regression values fitted for data from 100 to 600 accumulated degree-days. A linear response was observed between 100 to 600 accumulated degree-days; however, after 600 accumulated degree-days the rate of development of P. penetrans leveled off at 21 and 28 °C, whereas at 35 °C the rate decreased. Results suggest that accumulated degree-days may be useful only in predicting early-developmental stages of P. penetrans.     

Quantification of Endospore Concentrations of Pasteuria penetrans in Tomato Root Material

Six methods for quantification of the endospore concentrations of Pasteuria penetrans from tomato roots are described. Mortar disruption and machine disruption methods gave the highest estimations (endospores per gram of root material) of 83.7 and 79.0 million, respectively. These methods were significantly superior to incubation bioassay (47.7 million), enzymatic disruption (32.1 million), and enzymatic disruption + flotation (25.8 million) methods. A centrifugation bioassay method gave the lowest estimation of 12.7 million.