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A question regarding trichomes in plants

A question regarding trichomes in plants


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Trichomes in general are features of xerophytic leaves, which reduce water loss by evaporation by trapping water vapor and increasing humidity (as a result lowering the water potential gradient). BUT I wanted to know whether trichomes also reduce water loss by decreasing "surface area" of the leaf, so that less water loss occurs (considering they occur as finger-like projected structures that occur in large numbers)?


Trichomes can serve the purpose you ask about, but certainly not only that. They also appear on a great many other, some very delicate, plants from many different habitats. Decreasing water loss is only one of a number of different functions they might serve.

But with respect to xerophytic plants, trichomes are still a projection from the leaf surface and so, by definition, increase the surface area of a leaf by a significant amount. However, it's not the surface area per sé that leads to water loss, it's increased evapotranspiration which can be caused by and mitigated by many different factors, one of which is, indeed, trichomes.

Some trichomes, such as those on Tillandsia species, occlude or reflect light. This reduction in incident light is primarily what protects the leaf tissue from damage and helps reduce water loss due to heat. Such trichomes may also function, as you mention in your question, by creating a insulating layer of still air around the leaf, thereby reducing diffusion of water vapor away from the leaf and creating a smaller gradient, though this is usually not their primary function.


Background

Genetic variation in plants alters insect abundance and community structure in the field however, little is known about the importance of a single gene among diverse plant genotypes. In this context, Arabidopsis trichomes provide an excellent system to discern the roles of natural variation and a key gene, GLABRA1, in shaping insect communities. In this study, we transplanted two independent glabrous mutants (gl1𠄱 and gl1𠄲) and 17 natural accessions of Arabidopsis thaliana to two localities in Switzerland and Japan.

Results

Fifteen insect species inhabited the plant accessions, with the insect community composition significantly attributed to variations among plant accessions. The total abundance of leaf-chewing herbivores was negatively correlated with trichome density at both field sites, while glucosinolates had variable effects on leaf chewers between the sites. Interestingly, there was a parallel tendency for the abundance of leaf chewers to be higher on gl1𠄱 and gl1𠄲 than on their different parental accessions, Ler-1 and Col-0, respectively. Furthermore, the loss of function in the GLABRA1 gene significantly decreased the resistance of plants to the two predominant chewers flea beetles and turnip sawflies.

Conclusions

Overall, our results indicate that insect community composition significantly varies among A. thaliana accessions across two distant field sites, with GLABRA1 playing a key role in altering the abundance of leaf-chewing herbivores. Given that such a trichome variation is widely observed in Brassicaceae plants, the present study exemplifies the community-wide effect of a single plant gene on crucifer-feeding insects in the field.

Electronic supplementary material

The online version of this article (10.1186/s12870-019-1705-2) contains supplementary material, which is available to authorized users.


The 10 questions most important to society

A1. How do we feed our children’s children?

By 2050 the world population will have reached c. 9 billion people. This will represent a tripling of the world population within the average lifetime of a single human being. The population is not only expanding, but also becoming more discerning, with greater demands for energy-intensive foods such as meat and dairy. Meeting these increasing food demands over the years to come requires a doubling of food production from existing levels. How are we going to achieve this? Through the cultivation of land currently covered in rainforests, through enhanced production from existing arable land or by changing people’s habits to change food consumption patterns and reduce food waste? The reality is probably a combination of all three. However, if we are to reduce the impact of food production on the remaining wilderness areas of the planet then we need significant investment in agricultural science and innovation to ensure maximum productivity on existing arable land.

A2. Which crops must be grown and which sacrificed, to feed the billions?

The majority of agricultural land is used to cultivate the staple food crops wheat (Triticum aestivum), maize (Zea mays) and rice (Oryza sativa), the oil-rich crops soy (Glycine max), canola (Brassica napus), sunflower (Helianthus spp.) and oil palm (Elaeis guineensis) and commodity crops such as cotton (Gossypium spp.), tea (Camellia sinensis) and coffee (Coffea spp.). As the world population expands and meat consumption increases, there is a growing demand for staples and oil-rich crops for both human needs and animal feed. Without significant improvements in yields of these basic crop plants, we will experience a squeeze on agricultural land. It is therefore essential that we address the yield gap the difference between future yield requirements and yields available with current technologies, management and gene pools. Otherwise we may be forced to choose between production of staple food crops to feed the world population and the production of luxury crops, such as tea, coffee, cocoa (Theobroma cacao), cotton, fruits and vegetables.

