Phytophthora foot rot control in citrus with Myrothecium roridum1

Foot rot is the most important disease in citrus in Spain. Phytophthora nicotianae var. parasitica is the major cause of this disease. Field treatments with classic contact fungicides have been extensively used for many years to combat stem and trunk Phytophthora diseases in citrus, while, more recently, foliar application of the systemic fungicide fosetyl-A1 has been used with great success. It is now also possible to use a biological control method for foot rot disease. Myrothecium roridum, a cosmopolitan soil hyphomycete with high cellulolytic activity, produces toxins which are very active against Phytophthora fungi. Culture filtrates of M. roridum cultured in liquid medium with low sugar content strongly inhibit in vitro development of P.n. parasitica, P. syringae and P. capsici. In glasshouse trials employingcross inoculations of M. roridum and P.n. parasitica in stems of sweet orange and willowleaf mandarin seedlings, the bark lesions produced by Phytophthora were clearly stopped at 7 or 10 days after inoculation and the cankers subsequently did not show evident activity. In field trials, M. roridum inoculated into the roots of several citrus species limited development of cankers due to P.n. parasitica (artificial or natural infection). The good results open a new way for control of Phytophthora diseases in citrus.     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

Immunodetection of elicitins from Phytophthora spp. using monoclonal antibodies

Ten monoclonal antibodies were selected from mice immunized with a highly purified elicitin secreted by Phytophthora cryptogea , termed cryptogein. These antibodies could be classified into five groups according to their cross-reactivity to heterologous elicitins from other Phytophthora species, from strict specificity (reacting solely with cryptogein) to broad reactivity (reacting with all four elicitins under study). When examined on BIA core (a real-time biospecific interaction analyser), these monoclonal antibodies were found to recognize at least three different epitopes on the cryptogein molecule. Their use in elicitin detection and quantification was optimized in several ELISA protocols. A mixed monoclonal-polyclonal antibody, indirect DAS-ELISA procedure detected as little as 20 pg of purified elicitin per well (100 μl). The four elicitins could be detected with the aid of one of couple of polyvalent reagents, whilst each one could be detected separately using appropriate monoclonal antibodies. These protocols have been used to detect elicitins secreted by Phytophthora spp. into culture medium as well as in planta following plant inoculation.     

Effect of irrigation on the dynamics of Phytophthora citrophthora populations in soil of citrus orchards1

Effects of irrigation on the dynamics of soil populations of Phytophthora citrophthora have been studied in three citrus orchards in East Sicily (Italy). Significant increases of inoculum levels were detected 24 h after irrigation. In one orchard throughout summer, population of P. citrophthora beneath the tree canopy was more than 15 propagules g-¹ soil, a value which is considered a threshold level for root rot infections.     

Synergism between fungicides with different mechanisms of action against acylalanine-resistant strains of Phytophthora spp.1

The authors have studied the efficacy of two systemic compounds, cymoxanil and propamocarb, used as partners of acylalanines, in limiting mycelial growth of acylalanine-sensitive and resistant strains of Phytophthora capsici and P. nicotianae var. parasitica. The fungicidal activity of the single components was compared to that of the different mixtures, at various ratios. The degree of synergy was calculated using the method of Wadley. The results obtained indicate synergistic interactions of various intensities: synergistic factors (SF) up to seven were found. The highest synergistic effects were observed in the case of resistant Phytophthora strains.     

Involvement of Phytophthora cryptogea in sweet cherry decline in Turkey

Sweet cherry is a major commercial crop in Turkey, the most important producer of the fruit worldwide. Sweet cherry decline was observed in an orchard in Ankara province of Turkey. Affected young trees exhibited reduced tree vigor, yellowing and wilting of leaves, and dieback symptoms resulted in tree death. A Phytophthora sp. was consistently isolated from necroses that appeared on taproots and crowns. The pathogen was identified as Phytophthora cryptogea based on several morphological features and DNA sequence of the internal transcribed spacer (ITS) region. P. cryptogea was pathogenic on excised shoots and 1-year-old cherry rootstocks. This is the first report of P. cryptogea causing disease of sweet cherry in Turkey.     

Phytophthora oleae sp. nov. causing fruit rot of olive in southern Italy

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.     

Characterization of Phytophthora spp. Causing Outbreaks of Citrus Brown Rot in Florida

ABSTRACT Epidemics of citrus brown rot from 1994 to 1997 in the south-central and east-coast citrus areas of Florida were characterized and the causal Phytophthora spp. identified. Two species of Phytophthora, P. palmivora and P. nicotianae, were consistently associated with brown rot. Epidemics caused by P. palmivora appeared to be initiated on immature fruit dropped on the orchard floor. The soilborne fungus infected and sporulated on these fruit and was then disseminated to fruit above 1 m in the canopy. In contrast, infection by P. nicotianae, the common cause of root rot, was confined to the lowest 1 m of the canopy. Fruit infected by P. palmivora produced large amounts of ellipsoidal sporangia available for splash dispersal, whereas those infected by P. nicotianae produced far fewer spherical sporangia. Isolates from brown rot epidemics were compared with P. nicotianae from citrus in Florida and Texas, P. citrophthora in California, P. palmivora, and selected Phytophthora spp. from other hosts. Brown rot symptoms produced by the different pathogenic citrus isolates on inoculated fruit were indistinguishable. Morphology, mating behavior, and isozyme patterns of brown rot isolates from 1988 to 1997 matched P. palmivora from citrus roots, other host plants, and other locations, but were different from characterized isolates of P. citrophthora in California and P. nicotianae in Florida and Texas. Cellulose acetate electrophoresis of the isozyme glucose-6-phosphate isomerase rapidly identified the causal citrus pathogen from infected fruit and soil isolation plates. Although P. palmivora is an aggressive pathogen of citrus roots, bark, and fruit, populations in orchard soils were low compared with P. nicotianae.     

