Morphological and molecular characterization of Phytophthora species associated with root and crown rot of pomegranate in Iran

The purpose of this research was to identify the pathogens causing root and crown rot in major pomegranate-growing areas of Iran. Infected tissue samples were collected from trees with symptoms from 49 pomegranate orchards in four provinces of Iran: Fars, Markazi, Isfahan, and Kohgiluyeh va Boyer-Ahmad. In total, 23 Phytophthora spp. isolates were obtained, which were identified as P. cryptogea species complex (12 isolates) and P. cinnamomi (11 isolates) based on morphological characters. Molecular confirmation of identification was performed by inference of phylogeny of ITS-rDNA regions, β-tubulin gene, and the mitochondrial gene cytochrome c oxidase subunit 1. The results of our phylogenetic analysis confirmed the morphological identification of P. cinnamomi isolates and placed them in Clade 7c of Phytophthora. In addition, the P. cryptogea species complex isolates, despite morphological similarities, were in fact four distinct species including P. cryptogea sensu stricto (two isolates), P. pseudocryptogea (one isolate), P. sp. kelmania (one isolate), and P. erythroseptica (six isolates). This is the first report of pomegranate root and crown rot caused by P. cinnamomi and P. cryptogea species complex.     

Phytophthora species causing crown and root rot of tomato in southern Italy*

Phytophthora capsici, Phytophthora cryptogea and Phytophthora nicotianae were isolated from tomato plants with symptoms of crown and root rot in plastic‐house crops in Sicilia and Calabria (southern Italy). The species were identified primarily on the basis of morphological and cultural characteristics. The identification was confirmed using molecular methods, polyacrylamide gel electrophoresis (PAGE) of mycelial proteins and polymorphism of DNA sequences amplified by polymerase chain reaction using random primers (RAPD‐PCR). P. capsici caused significant losses in tomato crops that had succeeded capsicum crops. P. cryptogea was found to be the most frequent species causing basal stem rot of tomato, a disease of increasing importance in commercial tomato crops in plastic houses in Sicilia. P. nicotianae was common in plastic houses where poor drainage resulted in standing water.     

A strain ofPseudomonas syringae which does not belong to pathovarphaseolicola produces phaseolotoxin

A bacterial strain, CFBP 3388, isolated from Vetch (Vicia sativa, L.) was identified asP. s. pv.syringae on the basis of nutritional and biochemical patterns which were obtained with classical tests and the Biolog system. It caused necrotic symptoms typical ofP. s. pv.syringae on bean leaves and pods after artificial inoculation. However, the isolate caused a citrulline-reversible inhibition ofE. coli in phaseolotoxin bioassay. Furthermore, with CFBP 3388 DNA as template a 1900 bp DNA fragment, specific for the phaseolotoxin DNA cluster ofP. s. pv.phaseolicola, was amplified by PCR. This is the first demonstration that an isolate ofP. syringae that is not pv.phaseolicola can produce phaseolotoxinAbbreviations bp base pair - kb kilobase - OCT Ornithine Carbamoyl Transferase     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Electrophoretic study of three related Phytophthora species1

Morphological and cultural characters currently used for the classification of species of Phytophthora are inadequate to distinguish P. cryptogea, P. drechsleri and P. erythroseptica. In this study isolates of the above-mentioned species, including type and/or authentic cultures of P. cryptogea and P. drechsleri were compared. Morphological and cultural characters, together with electrophoretic patterns of both total mycelial proteins and isozymes (arylesterase and malate dehydrogenase), allowed the distinction of P. erythroseptica from the others. Authentic cultures of P. cryptogea and P. drechsleri showed distinctly different electrophoretic patterns of arylesterase and malate dehydrogenase isozymes and of total mycelial proteins. Some Italian isolates previously identified as P. cryptogea or P. drechsleri showed features of both species, therefore it was impossible to discriminate the two species solely on the basis of the electrophoretic patterns of mycelial proteins.     

Evaluation of plant growth-promoting rhizobacteria for biological control of pythium root rot of cucumbers grown in rockwool and effects on yield

Three strains ofPseudomonas fluorescens (63-49, 63-28, and 15), one strain ofPseudomonas corrugata (13) and one strain ofSerratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused byPythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicatedPythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect ofPythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation withP. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments ofPythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to thePythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to thePythium-inoculated control, but the increase was less than with 63-49. The use ofPseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.     

