Rearing Migratory Endoparasitic Nematodes in Citrus Callus and Roots Produced from Citrus Leaves

Radopholus similis and Pratylenchus coffeae were reared on callus and roots developed from citrus leaves. Callus formed best when leaf petioles were immersed in Astatula fine sand and the leaves were sprayed daily with 4 ppm 2,4-D solution and maintained at 25 or 30 C. The nematodes completed one generation in 20 days at 25 C. Highest populations of R. similis (1,127) occurred after 50 days, and the highest for P. coffeae (619) after 70 days. Leaf-callus cultures from R. similis-resistant citrus rootstocks showed the same degree of infection as susceptible rough lemon callus after 30 days.     

Population Fluctuation of Three Parasitic Nematodes in Florida Citrus

In Florida, Tylenchulus semipenetrans on citrus has two high and two low population levels each year. High levels occur in April-May and November-December, and low levels, in February-March and August-September. Population increases occur about 4-5 weeks after the spring and fall flush of root growth. Populations of Pratylenchus coffeae on citrus varied widely, and were not related to season. Populations of P. brachyurus showed seasonal variation with a high in June-July and a low in March-May. Males of T. semipenetrans and P. coffeae were found throughout the year, whereas males of P. brachyurus were rare and were found only during November and December.     

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.     

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.     

A Method for Demonstrating Prophenoloxidase after Electrophoresis

The demonstration of prophenoloxidase after electrophoresis is based on its activation by sodium dodecyl sulfate (SDS) or sodium oleate and staining the activated phenoloxidase with dopamine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). A rapid method is presented for demonstrating the presence of activated phenoloxidase using polyacrylamide gel electrophoresis followed by staining in the presence of SDS or sodium oleate.     

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.     

Ammonium and Potassium Uptake by Citrus Roots

Multiphasic kinetics were indicated for uptake of ammonium and potassium by intact cirtrus seedlings as measured by a continuous flow technique. Uptake at low concentration, below 10^?3M, was biphasis. The patterns for ammonium uptake by 60-day-old and 180-day-old seedlings were similar, but the rates of uptake per g dry weight of root were greater for the younger plants.     

A Reliable Method for Demonstrating Gram-Positive and Gram-Negative Bacteria

Combined Gram techniques have been reviewed in the interest of improving technical safety and reliability in the demonstration of bacteria, particularly the Gram-negative type. The many modifications of the technique present various difficulties (Brown and Brenn 193 1, Humberstone 1963, Taylor 1966, Luna 1968, Brown and Hopps 1973, Engbaek et al. 1979, Bancroft and Stevens 1982, Churukian and Schenk 1982).     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A Reliable Method for Demonstrating Axonal Degeneration Shortly After Axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on a reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

A Method for Demonstrating Bacterial Proteolytic Isoactivities after Electrophoresis in Acrylamide Gels

S ummary . A method for detecting bacterial proteinases in gel slabs after electrophoresis is described. It consists in placing the slabs on a layer of gelatin-agar buffer, allowing hydrolysis of the gelatin to occur and then developing the areas of hydrolysis with acid mercuric chloride. To illustrate this method we chose extracellular preparations of strains of Brevibacterium linens and Micrococcus spp.     

Effect of Paraquat on Tomato Roots Cultured in Vitro Susceptible and Resistant to Nematodes

Roots from two tomato cultivars, one resistant (Rossol) andthe other susceptible (Roma VF) to root-knot nematodes, werecultured in vitro and tested for their response to paraquat,an herbicide which generates superoxide in cells. The effectof paraquat on the permeability of root membranes was testedby the safranine method. Membranes from resistant roots wereinjured more markedly than susceptible ones. A statistical analysisof the variations on a series of enzyme activities from rootsincubated for 2 days with paraquat was carried out. Superoxidedismutase was repressed in treated resistant roots togetherwith ascorbate and glutathione peroxidases. In susceptible rootsthese enzymes were not significantly affected by paraquat treatmentexcept for ascorbate peroxidase which increased slightly. Syringaldazineand polyphenol oxidases, two enzymes active in the hypersensitiveresponse to nematodes, increased in resistant but not in thesusceptible cultivar. p-Phenilendiamine-pyrocathecol oxidasewas not affected while catalase increased markedly by paraquattreatment in both cultivars. (Received January 21, 1991; Accepted August 6, 1991)     

Isolation and Characterization of Beneficial Bacteria Associated with Citrus Roots in Florida

Cultivable diversity of bacteria associated with citrus was investigated as part of a larger study to understand the roles of beneficial bacteria and utilize them to increase the productive capacity and sustainability of agro-ecosystems. Citrus roots from Huanglongbing (HLB) diseased symptomatic and asymptomatic citrus were used in this study. A total of 227 and 125 morphologically distinct colonies were isolated and characterized from HLB asymptomatic and symptomatic trees, respectively. We observed that the frequency of bacterial isolates possessing various plant beneficial properties was significantly higher in the asymptomatic samples. A total of 39 bacterial isolates showing a minimum of five beneficial traits related to mineral nutrition [phosphate (P) solubilization, siderophore production, nitrogen (N) fixation], development [indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relief [production of 1-amino-cyclopropane-1-carboxylate deaminase] and production of quorum sensing [N-acyl homoserine lactones] signals were characterized. A bioassay using ethidium monoazide (EMA)-qPCR was developed to select bacteria antagonistic to Candidatus Liberibacter asiaticus. Using the modified EMA-qPCR assay, we found six bacterial isolates showing maximum similarity to Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans, and Serratia plymutica could significantly reduce the population of viable Ca. L. asiaticus in HLB symptomatic leaf samples. In conclusion, we have isolated and characterized multiple beneficial bacterial strains from citrus roots which have the potential to enhance plant growth and suppress diseases.     

一种适合AFLP分析的柑橘DNA提取方法

目的：通过对传统用于AFLP分析的DNA提取方法进行改善调和整得到一种效率较高,DNA损失较少适合于柑橘AFLP分析的DNA提取方法。方法：利用改善后的方法提取了13份参试柑橘材料（无病虫害的鲜叶）的DNA基因组,后用0．8％琼脂糖进行电泳检测,并进行了AFLP试验。结论：结果分别得到了清晰的DNA模板检测图和AFLP指纹图谱,表明该方法提取的DNA无降解且较纯,其质量完全满足AFLP技术的要求,并且在提取效率,减少DNA损失等方面体现出了优势。     

Distribution of Field Corn Roots and Parasitic Nematodes in Subsoiled and Nonsubsoiled Soil

A field trial was conducted for 2 years in an Arredondo fine sand containing a tillage pan at 15-20 cm deep to determine the influence of subsoiling on the distribution of corn roots and plant-parasitic nematodes. Soil samples were taken at various depths and row positions at 30, 60, and 90 days after planting in field corn subsoiled under the row with two chisels and in non-subsoiled corn. At 30 and 60 days, in-row nematode population densities to 60 cm deep were not affected by subsoiling compared with population densities in nonsubsoiled plots. After 90 days, subsoiling had not affected total root length or root weight at the 20 depth-row position sampling combinations, but population densities of Meloidogyne incognita and Criconemella spp. had increased in subsoiled corn. Numbers of Pratylenchus zeae were not affected. Subsoiling generally resulted in a change in distribution of corn roots and nematodes in the soil profile but caused little total increase in either roots or numbers of nematodes. Corn yield was increased by subsoiling.