Diclofop-methyl resistance in populations of Lolium spp. from central Italy

Populations of Lolium spp. collected in central Italy were screened for resistance to acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides and compared with known susceptible and resistant Lolium rigidum (Gaud.) populations from Australia. Populations Roma'94 and Tuscania'97 were up to 8- and 7.5-fold more resistant to diclofop-methyl, respectively, than susceptible populations in pot experiments. However, populations Tarquinia'97 and Vetralla'94 were not resistant. Diclofop-methyl resistance levels in the Italian populations were lower than in the Australian populations SLR31 and WLR96 (16.5 and > 64 times more resistant than S respectively). In an agar germination test, Tuscania'97 showed low levels of cross-resistance to fluazifop-p-butyl, whereas no cross-resistance was found in the Roma'94 population.     

Observations of Phytophthora spp. in Water Recirculation Systems in Commercial Hardy Ornamental Nursery Stock

Samples of water and sediment were taken from drains, reservoirs and wells from four commercial hardy ornamental nurseries with water recirculation systems. The samples were taken on seven different dates throughout a single year from August 1994 to July 1995. The samples were screened for Phytophthora species using five different methods: direct plating, three bait tests (using lupin seedlings, apples and Rhododendron leaves) and a DAS-ELISA (double-antibody sandwich enzyme-linked immunosorbent-assay) with two antisera. In the nurseries with old water recirculation systems, Phytophthora species were detected in the drains and in the reservoirs. In the nursery with a new recirculation system, the pathogens were only present in the drains. None of the water samples from wells in any of the nurseries were contaminated. Phytophthora species were present in the water as well as in the sediment samples from drains and reservoirs. They were detected in the water recirculation systems irrespective of the season. The number of isolates increased about sevenfold between late summer and spring. At least 12 different Phytophthora species were identified: some isolates were previously unrecorded species. The epidemiology of the pathogens in outdoor water recirculation systems as well as the importance of the results for commercial nurseries is discussed.     

Control of Zebra Disease of Agave Hybrids by Breeding for Resistance to Phytophthora spp.

Abstract

A zebra disease resistance factor has been incorporated into the high-yielding Agave hybrid no. 11648 by crossing this hybrid with A. lespinassei. The progeny of three crosses have been screened for resistance to Phytophthora nicotianae, the chief pathogen of zebra disease and results show that 56.7% of the plants have sufficient resistance to be of economic value. The two screening techniques used are described, and the nature of the resistance factor discussed.     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

Two Phytophthora species causing decline of wild olive (Olea europaea subsp. europaea var. sylvestris)

Since 2009, a severe decline leading to mortality has been observed affecting nearly 5 ha of a wild olive woodland of high ecological value in Seville, southern Spain. Phytophthora cryptogea and P. megasperma were consistently isolated from roots and rhizosphere of trees with symptoms sampled in 2009, 2011 and 2013. The isolates were identified on the basis of colony and reproductive structure morphology as well as temperature–growth relationships, and identification was further corroborated by their ITS and β‐tubulin sequences. Koch's postulates were demonstrated for both species on 1‐year‐old wild olives. Pathogenicity tests showed that both Phytophthora spp. are highly aggressive pathogens, although temperature–growth requirements for each species were distinct. As a consequence, the two species may be active in different seasons and their epidemiology may be differently influenced by global climate change, and they may show their active periods in different climatic scenarios. The climate change models for the Mediterranean Basin forecast a global temperature increase that favours the more thermophilic P. cryptogea. The high susceptibility to phytophthora root rot should not be disregarded in olive breeding programmes where wild olive is used as a source of resistance to verticillium wilt.     

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.     

Real-time assays for detection of Phytophthora spp. and identification of an avr3a gene variant

Journal of Plant Diseases and Protection - A study was carried out for identification and detection of Phytophthora spp. from soil and plant samples, collected from Solanaceous crops in Egypt and...     

Haplotype diversity of Tilletiopsis spp. causing white haze in apple orchards in Northern Italy

White haze (WH), a manifestation of extensive colonization of anamorphic smut fungi of the genus Tilletiopsis on the surface of apple fruit, has recently been described as a new postharvest disorder of the cultivar Elstar. This study shows that these fungi can also affect apple fruit of different cultivars prior to harvest, and therefore seriously compromise the quality of the produce in Northern Italy. In order to identify the Tilletiopsis taxa involved in the induction of WH in this region, 48 isolates, mainly derived from fruit samples, were analysed by DNA sequencing of the internal transcribed spacer region. Six haplotypes were identified that could be assigned to three different phylogenetic lineages, the T. washingtonensis group, the T. pallescens group and the Entylomataceae. More than 40% of the isolates were classified as T. washingtonensis which had not previously been associated with WH. Furthermore, this is the first study that reports the occurrence of this species on field‐grown fruit samples. The extensive overgrowth of apple fruit by Tilletiopsis spp., appearing erratically in the last decade, may be a consequence of moist weather conditions and novel cultural practices, such as the use of hail nets and the intensive application of foliar nutrients.