Populations of bacteria and actinomycetes associated with sclerotia ofPhymatotrichum omnivorum buried in Houston black clay

Sclerotia ofPhymatotrichum omnivorum (Shear) Duggar (the causal agent of root rot of cotton) were produced in the laboratory and then buried at a depth of 45 cm at three sites in Texas situated on Houston black clay soils with various cropping histories. The sites included a native grassland prairie, a field in continuous cotton production, and a field in which cotton, corn, and sorghum were grown in rotation. Samples of sclerotia were retrieved monthly over a 12 month period. Populations of bacteria and actinomycetes were enumerated using dilution-plate techniques and isolates were screened (in vitro) for their ability to produce substances inhibitory toP. omnivorum. The sclerotia supported large numbers of bacteria (including fluorescent pseudomonads) and actinomycetes. Numbers associated with sclerotia ranged from 10⁶–10⁹ cells per gram of sclerotia plus adherent soil and were 2–3 orders of magnitude greater than numbers from soil at the same depth but free of sclerotia. Bacteria and actinomycetes antagonistic toP. omnivorum were isolated from sclerotia buried at each of the three sites. Up to 26% of the isolates inhibited growth ofP. omnivorum.     

Zinc influences nodulation,disease severity,leaf drop and herbage yield of alfalfa cultivars

Zinc deficiency is an important limiting factor in sustainable crop production and is a factor often overlooked in determining the benefits and overall success of alfalfa pastures in rotations. A field experiment was conducted to investigate the effects of zinc and alfalfa cultivars on nodulation, herbage yield, leaf drop and disease severity (Phytophthora root rot disease and common leaf spot disease) in alfalfa (Medicago sativa L.). Ten cultivars of alfalfa (Hunter River, Hunterfield, Sceptre Aurora, Genesis, Aquarius, Venus (Y8622), PL69, P5929 and PL34HQ) were tested at two levels of zinc (+Zn: 4 kg ha^–1, -Zn: no zinc added) on a Zn-deficient soil (DTPA zinc of top 0–15 cm soil was 0.4 mg kg^–1 soil, while 15–30 cm subsoil Zn was 0.1 mg kg^–1 soil) under field conditions. Zinc application significantly increased number and dry weight of nodules, herbage yield and leaf to stem ratio of alfalfa plants. There was a significant reduction in leaf drop, and occurrence of Phytophthora root rot (caused by fungus Phytophthora megasperma f. sp. medicaginis) and common leaf spot (caused by fungus Pseudopeziza medicaginis) diseases with Zn application. Alfalfa cultivars had a differential response to low Zn. Hunter River and Hunterfield were the most affected by omitting zinc application, while Sceptre, PL34HQ and Aquarius were comparatively less affected. The present study suggest that Zn nutrition effects nodulation, leaf drop, disease occurrence and production potential of alfalfa. The alfalfa cultivars have differential ability to low Zn supply. Growing of Zn-efficient cultivars and adequate Zn nutrition may also improve the N₂-fixation by alfalfa on low-Zn soils.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa

Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon‐tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down‐regulated or up‐regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down‐regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down‐regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.     

Intercrop movement of convergent lady beetle,Hippodamia convergens (Coleoptera: Coccinellidae), between adjacent cotton and alfalfa

A 2‐year study was conducted to characterize the intercrop movement of convergent lady beetle, Hippodamia convergens Guerin‐Meneville (Coleoptera: Coccinellidae) between adjacent cotton and alfalfa. A dual protein‐marking method was used to assess the intercrop movement of the lady beetles in each crop. In turns field collected lady beetles in each crop were assayed by protein specific ELISA to quantify the movement of beetles between the crops. Results indicated that a high percentage of convergent lady beetles caught in cotton (46% in 2008; 56% in 2009) and alfalfa (46% in 2008; 71% in 2009) contained a protein mark, thus indicating that convergent lady beetle movement was largely bidirectional between the adjacent crops. Although at a much lower proportion, lady beetles also showed unidirectional movement from cotton to alfalfa (5% in 2008 and 6% in 2009) and from alfalfa to cotton (9% in 2008 and 14% in 2009). The season‐long bidirectional movement exhibited by the beetles was significantly higher in alfalfa than cotton during both years of the study. The total influx of lady beetles (bidirectional and unidirectional combined) was significantly higher in alfalfa compared with that in cotton for both years. While convergent lady beetles moved between adjacent cotton and alfalfa, they were more attracted to alfalfa when cotton was not flowering and/or when alfalfa offered more opportunities for prey. This study offers much needed information on intercrop movement of the convergent lady beetle that should facilitate integrated pest management decisions in cotton utilizing conservation biological control.     