珠海非洲菊疫病的鉴定和防治

１９９６年,珠海非洲菊生产基地非洲菊出现萎蔫症状,大量植株死亡,感病品种年发病率达４０％～５０％。经病情调查和病原菌分离鉴定明确,该病由ＰｈｙｔｏｐｈｔｈｏｒａｃｒｙｐｔｏｇｅａＰｅｔｈｙｂｒｉｄｇｅ＆Ｌａｆｅｒｔｙ和Ｐ．ｃａｃｔｏｒｕｍＳｃｈｒｏｔｅｒ所致,前者为主要病原。发病程度与温度、田间湿度及品种抗病性密切相关。室内药剂筛选试验表明,甲霜灵－锰锌抑菌效果最好;杀毒矾次之。田间选种抗病品种、作高畦、改喷灌为滴灌、拔除病株、药剂防治等措施,防效较好。     

Identification and pathogenicity of<Emphasis Type="Italic">Fusarium</Emphasis> spp. from stem bases of winter wheat in Erzurum,Turkey

Four-hundred-sixty-eightFusarium andFusarium-like isolates were obtained from crowns and subcrown internodes of winter wheat grown in Erzurum, Turkey. Of these isolates, 34.8% wereFusarium acuminatum, 32.3% wereF. equiseti, 16.9% wereF. oxysporum, 15.0% wereMicrodochium nivale (formerlyFusarium nivale), 0.6% wereF. tabacinum and 0.4% wereF. solani. In pathogenicity tests on wheat, the highest disease severity was caused by isolates ofM. nivale, whereas isolates ofF. acuminatum, F. equiseti, F. oxysporum andF. solani were slightly virulent; isolates ofF. tabacinum were nonpathogenic. This is the first report ofM. nivale andF. tabacinum from wheat in Turkey. http://www.phytoparasitica.org posting Jan. 29, 2003.     

<Emphasis Type="Italic">Ulocladium atrum</Emphasis> as a biological control agent for white mold of bean caused by<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m⁻² of 10⁶ spores ml⁻¹ suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha⁻¹ per application in 2001, or Lance (boscalid) at the rate of 750 g ha⁻¹ per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Vegetative compatibility among isolates ofColletotrichum gloeosporioides from almond in Israel

Anthracnose, caused byColletotrichum gloeosporioides, is the major disease of almond in Israel. Pathogen attack of young fruit results in fruit rot and leaf wilting. Seventy isolates ofC. gloeosporioides were obtained from affected almond fruits collected at 11 sites during 1991–2 and 1994. Chlorate-resistant nitrate-nonutilizing (nit) mutants were generated from each isolate and used in complementation (heterokaryon) tests. The formation of complementary stable heterokaryons between mutants from different isolates showed that all the isolates belonged to a single vegetative compatibility group. Representative isolates ofC. gloeosporioides from almond did not form heterokaryons with local isolates ofColletotrichum from anemone and avocado, indicating that the almond isolates constitute a distinct subspecific group withinC. gloeosporioides.     

Natural occurrence of and inoculation experiments withConidiobolus coronatus andConidiobolus sp. in glasshouse populations ofBemisia tabaci

Bemisia tabaci (Gennadius), the sweetpotato whitefly, has only one known entomophthoralean pathogen,Erynia radicans (Entomophthoraceae). Two new pathogens have been isolated recently from a glasshouse population of this pest:Conidiobolus coronatus and another, undescribed species ofConidiobolus (Entomophthorales: Ancylistaceae). Artificial inoculation experiments revealed that eggs ofB. tabaci are practically immune to infection by either species. Second-instar larvae are highly resistant, only high doses of conidia causing between <1% and 4.6% mortality. Adults were found to be much more susceptible. Doses of 60 conidia/mm² ofC. coronatus caused average mortalities ofca 95%. The maximum mortality of adults caused byConidiobolus sp. was much lower,ca 30%, at a dose of 210 conidia/mm². The incubation period (inoculation to death) for both species, under our experimental conditions, is very short: 18–24 h forC. coronatus and 30 h forConidiobolus sp. Both fungi produced loricoconidia (conidia metamorphosed into resting spores) on cotton leaves and other dry surfaces. This ability allowedConidiobolus sp. to remain viable for 17–21 days on cotton leaves, in glasshouse conditions and in the absence of hosts, whileC. coronatus persisted for 10–14 days.     