A3. When and how can we simultaneously deliver increased yields and reduce the environmental impact of agriculture?

The first green revolution of the late 1950s and early 1960s generated unprecedented growth in food production. However, these achievements have come at some cost to the environment, and they will not keep pace with future growth in the world population. We need creative and energetic plant breeding programmes for the major crops world-wide, with a strong public sector component. We need to explore all options for better agronomic practice, including better soil management and smarter intercropping, especially in the tropics. Finally, we need to be able to deploy existing methods of genetic modification that reduce losses to pests, disease and weeds, improve the efficiency of fertilizer use and increase drought tolerance. We also need to devise methods to improve photosynthetic efficiency, and move the capacity for nitrogen fixation from legumes to other crops. These are all desirable and, with public support, feasible goals.

A4. What are the best ways to control invasive species including plants, pests and pathogens?

Invasive species are an increasingly significant threat to our environment, economy, health and well-being. Most are nonindigenous (evolved elsewhere and accidentally introduced) and have been removed from the constraints regulating growth in their native habitat. The best method of control is to prevent establishment in the first place or to quickly identify establishment and adopt an eradication programme. However, if an invasive species becomes established many of the options for removal can cause environmental damage, for example chemical control or mechanical excavation. Biological control (introduction of a natural predator ⁄ pathogen) can work well as long as the control organism targets only the invasive species. Otherwise there is a risk that the control organism might also become an invasive species. Alternatives, such as manipulating existing natural enemies and ⁄ or the environment to enhance biological control, are also being developed. Sustainable solutions are required if we are to deal with the continually growing problem of invasive species.

A5. Considering two plants obtained for the same trait, one by genetic modification and one by traditional plant breeding techniques, are there differences between those two plants that justify special regulation?

The products of traditional plant breeding are subject to no special regulations, even though the wild sources of germplasm often used by breeders may contain new components that have not been assessed before. A plant derived by genetic modification, however, is highly regulated, even though the target genotype and the modification itself may both be highly characterized and accepted as innocuous for their intended use. This is a major exception to the norm for safety regulation in food and other areas, which is normally based on the properties of the object being regulated. It is important for food safety and for the public’s perception of science and technology in general to establish whether there are any objective differences between these groups of products that justify the different approaches to their regulation.

A6. How can plants contribute to solving the energy crisis and ameliorating global warming?

Plants use solar energy to power the conversion of CO2 into plant materials such as starch and cell walls. Plant material can be burnt or fermented to release heat energy or make fuels such as ethanol or diesel. There is interest in using algae (unicellular aquatic plants) to capture CO2 emissions from power stations at source. Biomass cellulose crops such as Miscanthus giganteus (Poaceae) are already being burnt with coal at power stations. There is understandable distaste for using food crops such as wheat and maize for fuel, but currently 30% of the US maize crop is used for ethanol production, and sustainable solutions are being found. Sugarcane (Saccharum officinarum) significantly reduces Brazil’s imports of fossil fuels. Agave (Agavea fourcroydes) in hot arid regions can provide very high yields (> 30 T ha)1) of dry matter with low water inputs compared with other crops. To ameliorate global warming, CO2 must be taken out of the air and not put back. There is considerable interest in ‘biochar’ in which plant material is heated without air to convert the carbon into charcoal. In this form, carbon cannot readily re-enter the air, and, if added to the soil, can increase fertility. Carbon markets do not currently provide sufficient incentive for farmers to grow crops simply to take CO2 out of the air.

A7. How do plants contribute to the ecosystem services upon which humanity depends?

Ecosystem services are those benefits we human beings derive from nature. They can be loosely divided into supporting (e.g. primary production and soil formation), provisioning (e.g. food, fibre and fuel), regulating (e.g. climate regulation and disease regulation) and cultural (e.g. aesthetic and recreational) services. Plants are largely responsible for primary production and therefore are critical for maintaining human well-being, but they also contribute in many other ways. The Earth receives virtually no external inputs apart from sunlight, and the regenerative processes of biological and geochemical recycling of matter are essential for life to be sustained. Plants drive much of the recycling of carbon, nitrogen, water, oxygen, and much more. They are the source of virtually all the oxygen in the atmosphere, and they are also responsible for at least half of carbon cycling (hundreds of billions of metric tons per year). The efficiency with which plants take up major nutrients, such as nitrogen and phosphorus, has major impacts on agricultural production, but the application of excess fertilizers causes eutrophication, which devastates acquatic ecosystems. Plants are already recognized as important for sustainable development (e.g. plants for clean water) but there are many other ways that plants might contribute. A combined approach of understanding both the services provided by ecosystems and how plants contribute to the functioning of such ecosystems will require interdisciplinary collaboration between plant scientists, biogeochemists, and ecologists.

