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."     

Infectivity of Two Biotypes of the Citrus Nematode on Citrus and on Some Other Hosts

The infectivity and development of two biotypes of citrus nematode (Tylenchulus semipenetrans) were compared on highly resistant Poncirus trifoliata selection ''Pomeroy,'' moderately susceptible ''Troyer'' citrange, and highly susceptible sweet orange selection ''Homosassa'' small seedlings in a glasshouse. Biotype-1 was more infective on the above hosts and developed faster on sweet orange and on ''Troyer'' citrange than Biotype-2. The differences in infectivity were interpreted to reflect differences in the ability of the nematodes to penetrate the epidermis and hypodermis and become established in host roots. Poncirus selections ''Pomeroy,'' ''Webber-Fawcett,'' and ''Rubidoux'' seedlings were highly resistant to the citrus nematode in California, but seedlings of ''Pomeroy'' and ''Rubidoux'' were only moderately resistant in Japan. These differences in degree of infection may indicate different biotypes of the nematode. Host range tests with California Biotype-1 indicate that it differs from those occurring in Israel.     

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.     

Influence of Metalaxyl on Three Nematodes of Citrus

Metalaxyl significantly reduced population of Pratylenchus coffeae, Radopholus similis, and Tylenchulus semipenetrans in roots of Citrus limon (rough lemon) under greenhouse conditions. Postinoculation treatment of rough lemon seedlings was not as effective i n reducing nematode populations as was treatment before inoculation. Fewer nematodes infected metalaxyl-treated roots than nontreated roots. However, incubation of nematodes in metalaxyl did not inhibit nematode motility or their ability to locate and infect roots. Cellular responses to nematode injection differed between treated and nontreated tissues. Metalaxyl appeared to confer nematode contraol by modifying citrus roots such that a normally susceptible rootstock became tolerant.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Pathogenicity and Interaction of Three Nematode Species on Six Bermudagrasses

The ring nematode (Criconemoides ornatus), stunt nematode (Tylenchorhynchus martini), and sting nematode (Belonolaimus Iongicaudatus) reproduced readily on six bermudagrasses (Common, ''U-3'', ''Tufcote'', ''Continental'', ''Tiffine'', and ''Tifdwarf''). Populations of a single nematode species influenced the population development of a second and third parasitic nematode species on a particular host plant. Activity of most nematodes adversely affected reproduction of other nematode species in mixed cultures. Generally, the number of fibrous roots produced by plants decreased as the number of nematode species in the treatments increased. Tifdwarf bermudagrass appeared to be more tolerant to C. ornatus and T. martini than other grasses tested.     

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.     

Identification of Seed Gall Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

A molecular analysis of eight described species of seed gall nematode, along with six undescribed isolates from different hosts, has revealed a strong association between nucleotide sequence polymorphism and host status. Each anguinid nematode associated with a unique host produced a unique PCR-RFLP pattern for the ITS1 region. Anguina species that had been synonymized in the past, Anguina agrostis, A. funesta, and A. wevelli (Afrina wevelli), were readily discriminated. Two undescribed species from northern New South Wales and southeastern South Australia, reported to be vectors of Rathyaibacter toxicus in the disease called ''''floodplain staggers,'''' were differentiated by a single restriction enzyme, and both could be separated easily from A. funesta, the vector of R. toxicus in annual ryegrass toxicity. Other species differentiated in this study include A. agropyronifloris, A. graminis, A. microlaenae, A. pacificae, and undescribed species from host species Dactylis glomerata, Agrostis avenacea, Polypogon monospeliensis, Stipa sp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITS1 region suggests that considerable anguinid genetic diversification has accompanied specialization on different host species.     

