Characterization of fungi (Fusarium and Rhizoctonia) and oomycetes (Phytophthora and Pythium) associated with apple orchards in South Africa

Several species of fungi and oomycetes including Fusarium, Rhizoctonia, Phytophthora and Pythium have been reported as root pathogens of apple where they contribute to a phenomenon known as apple replant disease. In South Africa, little is known about specific species in these genera and their pathogenicity toward apple. Therefore, these aspects were investigated along with the development and optimization of qPCR tests for detection and quantification of the most virulent oomycete species. In eight investigated orchards, the oomycete Phythophthora cactorum was widely distributed, while nine Pythium species were differentially distributed among the orchards. Pythium irregulare was the most widely distributed and the most virulent species along with P. sylvaticum, P. vexans and Ph. cactorum. Seven binucleate Rhizoctonia anastomosis groups (AGs) were also differentially distributed among the orchards, with the majority appearing to be non-pathogenic while certain AG-I and AG-F isolates exhibited low virulence on apple. In the genus Fusarium, F. oxysporum was widely distributed, but isolates were non-pathogenic. Fusarium solani and F. avenaceum were less frequently encountered, with only some isolates having low virulence. qPCR data obtained from seedling roots inoculated with the most virulent Pythium species (P. irregulare, P. sylvaticum and P. vexans) and the genus Phytophthora were not always reproducible between trials, or isolates of the same species. In general, seedling growth inhibition was associated with the presence of a low amount of pathogen DNA (±40 fg μl⁻¹ to 2 pg μl⁻¹) in roots. Pythium irregulare, although having the lowest DNA concentrations in roots, was the only species for which a significant negative correlation was found between seedling weight and pathogen DNA concentration.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Diversity and populations of Phytophthora,Phytopythium and Pythium species recovered from sediments in an agricultural run‐off sedimentation reservoir

Agricultural run‐off sedimentation reservoirs are an emerging aquatic system of critical importance to plant biosecurity, water and environmental sustainability. Oomycete pathogens such as Phytophthora and Pythium species in irrigation water have been demonstrated to pose significant risks to ornamental crops, but little is known about their diversity and populations in sediments of agricultural irrigation systems. This study investigated the oomycete communities including Phytophthora (Ph.), Phytopythium (Pp.) and Pythium (Py.) species in sediments at various depths of an agricultural run‐off sedimentation reservoir in Virginia during the winters of 2011 and 2015. The recovery of these oomycetes declined sharply with sediment depth from surface to 0·8 m and none was recovered from sediments deeper than 1·4 m. A total of 47 oomycete species were recovered, with all four species of Phytophthora and five of Phytopythium exclusively from the surface. Recovered species included many important plant pathogens such as Ph. nicotianae, Ph. pini, Ph. tropicalis, Pp.  vexans, Py. irregulare and Py. monospermum. These results underline the importance of decontaminating sediments excavated from top layers (0–1·4 m) of the sedimentation reservoir before reuse in plant production.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

Prevalence of Phytophthora species in macadamia orchards in Australia and their ability to cause stem canker

In Australia, Phytophthora cinnamomi is the only species reported as the causal agent of stem canker and root rot in macadamia. In other countries, five Phytophthora species have been reported to cause diseases in macadamia, which led us to question if more than one Phytophthora species is responsible for poor tree health in macadamia orchards in Australia. To investigate this, samples were collected from the rhizosphere, stem, and root tissues of trees with and without symptoms, nurseries, and water sources from 70 commercial macadamia orchards in Australia. Phytophthora isolates were identified based on morphological characteristics and DNA sequencing. P. cinnamomi was the most predominant and widely distributed species, and was obtained from the different types of samples including symptomless root tissues. In addition to P. cinnamomi, only P. multivora was isolated from diseased tissue (stem canker) samples. Six other Phytophthora species were obtained from the rhizosphere samples: P. pseudocryptogea, P. citrophthora, P. nicotianae, P. gondwanense, P. sojae, and a new Phytophthora taxon. Only P. cinnamomi was obtained from macadamia nursery samples, while five Phytophthora species were obtained from water sources. Of the heterothallic Phytophthora species, mating type A2 isolates were dominant in P. cinnamomi isolates, whereas only mating type A1 isolates were obtained for P. nicotianae, P. pseudocryptogea, and P. citrophthora. Pathogenicity assays revealed that P. cinnamomi and P. multivora caused significantly larger stem and leaf lesions than P. citrophthora, P. nicotianae, and P. pseudocryptogea. Phytophthora sp. and P. sojae were nonpathogenic towards leaves and stems.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

<Emphasis Type="Italic">Pythium</Emphasis> and <Emphasis Type="Italic">Phytophthora</Emphasis> species associated with root and stem rots of kalanchoe

Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).     