A8. What new scientific approaches will be central to plant biology in the 21st Century?

Biologists now have a good general understanding of the principles of cell and developmental biology and genetics, and how plants function, change, and adapt to their environment. Addressing the questions in this list, including those related to generating crops that can deal with future challenges, will require detailed knowledge of many more processes and species. New high-throughput technologies for analysing genomes, phenotypes, protein complements, and the biochemical composition of cells can provide us with more detailed information in a week than has ever been available before about a particular process, organism or individual. This is delivering a deluge of information that is both exhilarating and daunting. The challenge is to develop robust ways of analysing and interpreting this mountain of data to answer questions and deliver new insights. The skill sets required to make full use of the new information extend far beyond those previously expected from biologists. There is general agreement that we need a new era of collaboration between all types of plant scientists, geographers, geologists, statisticians, mathematicians, engineers, computer scientists, and other biologists to evaluate complex data, find new relationships, develop and test hypotheses, and make discoveries. Challenges include understanding complex traits and interactions with the environment, generating ‘designer crops’, and using modelling to predict how different genotypes will cope with alterations in the climate.

A9. (a) How do we ensure that society appreciates the full importance of plants?

Plants are fundamental to all life on Earth. They provide us with food, fuel, fibre, industrial feedstocks, and medicines. They render our atmosphere breathable. They buffer us against extremes of weather and provide food and shelter for much of the life on our planet. However, we take plants and the benefits they confer for granted. Given their importance, should we not pay plants greater attention and give higher priority to improving our understanding of them? Awareness could be increased through the media, school education, and public understanding of science activities, but a major step-change in activity will be required to make a substantial difference.

A9. (b) How can we attract the best young minds to plant science so that they can address Grand Challenges facing humanity such as climate change, food security, and fossil fuel replacement?

Everyone knows that we need doctors, and the idea that our best and brightest should go into medicine is embedded in our culture. However, even more important than medical care is the ability to survive from day to day this requires food, shelter, clothes, and energy, all of which depend on plants. Beyond these essentials, plants are the source of many other important products. As is clear from the other questions on this list, plant scientists are tackling many of the most important challenges facing humanity in the 21st Century, including climate change, food security, and fossil fuel replacement. Making the best possible progress will require exceptional people. We need to radically change our culture so that ‘plant scientist’ (or, if we can rehabilitate the term, ‘botanist’) can join ‘doctor’, ‘vet’ and ‘lawyer’ in the list of top professions to which our most capable young people aspire.

A10. How do we ensure that sound science informs policy decisions?

It is important that policy decisions that can affect us all, for example environmental protection legislation, are based on robust and objective evidence underpinned by sound science. Without this, the risk of unintended consequences is severe. Ongoing dialogue between policy makers and scientists is therefore critical. How do we initiate and sustain this dialogue? How do we ensure that policy makers and scientists are able to communicate effectively? What new mechanisms are needed to enable scientists to respond to the needs of policy makers and vice versa?


RESULTS

The overexpression phenotype: In wild-type plants the initiation of trichomes in the first leaf pair is tightly coupled with the stages of leaf development (P yke et al. 1991 L arkin et al. 1996). Leaf primordia must reach a minimum size of

100 μm before trichome initiation begins (Figure 1A). Trichome formation at the leaf base and within intercalary zones between existing trichomes continued until the leaf reached

900 μm. The mature leaf had a mean of 28 ± 0.97 trichomes on its adaxial surface (Table 1). Trichome initiation in 35S::GL1 plants mirrored the behavior of wild type up to about the 400-μm stage, after which initiation was inhibited (Figure 1B). Trichome number at this stage is not significantly different from the trichome number on a fully expanded first leaf (Table 1). This behavior is very similar to that of RTNler in Col background (see Figure 7 in L arkin et al. 1996) and is consistent with 35S::GL1 inhibition throughout the entire leaf after a defined developmental stage. The spatial arrangement of trichomes on 35S::GL1 leaves represents the final position of the epidermal cells that had differentiated into trichomes at or before the 400-μm stage. The presence of 9.7 ± 1.0 trichomes within 0.3 m m of the leaf margin of 35S::GL1, compared to 15 ± 1.3 in wild type, is also consistent with inhibition of intercalary trichome initiation in the population of epidermal cells near the leaf margin. It is not known if the 35S::GL1-dependent inhibition is mediated by cell-cell contact and/or organ/tissue level processes.