Factors Affecting Population Trends of Plant-Parasitic Nematodes on Rangeland Grasses

The effects of environmental conditions on population trends of plant-parasitic nematodes were studied in experimental plots of five wheatgrasses in the western Utah desert. In a 3-year (1984-86) field study, soil water and temperature affected the population trends of the ectoparasites, Tylenchorhynchus acutoides and Xiphinema americanum, and the migratory endoparasite, Pratylenchus neglectus, on Fairway crested wheatgrass, Agropyron cristatum; ''Hycrest'' crested wheatgrass, A. cristatum X A. desertorura; ''Rosana'' western wheatgrass, Pascopyrum smithii; ''Oahe'' intermediate wheatgrass, Thinopyrum intermedium; and RS-1 hybrid (Elytrigia repens X Pseudoroegneria spicata). The largest soil populations of these nematode species were collected in 1984 under good plant-growth conditions. A reduction in nematode populations occurred in 1985 and 1986, possibly because of low soil-water conditions. There was a positive relationship between high soil water and maximum population densities of T. acutoides in the spring and fall of 1984, and between low soil water and minimum population densities of the nematode in 1985 and 1986. Pratylenchus neglectus populations were affected by soil water, although to a lesser degree than the ectoparasitic nematodes. Population densities of the three nematode species were significantly lower in the drier years of 1985 and 1986 than in 1984. Nematode populations were greater at the lower soil depths in the fall than in the spring or summer.     

Influence of Four Nematodes on Root and Shoot Growth Parameters in Grape

Two grape cultivars, susceptible French Colombard and tolerant Rubired, and four nematodes, Meloidogyne incognita, Pratylenchus vulnus, Tylenchulus semipenetrans, and Xiphinema index, were used to quantify the equilibrium between root (R) and shoot (S) growth. Root and shoot growth of French Colombard was retarded by M. incognita, P. vulnus, and X. index but not by T. semipenetrans. Although the root growth of Rubired was limited by all the nematodes, the shoot growth was limited only by X. index. The R:S ratios of Rubired were higher than those of French Colombard. The reduced R:S ratios of Rubired were primarily an expression of reduction in root systems without an equal reduction in shoot growth, whereas in French Colombard the reduced R:S ratios were due to a reduction in both shoot growth and root growth and to a greater reduction in root growth than shoot growth. All nematodes reproduced equally well on both cultivars. Both foliage and root growth of French Colombard were significantly reduced by M. incognita and P. vulnus. Nematodes reduced the shoot length by reducing the internode length. Accumulative R:S ratios in inoculated plants were significantly smaller than those in controls in all nematode treatments but not at individual harvest dates. Bud break was delayed by X. index and was initiated earlier by P. vulnus and M. incognita. All buds in nematode treatments were less vigorous than in controls.     

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.     

Eggs of Tylenchulus semipenetrans Inhibit Growth of Phytophthora nicotianae and Fusarium solani in vitro

In previous greenhouse and laboratory studies, citrus seedlings infested with the citrus nematode Tylenchulus semipenetrans and later inoculated with the fungus Phylophthora nicotianae grew larger and contained less fungal protein in root tissues than plants infected by only the fungus, demonstrating antagonism of the nematode to the fungus. In this study, we determined whether eggs of the citrus nematode T. semipenetrans and root-knot nematode Meloidogyne arenaria affected mycelial growth of P. nicotianae and Fusarium solani in vitro. Approximately 35,000 live or heat-killed (60°C, 10 minutes) eggs of each nematode species were surface-sterilized with cupric sulfate, mercuric chloride, and streptomycin sulfate and placed in 5-pl drops onto the center of nutrient agar plates. Nutrient agar plugs from actively growing colonies of P. nicotianae or F. solani were placed on top of the eggs for 48 hours after which fungal colony growth was determined. Live citrus nematode eggs suppressed mycelial growth of P. nicotianae and F. solani (P ≤ 0.05) compared to heat-killed eggs and water controls. Reaction of the fungi to heat-killed eggs was variable. Root-knot nematode eggs had no effect on either P. nicotianae or F. solani mycelial growth. The experiment demonstrated a species-specific, direct effect of the eggs of the citrus nematode on P, nicotianae and F. solani.     

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.     