Root hair infection by Plasmodiophora brassicae in clubrootresistant and susceptible genotypes of Brassica oleracea,B. rapa and B. napus

The pathogenesis of clubroot, a disease of cruciferous crops caused by the fungusPlasmodiophora brassicae, starts with infection of the root hairs. This process was studied in 13 accessions ofBrassica oleracea, B. napus and B. rapa with varying levels of plant resitance toP. brassicae. Seedlings were grown in a mineral solution, inoculated with resting spores ofP. brassicae, and the number of plasmodia developing in root hairs was recorded. When compared with the standard susceptible cultivar Septa, both higher and lower resistance to root hair infection was found in the accessions of the differentBrassica species. No complete resistance to root hair infection was found. Over the accessions studied, there was no correlation between the plant resistance estimated from greenhouse tests and the resistance to root hair infection of seedlings. The resistance of all accessions must at least partly be caused by other mechanisms which operate after the root hair plasmodia are formed.     

Biological control of damping-off caused byPythium ultimum andRhizoctonia solani usingTrichoderma spp. applied as industrial film coatings on seeds

Conidia of sevenTrichoderma strains were applied on cucumber or radish seeds as a simple methyl cellulose coating or through an industrial film coating process. The seeds were sown in a peat-based soil artificially infested byR. solani orP. ultimum. Four strains controlled damping-off caused byR. solani when applied as a simple coating or as an industrial film-coating. Also, four strains significantly reduced damping-off caused byP. ultimum in cucumber. A correlation was found between production of volatile antibiotics in vitro and control ofP. ultimum. Survival during storage varied according to the strain. Better survival was observed for two strains, with a decrease in conidial viability of one order of magnitude after storage for three and five months at 15 ° C and 4 ° C, respectively. The results show the feasibility of biocontrol of seedling diseases by some antagonists applied onto seeds through an industrial film-coating process.     

Note: Tachinid parasitoids of<Emphasis Type="Italic">Ancyrosoma leucogrammes</Emphasis> and notes on parasitization rates of<Emphasis Type="Italic">Clytiomya dupuisi</Emphasis>

The study deals with the tachinid parasitoids ofAncyrosoma leucogrammes (Gmelin) (Heteroptera: Pentatomidae):Clytiomya dupuisi Kugler,Clytiomya sola (Rondani) andGymnosoma clavatum (Rohdendorf) (Diptera: Tachinidae). All species are recorded for the first time as parasitoids ofA. leucogrammes, andC. dupuisi was reared for the first time from a host. The parasitization rates ofC. dupuisi on adults ofA. leucogrammes varied from 7% to 9% between 1994 and 1999.C. sola andG. clavatum were reared in only small numbers. http://www.phytoparasitica.org posting July 10, 2003.     

Interaction ofPseudomonas fluorescens withRhizobium for their effect on the management of peanut root rot

A biocontrol agent (Pseudomonas fluorescens) and a phytostimulator (Rhizobium) have been shown to have beneficial effects on plant growth and health. The study of plants inoculated withPseudomonas andRhizobium requires special attention because of the possibility that these agents may influence each other. Our study was conducted to test the effect of these inoculants on co-inoculation in peanut to control root rot, a severe soilborne disease caused byMacrophomina phaseolina. One fluorescent pseudomonad strain, Pf 1, which effectively inhibited the mycelial growth ofM. phaseolina underin vitro conditions, was studied for its compatibility with the biofertilizer bacterial strainRhizobium TNAU 14. Dual culture and colorimetric studies indicated the existence of a positive interaction between the microbial inoculants. However, glasshouse and field studies showed seed treatment and soil application ofPseudomonas fluorescens Pf 1 to be the most effective treatment in reducing root rot incidence and improving the crop vigor index, in comparison with treatments in which both inoculants were applied. http://www.phytoparasitica.org posting Feb. 11, 2002.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Pathogenicity ofVerticillium lecanii to different developmental stages of the silverleaf whitefly,Bemisia argentifolii

Thirty-five strains ofVerticillium lecanii which originated from different hosts and geographical locations were tested as potential biocontrol agents against silverleaf whitefly,Bemisia argentifolii Bellows & Perring. All strains were tested for their pathogenicity to third-instar nymphs. Several isolates which exhibited high pathogenicity to nymphs were also tested against eggs, pupae and adults ofB. argentifolii. Eggs were found to be immune to infection, but mortality of hatching nymphs reached 95–98%. The rate of hatching nymphs’ infection depended on the age at which the eggs were inoculated and the strain’s virulence. Mortality of nymphs recorded on day 4 after inoculation varied from 0.5±0.3% to 83±2.4%; that of the control ranged from 2.5% to 10.2%. The most virulent strains, with LT₅₀ ranging between 3.2 and 3.8 days, were isolated from aphids in Israel and probably have a similar origin. The pathogenicity ofV. lecanii strains to pupae 6 days after inoculation varied between 59± 12.1 % and 72.5± 13.1 %, as compared with natural mortality of 13.5±4%. The maximum adult mortality caused byV. lecanii strains was between 34.1±5.1% and 52.6±3.8%.     