Green manures and crop sequences influence alfalfa root rot and pathogen inhibitory activity among soil-borne streptomycetes

A two-year trial was conducted to determine the effects of green manures and crop sequences on plant disease, streptomycete and bacterial densities, and inhibitory activity of indigenous streptomycetes against four target pathogens. Green manure treatments, buckwheat (Fagopyrum esculentum L.), canola (Brassica napus L.), sorghum-sudangrass (Sorghum bicolor) (L.) Moench × Sorghum sudanense (Piper) Stapf.), and fallow control were tested in conjunction with three crop sequences in a Phytophthora-infested soil placed in containers. Alfalfa (Medicago sativa L.), potato (Solanum tubersoum L.), or corn (Zea mays L.) was grown in the first year, and alfalfa was grown in all containers in the second year. Compared to fallow controls, alfalfa grown in sorghum-sudangrass- or buckwheat-treated soil had significantly greater stand counts and total biomass, respectively. In addition, alfalfa grown in fallow-treated soils had the greatest Phytophthora root rot as a function of stand count. Crop rotation also had a significant effect on alfalfa root rot and yield. Potato scab disease intensity was greatest on tubers grown in fallow-treated soils, while tubers grown in canola-treated soils had the highest yields (total tuber weight). Green-manure-treated soils tended to have greater streptomycete and bacterial densities than fallow-treated soils. In addition, buckwheat- or sorghum-sudangrass-treated soils had greater proportions of streptomycetes that were antagonistic against the target pathogens than fallow-treated soils. The proportion of antagonists in soil was negatively correlated with alfalfa root rot, and positively correlated with alfalfa stand counts. Inhibitory activity of the streptomycetes was also negatively correlated with potato scab and positively correlated with potato yield. These data suggest that green manures may provide a strategy for increasing pathogen inhibitory activity within the streptomycete community in soil, and, in conjunction with crop rotation, may contribute to the control of a diverse collection of soil-borne plant pathogens on multiple crop species.     

Genotyping‐by‐sequencing‐based genome‐wide association studies on Verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa L.)

Verticillium wilt (VW) is a fungal disease that causes severe yield losses in alfalfa. The most effective method to control the disease is through the development and use of resistant varieties. The identification of marker loci linked to VW resistance can facilitate breeding for disease‐resistant alfalfa. In the present investigation, we applied an integrated framework of genome‐wide association with genotyping‐by‐sequencing (GBS) to identify VW resistance loci in a panel of elite alfalfa breeding lines. Phenotyping was performed by manual inoculation of the pathogen to healthy seedlings, and scoring for disease resistance was carried out according to the standard test of the North America Alfalfa Improvement Conference (NAAIC). Marker–trait association by linkage disequilibrium identified 10 single nucleotide polymorphism (SNP) markers significantly associated with VW resistance. Alignment of the SNP marker sequences to the M. truncatula genome revealed multiple quantitative trait loci (QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and 8, respectively. Resistance loci found on chromosomes 7 and 8 in the present study co‐localized with the QTLs reported previously. A pairwise alignment (blastn ) using the flanking sequences of the resistance loci against the M. truncatula genome identified potential candidate genes with putative disease resistance function. With further investigation, these markers may be implemented into breeding programmes using marker‐assisted selection, ultimately leading to improved VW resistance in alfalfa.     

Physiological studies on Phymatotrichum omnivorum. IX. Synthesis of indole acetic acid in vitro.

The cotton root rot fungus, Phymatotrichum omnivorum, synthesized indole acetic acid (IAA) in culture medium containing tryptophan as the only source of nitrogen. Synthesis of IAA was not affected by illumination, and maximum amounts of the auxin were produced at 30 days. The optimum hydrogen ion concentration and temperature for IAA production were 6.5 and 28 degrees C, respectively. Indole ethanol (IEA) was also detected in tryptophan media, primarily during the active growth of the organism.     