Pathogenicity of 21 newly described Phytophthora species against seven Western Australian native plant species

The pathogenicity of some Phytophthora species recently described from Western Australia, together with P. cinnamomi as a control, was tested against seven Western Australian native plant species in the glasshouse. Host species were Banksia grandis, B. littoralis, B. occidentalis, Casuarina obesa, Corymbia calophylla, Eucalyptus marginata and Lambertia inermis. Twenty‐two Phytophthora species were grown on a vermiculite, millet seed and V8 substrate and used as soil inoculum when the plant hosts were approximately 3 months old. Pathogenicity was assessed after 6 weeks and plants were scored for death, root damage, and percentage reduction of shoot growth compared with control plants. The pathogenicity of P. cinnamomi was confirmed. Phytophthora niederhauserii was shown to be similar to P. cinnamomi in pathogenicity and of concern ecologically. Other species that killed one or more hosts were P. boodjera, P. constricta, P. elongata, P. moyootj and P. rosacearum, while P. condilina, P. gibbosa, P. gregata, P. litoralis and P. ‘personii’ caused significant reduction to shoot and/or root growth, but did not kill plants. Host species susceptible to the highest number of Phytophthora species were B. grandis, B. littoralis, B. occidentalis and E. marginata. No Phytophthora species tested killed C. calophylla.     

Age‐related susceptibility of Eucalyptus species to Phytophthora boodjera

Phytophthora boodjera is a newly described pathogen causing damping off and mortality of Eucalyptus seedlings in Western Australian nurseries. This study evaluated the age‐related susceptibility of several taxa of mallee Eucalyptus to P. boodjera in sterilized washed river sand‐infestation pot trials. Phytophthora cinnamomi and P. arenaria were included for comparison. Seedlings of Eucalyptus taxa were inoculated at 0, 2, 4, 12 and 88 weeks with individual Phytophthora isolates. Pre‐emergent mortality in the presence of Phytophthora was almost 100%. Post‐emergent mortality was 50–100%, depending on isolate, compared to 0% for the control. Mortality was also high for inoculated 1 month‐old seedlings (46–68%) and root length of surviving seedlings was severely reduced. Death from root infection was not observed for seedlings inoculated at 12 and 88 weeks, but they developed root necrosis and reduced root dry weight compared to non‐inoculated controls. Phytophthora boodjera is a pre‐ and post‐emergent pathogen of mallee eucalypts. These eucalypts are susceptible to P. boodjera at all life stages tested, but the mortality rates declined with plant age. Similar results were obtained for P. cinnamomi and P. arenaria. The events leading to its recent appearance in the nurseries remain unknown and further investigations are underway to determine if this is an introduced or endemic pathogen. The approach used here to understand the impact of a Phytophthora species on multiple hosts at different seedling ages is novel and sets a benchmark for future work.     

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.     

Occurrence and distribution of <Emphasis Type="Italic">Phytophthora</Emphasis> species in European chestnut stands,and their association with Ink Disease and crown decline

The Phytophthora complex associated with Castanea sativa Mill. was investigated in five European countries in 35 regions and with respect to various domestication levels. Annual precipitation and length of drought season were the main parameters that regulated the presence of Phytophthora species in the chestnut stands. Seven species of Phytophthora were detected; three of these, P. megasperma, P. cryptogea and P. syringae had not been previously reported on sweet chestnut. P. cinnamomi. P. cambivora and P. citricola were most frequently isolated. P. cinnamomi and P. cambivora were the species significantly associated with declining trees with symptoms of Ink Disease. P. cinnamomirequired distinct ecological conditions compared to the other species. P. cinnamomi was never detected in sites characterized by minimum temperatures below 1.4 °C, maximum temperature above 28 °C, or soil pH below 5.4. The results obtained provide useful information for modeling the probability of Ink Disease, crown decline and associated Phytophthora species in chestnut groves in global climatic change scenarios.     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.     