Histological study of giant cells formed by the root-knot nematodeMeloidogyne artiellia as compared withM. hapla andM. javanica in cabbage, turnip and barley

Feeding sites induced by the root-knot nematodeMeloidogyne artiellia in turnip (Brassica rapa), cabbage (Brassica oleracea) and barley (Hordeum vulgare) were examined by light and electron transmission microscopy, and compared with those formed byM. javanica andM. hapla. The feeding cells ofM. artiellia in turnip and cabbage showed hypertrophy, hyperplasia and vacuolization, and became multinucleate giant cells. In contrast to the typical simple-shaped giant cells ofM. javanica andM. hapla, those ofM. artiellia had an amoeboid structure containing ‘protuberances’, which are distinct from previously reported ‘projects’ of giant cells induced byM. incognita in other plants. Protuberances expanding between vascular cells were observed in young and developed giant cells. Since no cell-wall fragments were found at the bases of the protuberances, either by light or electron microscopy, it is strongly indicated that these structures were caused by local expansion of the giant cells rather than by fusion with adjacent cells. The giant cells ofM. artiellia in barley had regular shapes of giant cells without protuberances, and resembled those induced byM. javanica orM. hapla in turnip and cabbage. http://www.phytoparasitica.org posting Sept. 17, 2006.     

Parasitoid community of the loreyi leaf wormMythimna (Acantholeucania) loreyi: Novel host-parasitoid associations and their efficiency in the eastern mediterranean region of Turkey

The loreyi leaf worm,Mythimna (Acantholeucania) loreyi Duponchel (Lepidoptera, Noctuidae), is a pest of gramineous crops and causes significant economic damage to maize. In field surveys on maize to determine the parasitoid community and its impact on the pest in the eastern Mediterranean region of Turkey, nine parasitoid species were found associated with immature stages ofM. loreyi: The hymenopteran (Braconidae and Ichneumonidae) parasitoidsCotesia (=Apanteles) ruficrus (Haliday),Chelonus oculator Panzer,Meteorus ictericus Nees,Hyposoter didymator (Thunberg),Sinophorus sp.; and the dipteran (Tachinidae) parasitoidsPseudogonia rufifrons Wiedeman,Exorista rossica Mesnil,Gonia picea (Robineau-Desvoidy) andLinnaemya vulpina (Fallen) — the last three recorded for the first time as parasitoids ofM. loreyi in Turkey.C. ruficrus was the dominant parasitoid species, being recovered from 38.5% of the larvae collected and was also the most prevalent species, existing in 91.0% of the fields in whichM. loreyi was found. Total parasitism levels achieved by braconid species was 41.4%, by ichneumonid parasitoids 4.8%, and by tachinid parasitoids 1.9%. In a separate field experiment, seasonal population fluctuations and natural efficiency ofC. ruficrus onM. loreyi were found to be 35.1% and 42.4%, respectively. Population levels ofC. ruficrus were closely related to fluctuations in the population ofM. loreyi, with parasitism ranging between 0 and 77.3% during the study. http://www.phytoparasitica.org posting Aug. 28, 2005.     

Effects of fertilization with ammonium sulphate and potassium sulphate on the development of Sphaeropsis sapinea in Corsican pine

Potted plants ofPinus nigra subsp.laricio were fertilized with ammonium sulphate (AS) and potassium sulphate (PS) each year during 3 consecutive years. The then 5-year-old plants were artificially inoculated withSphaeropsis sapinea. The fungus caused much bigger bark necroses in plants that had been fertilized with AS, than in plants fertilized with PS. It is concluded thatP. nigra growing in poor sandy soil becomes more susceptible toS. sapinea when fertilized with an excess of AS. This may explain partly the increased incidence ofS. sapinea inP. nigra stands in areas with a high rate of atmospheric NH₄-deposition in the Netherlands.