Comparison of Five Populations of Tylenchulus semipenetrans to Citrus,Poncirus, and their Hybrids

The infectivity of five populations of Tylenchulus semipenetrans were compared and differentiated on 10 hosts (5 Citrus spp., 1 Poncirus trifoliata, and 4 hybrids of Citrus spp. X P. trifoliata). Differences in levels of infection and development (P = 0.01) occurred between Citrus spp. and P. trifoliata cv. ''Pomeroy'' and their three hybrids, C. paradisi X P. trifoliata cv. ''Swingle'' citruntelo and C. sinensis, cv. ''Ruby'' orange X P. trifoliata cv. ''Webber Fawcett 14-7'', and ''15-7''. Poncirus trifoliata cv. Pomeroy was susceptible to a California biotype 3 and highly resistant to the other citrus nematode populations. Low infection levels with California biotype 1, Arizona, and Florida populations on Swingle citrumelo, and the two Ruby orange hybrids indicated inherited resistance. Reproduction of the nematode population from Texas was greatest on the three hybrids, Swingle citrumelo, Ruby orange 14-7, and 15-7, from the California 1, Arizona, and Florida populations, but its comparable densities on P. trifoliata and Citrus spp. were not sufficiently different from these populations to consider it a separate biotype. California biotype 3 was sufficiently different from all other populations to be considered a different biotype, and it was named the "Poncirus biotype."     

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.     

Interactions of Concomitant Species of Nematodes and Fusarium oxysporum f. sp. vasinfectum on Cotton

Meloidogyne incognita, Hoplolaintus galeatus, and North Carolina and Georgia populations of Belonolaimus longicaudatus were introduced singly and in various combinations with Fusarium oxysporum f. sp. vasinfectum on wilt-susceptible ''Rowden'' cotton. Of all the nematodes, the combination of the N. C. population of B. longicaudatus with Fusarium promoted greatest wilt development. H. galeatus had no effect on wilt. With Fusarium plus M. incognito or B. longicaudatus, high nematode levels promoted greater wilt than low levels. The combination of either population of B. longicaudatus with M. incognita and Fusarium induced greater wilt development than comparable inoculum densities of either nematode alone or where H. galeatus was substituted for either of these nematodes. Nematode reproduction was inversely related to wilt development. Without Fusarium, however, the high inoculum level resulted in greater reproduction of all nematode species on cotton. Combining M. incognita with B. longicaudatus or H. galeatus gave mutually depressive effects on final nematode populations. The interactions of H. gateatus with B. longicaudatus varied with two populations of the latter.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.     

Penetration and Development of Meloidogyne hapla in Resistant and Susceptible Alfalfa Under Differing Temperatures

Studies were conducted to examine under differing temperatures (12, 16, 20, 24, 28, and 32 C) the penetration anti development of Meloidogyne hapla in resistant lines ''298'' and ''Nev. Syn XX'', and susceptible ''Lahontan'' and ''Ranger'' hardy-type alfalfas. The results indicated that resistance to M. hapla was similar to that previously described for M. incognita in nonhardy alfalfa. Although initial penetration in resistant seedlings was similar to that of susceptible seedlings, nematode larvae failed to establish and develop in root tissues and nematode numbers subsequently declined. In susceptible seedlings, nematode development proceeded rapidly, and egg production began after 5 weeks. Temperature had little influence on the nematode development except to slow the response at the lower temperatures. Other studies were conducted to verify a previously reported immune (no penetration) reaction to M. hapla by the ''Vernal'' selection ''M-4''. When compared to the resistant (penetration without nematode development) Vernal selection ''M-9'' under differing temperatures (20, 24, 28, and 32 C), each selection was equally penetrated by M. hapla but at a lower level than in susceptible Ranger cuttings. Generally, no root galling was observed in either M-4 or M-9; however, very slight galling was found 35 days after inoculation on about 50% of these cuttings when grown at 32 C.     

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.     

Feeding of the Nematode Acrobeloides nanus on Bacteria

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode''s intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode''s intestine.