Control of Phytophthora species in plant stock for habitat restoration through best management practices

Emergent plant pathogens represent one of the most significant threats to biodiversity, and exotic Phytophthora species have recently emerged as a serious problem in restored habitats in California and in nurseries producing the plant stock. It is hypothesized that ‘best management practices’ prescribed through a Phytophthora Prevention Programme (PPP) could be useful in minimizing phytophthora disease incidence. To understand the magnitude of the problem and the efficacy of the PPP, plants in restoration nurseries were evaluated for (i) the Phytophthora species assemblage present in the absence of the PPP, and (ii) the effectiveness of the PPP to reduce them. Sampling included 203 plants grown in the absence of the PPP, and 294 grown implementing the PPP. Only samples collected in the absence of the PPP were Phytophthora-positive, and cumulatively yielded 55 isolates from 13 different taxa, including 1 putative interspecific hybrid genotype. There were 21 novel Phytophthora–plant species combinations. The most common Phytophthora species was P. cactorum. Four plant species had the highest disease incidence, namely: Diplacus aurantiacus (50 ± 11.2%), Heteromeles arbutifolia (33 ± 9.6%), Ceanothus thyrsiflorus (30 ± 8.4%), and Frangula californica (30 ± 8.4%). Disease incidence in nurseries after the implementation of the PPP dropped to zero (P < 0.001), and was unaffected to any significant degree by nursery differences, or plant species tested. This study identifies a large number of novel ‘plant species × Phytophthora species’ combinations, and provides for the first time strong evidence that the PPP significantly reduced Phytophthora in plant stock for habitat restoration.     

Phytophthora diversity and the population structure of Phytophthora ramorum in Swiss ornamental nurseries

Invasive oomycete pathogens have been causing significant damage to native ecosystems worldwide for over a century. A recent well‐known example is Phytophthora ramorum, the causal agent of sudden oak death, which emerged in the 1990s in Europe and North America. In Europe, this pathogen is mainly restricted to woody ornamentals in nurseries and public greens, while severe outbreaks in the wild have only been reported in the UK. This study presents the results of the P. ramorum survey conducted in Swiss nurseries between 2003 and 2011. In all 120 nurseries subjected to the plant passport system, the main P. ramorum hosts were visually checked for above ground infections. Phytophthora species were isolated from tissue showing symptoms and identified on the basis of the morphological features of the cultures and sequencing of the ribosomal ITS region. Phytophthora was detected on 125 plants (66 Viburnum, 58 Rhododendron and one Pieris). Phytophthora ramorum was the most frequent species (59·2% of the plants), followed by P. plurivora, P. cactorum, P. citrophthora, P. cinnamomi, P. cactorum/P. hedraiandra, P. multivora and P. taxon PgChlamydo. The highest incidence of P. ramorum was observed on Viburnum × bodnantense. Microsatellite genotyping showed that the Swiss P. ramorum population is highly clonal and consists of seven genotypes (five previously reported in Europe, two new), all belonging to the European EU1 clonal lineage. It can therefore be assumed that P. ramorum entered Switzerland through nursery trade. Despite sanitation measures, repeated P. ramorum infections have been recorded in seven nurseries, suggesting either reintroduction or unsuccessful eradication efforts.     

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.     

Production of Indole-3-acetic Acid and Tryptophol by Pythium ultimum and Pythium Group F: Possible Role in Pathogenesis

Pythium group F is a minor pathogen which induces symptomless infections that occur frequently and results in yield losses in tomato soilless cultures. To elucidate the mode of action of this microorganism, the influence of culture filtrates of Pythium group F on tomato growth was investigated and compared to that of the pathogen Pythium ultimum. Depending on metabolite production by the fungus, marked differences were observed in plant response. Pythium group F crude culture filtrates or low molecular weight fractions (< 500) caused swelling behind the root tip and reduced root growth; the cohesion and adherence of cells within the cortical area were also affected. These symptoms were similar to those observed on plants treated with indole-3-acetic acid. By contrast, P. ultimum filtrates caused a marked distortion of cell shape accompanied with folding of host cell walls, particularly in the cortical area. These symptoms were characteristic of the activity of toxic compound(s) on host cells. Chemical analysis of the filtrates demonstrated that indole-3-acetic acid and tryptophol were produced by Pythium group F and P. ultimum. However, Pythium group F took up and metabolized more indole-3-acetic acid precursors, especially tryptophan, a key amino acid in the pathways leading to indole-3-acetic acid synthesis. The fact that Pythium group F and P. ultimum transformed tryptamine and indole-3-acetaldehyde into indole-3-acetic acid and tryptophol confirms the existence of a tryptamine pathway within both fungi. These results support the hypothesis that auxins facilitate Pythium group F infections. On the other hand, toxin(s) and hydrolytic enzymes are likely involved in P. ultimum pathogenesis.     