Relationship between leaf length and trichome number in wild-type Col and in homozygous lines of Col that were transformed with the 35S::GL1 transgene. Trichome number for individual leaves at defined lengths was measured and plotted on the histogram. (A) Col. (B) 35S::GL1. The mean trichome number ± SD for mature leaves is indicated with vertical error bars for each genotype. The mean final leaf length for Columbia is 5.4 ± 0.5 m m and for 35S::GL1 is 5.3 ± 0.6 mm.


Update: Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest

Multicellular glandular trichomes are epidermal outgrowths characterized by the presence of a head made of cells that have the ability to secrete or store large quantities of specialized metabolites. Our understanding of the transcriptional control of glandular trichome initiation and development is still in its infancy. This review points to some central questions that need to be addressed to better understand how such specialized cell structures arise from the plant protodermis. A key and unique feature of glandular trichomes is their ability to synthesize and secrete large amounts, relative to their size, of a limited number of metabolites. As such, they qualify as true cell factories, making them interesting targets for metabolic engineering. In this review, recent advances regarding terpene metabolic engineering are highlighted, with a special focus on tobacco (Nicotiana tabacum). In particular, the choice of transcriptional promoters to drive transgene expression and the best ways to sink existing pools of terpene precursors are discussed. The bioavailability of existing pools of natural precursor molecules is a key parameter and is controlled by so-called cross talk between different biosynthetic pathways. As highlighted in this review, the exact nature and extent of such cross talk are only partially understood at present. In the future, awareness of, and detailed knowledge on, the biology of plant glandular trichome development and metabolism will generate new leads to tap the largely unexploited potential of glandular trichomes in plant resistance to pests and lead to the improved production of specialized metabolites with high industrial or pharmacological value.


Structure of Epidermis in Plants (With Diagram)

In this article we will discuss about the structure of epidermis in plants. This will also help you to draw the structure and diagram of epidermis in plants.

The outermost layer or layers of cell covering all plant organs are the epidermis. It is in direct contact with the environment and so it modifies itself to cope up with the natural surroundings.

It thus protects the inner tissues from any adverse natural calamities like high temperature, desiccation, mechanical injury, external infection etc. In some plants the epidermis may persist throughout the life, while in others it is replaced by periderm when the epidermis is sloughed off along with underlying tissues.

The epidermis of all organs originates from the outermost layer of apical meristem. Haberlandt, Hanstein and Schmidt called this surface layer of meristem as protoderm, dermatogen and tunica respectively. In cryptogams epidermis originates from a single initial cell that also forms cortex and stele.

The epidermis of gymnospermous root originates, in association with root cap, from periblem. In dicotyledonous root, epidermis develops from initials of dermatogen, which are not distinct from those of root cap. The epidermis of monocotyledonous root owes its origin from the periblem along with the cortex.

Structure and Contents:

Usually the epidermis consists of one layer of cells. Several-layered epidermis, termed multiple epidermis, is found in the leaves of Ficus, Nerium and in the aerial roots of orchid. The initials of epidermis divide periclinally to form multiple epidermis. The multiple epidermis of orchid root has the special name —velamen.

The epidermis of aerial parts of a plant consists of living parenchyma cells whose shape, size and arrangement may differ. The epidermal cells are more or less tabular (=horizontally flattened) in cross sectional view. In leaves, the epidermal cell walls appear as sinuous in dicots and in monocots they appear as straight or sinuous in surface view. Usually the cells of epidermis are compactly set with none or few intercellular spaces (e.g. flower petals).

The epidermal cells are devoid of chloroplasts. The guard cells of stomata that are specialized epidermal cells contain chloroplastids. Other pigment like anthocyanin may occur in epidermal cells. In some plants silicon may be deposited in the epidermal cells cither in the lumen or wall. The wall of trichome may be silicified.

Silicon containing cell can be differentiated from the adjacent epidermal cells by its shape and size. This cell is solitary and may be either scattered over the leaf surface or situated over the veins in longitudinal rows. Silicon is deposited in the bracts of rice, in the marginal trichomes of oat, in the leaves of Cyperus, Avena etc.