Trichoderma and chitin mixture based bioformulation for the management of head rot (Sclerotinia sclerotiorum (Lib.) deBary)–root-knot (Meloidogyne incognita Kofoid and White; Chitwood) complex diseases of cabbage

The effect of application of different biocontrol agents (Trichoderma spp (eight isolates), Pseudomonas fluorescens (four isolates) and Bacillus subtilis (two isolates)) was tested against head rot fungus, Sclerotinia sclerotiorum and root-knot nematode, Meloidogyne incognita complex diseases. In vitro studies showed that three Trichoderma isolates (Tvc1, Tvc2 and Thc) were effective in inhibition of mycelial growth of the fungus as well as egg hatching ability of the nematode. Application of talc based formulation of biocontrol agents (bioformulations) individually as well as in mixtures with or without chitin was tested against head rot–root-knot disease complex under greenhouse conditions. The combined application of bioformulation mixture (Tvc1, Tvc2 and Thc) along with chitin reduced the incidence of the complex diseases and induced significantly increased activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and chitinase in cabbage. Activities of PAL and chitinase reached maximum levels within 10 – 20 d, while the activity of PAL continued to be maintained up to 40 d after the application of Tvc1 + Tvc2 + Thc + chitin. Isozyme analyses observed that unique PO (PO1, PO2 PO3 and PO4) and PPO (PPO1, PPO2 and PPO3) enzymes were induced after 10 d in cabbage plants treated with Tvc1 + Tvc2 + Thc + chitin upon challenge inoculation with head rot–root-knot pathogens. Similarly, the bioformulation mixture with chitin was successful at reducing the incidence of head rot–root-knot apart from enhancing the crop yield under field conditions. The mechanism associated with reduced incidence of head rot–root-knot in cabbage may be due to induction of defense proteins (PAL, PO, PPO and chitinase) in the crop.     

Genetic and physical localization of an anthracnose resistance gene in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Anthracnose of alfalfa, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. An improved understanding of the genetic and molecular mechanisms underlying host resistance will facilitate the development of resistant alfalfa cultivars, thus providing the most efficient and environmentally sound strategy to control alfalfa diseases. Unfortunately, cultivated alfalfa has an intractable genetic system because of its tetrasomic inheritance and out-crossing nature. Nevertheless, the model legume Medicago truncatula, a close relative of alfalfa, has the potential to serve as a surrogate to map and clone the counterparts of agronomically important genes in alfalfa—particularly, disease resistance genes against economically important pathogens. Here we describe the high-resolution genetic and physical mapping of RCT1, a host resistance gene against C. trifolii race 1 in M. truncatula. We have delimited the RCT1 locus within a physical interval spanning ∼200 kb located on the top of M. truncatula linkage group 4. RCT1 is part of a complex locus containing numerous genes homologous to previously characterized TIR-NBS-LRR type resistance genes. The result presented in this paper will facilitate the positional cloning of RCT1 in Medicago.     

Bacillus subtilis subsp. spizizenii MB29 controls alfalfa root rot caused by Fusarium semitectum

Medicago sativa L. is the most important forage legume in China. Reducing production losses caused by disease is an essential aspect of maximising alfalfa production. In the current study a Fusarium semitectum isolate collected from alfalfa roots exhibiting symptoms of root rot was proven to infect alfalfa by fulfilling Koch's postulates. A bacterial strain, MB29, also collected from alfalfa roots, was evaluated as a potential biocontrol agent against F. semitectum and a range of other alfalfa pathogens using in vitro tests. It was found that MB29 reduced the mycelia growth of all the pathogens assessed, and in the case of F. semitectum by as much as 84.47%. Furthermore, in vivo test showed that MB29 reduced the severity of rot symptoms in alfalfa seedlings resulting from F. semitectum infection. Strain MB29 was subsequently classified as Bacillus subtilis subsp. spizizenii using the Biolog MicroLog microbial identification system and sequence analysis of its 16S rDNA gene. Taken together these results indicate that B. subtilis subsp. spizizenii MB29 has great potential for the control of root rot diseases in alfalfa.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

A New Report of Fruit Rot of Lemon Caused by Ceratocystis radicicola from Iran

In the year 2000, a fungus was isolated from rotting lemon (Citrus limon) fruit in the Kahnouj region, south‐eastern Iran and subsequently identified as Ceratocystis radicicola (anamorph: Chalara sp.). A pathogenicity test was carried out to fulfil Koch's postulates. The pathogen could only enter the ripen lemon fruit and cause the rot disease through the wounds and cracks. The fungus was also pathogenic on fruit of other citrus species as well as date palm (Phoenix dactylifera). Symptom development was slow on fruits on the tree but accelerated after fruit harvest.     