Oomycete species associated with Theobroma cacao crops in Colombia

The study of oomycetes associated with crops is highly important due to the economic losses they might cause. In cacao, the genus Phytophthora has been extensively studied, but little is known about other genera and species of oomycetes associated with this plant. This study aimed to determine the oomycetes’ diversity present in Colombian cacao crops. A total of 146 isolates were obtained from diseased plants and soil in 11 departments. Analysis of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (coxI) sequences was performed along with the assessment of morphological characteristics. Nine species were identified, distributed in four genera: Phytophthora (P. palmivora, 54%; P. nicotianae, 1%), Phytopythium (Phy. chamaehyphon, 15%; Phy. cucurbitacearum, 9%; Phy. vexans, 7%; Phy. helicoides, 1%), Globisporangium (G. splendens, 3%), and Pythium (Py. delicense, 1%; Py. inflatum,1%). Additionally, an unidentified and possibly new species of Phytophthora (5%) and three unidentified species of Phytopythium (3%) were found. This is the first report of Globisporangium, Phytopythium, and Pythium in cacao crops of Colombia and the first report of the species Phy. chamaehyphon in the country. Interestingly, some isolates of Phytopythium spp. were isolated from necrotic leaves and vascular section of stems, which may suggest a role in cacao diseases traditionally associated with Phytophthora. Also, it is proposed that the new species of Phytophthora may be contributing significantly to black pod disease in Colombian cacao crops, and we highlight that the study of P. palmivora is urgent because of its distribution all over the country.     

Incidence and distribution of botryosphaeriaceous species in New Zealand vineyards

The aim of this study was to determine the identification, incidence and distribution of botryosphaeriaceous species in New Zealand vineyards. A field study of 43 vineyards across six wine growing regions was conducted. A total of 336 isolates of botryosphaeriaceous species were isolated from 238 diseased grapevine samples. Morphological identification and phylogenetic analysis of the ribosomal RNA gene region, partial sequence of the translation elongation factor 1-alpha gene (EF1-α) and the β-tubulin gene identified nine botryosphaeriaceous species: N. parvum, N. luteum, N. australe, N. ribis, D. mutila, D. seriata, B. dothidea, Do. iberica and Do. sarmentorum. These species have been reported in other grape growing regions worldwide. Eighty eight percent of vineyards and 68% of symptomatic vines sampled were positive for botryosphaeriaceous species. Incidence and distribution of the botryosphaeriaceous species populations varied between the North and South Islands with N. parvum being the predominant species. The variability in incidence and distribution of the botryosphaeriaceous species may be influenced by climatic conditions and different sources of inoculum in the regions sampled. The results of this research clearly identified botryosphaeriaceous species as the target pathogens for development of management strategies for grapevine decline in New Zealand.     

Zoospore chemotaxis of closely related legume‐root infecting Phytophthora species towards host isoflavones

Phytophthora niederhauserii, P. pisi, P. sojae and P. vignae are closely related species that are pathogenic to various legume plants. While P. sojae and P. vignae are reported to specifically infect soybean and cowpea, respectively, P. pisi is reported to attack pea and faba bean. Phytophthora niederhauserii is considered to have a broad host range. Zoospores of some Phytophthora species are chemotactically attracted to the isoflavones that are secreted by their host plants. The focus of the current study was to determine the chemotaxic behaviour of zoospores from closely related legume‐root infecting Phytophthora species and to investigate the correlation, if any, to host preference as determined by greenhouse pathogenicity tests. The results showed that P. sojae and P. vignae were attracted to the non‐soybean isoflavone prunetin as well as to the soybean isoflavones genistein and daidzein, which is in contrast with their host specificity on soybean and cowpea, respectively. On the other hand, P. pisi and P. niederhauserii were only attracted to prunetin, previously reported to be produced by pea, but not to the isoflavones associated with the non‐host soybean. The lack of responsiveness to genistein and daidzein in P. pisi may represent a recent adaptation to the host specialization towards pea. However, the affinity of P. niederhauserii to prunetin shows that this trait can also be present in taxa not specifically associated with legume hosts.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).