Immobilization ofPhanerochaete chrysosporium on porous polyurethane particles with application to biodegradation of 2-chlorophenol

Porous polyurethane particles were prepared and used for the immobilization of white rot fungusPhanerochaete chrysosporium. The immobilized cells were employed for the production of lignin peroxidase. Polyurethane immobilized spores, or mycelial pellets ofPhanerochaete chrysosporium as well as freely suspended mycelial pellets of fungus were used as biocatalyst for the degradation of 2-chlorophenol. The polyurethane carriers appear to be superior to the other carriers already used for the immobilization of fungus.     

Seed Rot,a New Disease of Beech Tree Caused by Neonectria ramulariae (anamorph: Cylindrocarpon obtusiusculum)

During a survey of seed diseases of Fagus crenata, a new fungal disease of the seeds was found with high frequency in Akita, northern Japan. Main symptoms are often expressed as browning of the cracked parts from exposed cotyledons and complete loss of viability of infected seeds. Reddish perithecia and whitish yellow sporodochia were occasionally observed symptoms and determined that they were anamorph–telemorph relationship on the basis of both cultural observations. Inoculation studies confirmed that this fungus was the cause of seed rot. The fungus is morphologically identical with Neonectria ramulariae (anamorph: Cylindrocarpon obtusiusculum) that is well known as the soil‐born fungi around the world. Sequences between the authentic isolate of Neo. ramulariae (CBS 151.29) and the pathogenic fungus based on ITS, LSU and tub showed high similarity. Thus, ‘seed rot’, the new disease of beech seeds caused by Neo. ramulariae (anamorph: Cyl. obtusiusculum) was proposed in this study.     

Die Beurteilung des Einflusses qualitativer Prüffaktoren (Infektionstermine,Sorten) auf den Befall mit Hilfe der Kontingenztafelanalyse

Mycelial growth of some wood‐rotting fungi was studied on a solid modified medium MS (Murashige and Skoog, 1962) with indole‐3‐acetic acid at concentrations of 10^‐6 to 10^‐3 M. The IAA concentrations of 10^‐6 M and 10^‐5 M inhibited mycelial growth of the fungus Phaeolus schweinitzii, Laetiporus sulphureus and Pleurotus ostreatus while the same concentrations stimulated mycelial growth of the fungus Stereum rugosum. The IAA concentrations of 10^‐6 M stimulated mycelial growth in Piptoporus betulinus and temporarily stimulated mycelial growth in Heterobasidion annosum. The IAA concentration of 10^‐4 M appeared critical for wood‐rotting fungi. The IAA concentration of 10^‐3 M inhibited mycelial growth in all the fungi under study.     

Ontogenetic and trans-generational dynamics of a vertically transmitted fungal symbiont in an annual host plant in ozone-polluted settings

Tropospheric ozone is an abiotic stress of increasing importance in the context of global climate change. This greenhouse gas is a potent phytotoxic molecule with demonstrated negative effects on crop yield and natural ecosystems. Recently, oxidative stress has been proposed as a mechanism that could regulate the interaction between cool-season grasses and Epichloë endophytes. We hypothesized that exposure of Lolium multiflorum plants, hosting endophytes to an ozone-polluted environment at different ontogenetic phases, would impact the trans-generational dynamics of the vertically transmitted fungal symbiont. Here, we found that the ozone-induced stress on the mother plants did not affect the endophyte vertical transmission but it impaired the persistence of the fungus in the seed exposed to artificial ageing. Endophyte longevity in seed was reduced by exposure of the mother plant to ozone. Although ozone exposure did not influence either the endophyte mycelial concentration or their compound defences (loline alkaloids), a positive correlation was observed between host fitness and the concentration of endophyte-derived defence compounds. This suggests that fungal defences in grass seeds were not all produced in situ but remobilized from the vegetative tissues. Our study reveals ozone trans-generational effects on the persistence of a beneficial symbiont in a host grass.