In the internode of Avena sativa, the epidermal cells at the intercostal position form cork-silica cell pairs, i.e. cork and silica containing cells are in close contact with each other. In the leaf of Ficus, some of the epidermal cells contain crystals of calcium carbonate, known as cystolith. These cells are easily distinguishable from the other epidermal cells by their large size and these specialized epidermal cells containing cystolith are called lithocyst.

Usually the walls of epidermal cells are thin. Thick walled lignified epidermal cells occur in some gymnosperms. Cutin, a fatty substance, is very often deposited on the outer surface of the epidermal cell wall to form cuticle over which wax may also be deposited. The cuticle is resistant to decay and is well preserved in fossils.

The cuticle often preserves the characteristic features of the epidermal surfaces such as the types and distribution of hairs and stomata. Thus the fossil plants may be identified by cuticular studies. Palmer (1976) used the fossil grass cuticle instead of grass pollen, as a new palaeoecological tool to reconstruct the nature of past vegetation of East African lake sediments.

Now a days cuticular pattern is used in recognizing small fragments of plants, which are necessary in forensic medicine, animal nutrition, pharmacognosy etc. Cuticular pattern is also taxonomically useful to characterize genus and species. The cuticle is impervious to water but in grapes water diffuses out when it is transformed to sultana. It has protective function. Cutin is resistance to microorganisms and prevents the entry of the pathogen.

In many plants wax is deposited on the surface of the cuticle. This forms a powdery coating on various fruits, e.g. plum, grapes etc. and on leaves. This gives a glossy appearance to the surface of leaves and fruits (e.g. grapes). Wax is deposited either in the form of granules, rods or tubes, which form various specific patterns on the surface.

The deposition may also be in the form of projections and folds. Wax is also deposited on the inside of the pitcher of Nepenthes in the form of overlapping scales. The scales adhere to the feet of insects, which fall inside the pitcher. So the insects cannot climb out of the pitcher. The morphological form of the deposition of wax is typical for the species.

With the aid of scanning electron microscope the wax and cuticular pattern can be observed directly. The study of wax pattern on the epidermal surface is extremely useful in agricultural practices. Waxy epidermis is not wetted. So the effectiveness of fungicide and herbicide can be obtained by studying the extent of wax deposition. Wax obtained from the wax palm Copernicia cerifera is commercially used in making polishes and phonographs records.

Hairs are present all over the surfaces of plant organs namely — roots, stems, leaves, floral parts, seeds (e.g. Gossypium) and stamens (ex. Tradescantia).

They are present at a short distance behind the root tip of most monocots and dicots. Root hairs in some species are formed from distinct epidermal cells termed trichoblast.

Trichoblasts may be morphologically similar to other epidermal cells or they are distinguishable by their smaller size with dense cytoplasm. Trichoblast prolongs to form unicellular root hairs. The cell wall is generally thin and is covered by a thin layer of cuticle mucilage may also occur on the surface (Fig. 12.1).

Apart from anchorage the other main function of root hairs is absorption. It has been found that the rate of absorption in the epidermal surface with and without root hairs is same. Moreover the short root hairs are more efficient in absorption than longer ones. So it appears that the longer hairs absorb the distantly situated water. The root hairs synthesize cellulose at their tips.

Hairs other than root hairs:

Hairs or trichomes are the outgrowths of epidermal cells. They are either unicellular or multicellular. Multicellular hairs may be composed of a single linear row of cells or several rows.

Trichomes, either unicellular or multicellular, are classified into glandular and non-glandular hairs. The former is secretory in function and the latter is the covering hair and does not secrete. It is believed that non-glandular trichomes are protective in function and may prevent undue water loss (Fig. 12.2).

The covering trichomes may have a star like appearance (stellate hair) or a miniature tree (dendroid hair, e.g. Verbascum). In Hamamelis the covering hairs occur in tufts. Hair like projections are present in may flower petals termed papillae. The glandular hairs consist of a stalk and a head that may be unicellular or multicellular. A cuticle like structure covers the head.

The secretory substances accumulate in the sac formed between the cuticle and the cell of head. Example: Oils, resins, camphor, peppermint (e.g. Mentha) etc. The leaves of Olea have scales, which are composed of a short stalk and head, consisting of discoid plate of cells.

Some of the epidermal cells of Mesembryanthemnm may enlarge where water accumulates. These specialized epidermal cells are termed as vesicles or bladders. Most trichomes have thin and cellulosic cell wall lignified cell walls also occur (ex. seed coat of Strychnos nuxvoniica).

Trichomes with their different types may be of taxonomic significance. The types of trichome can identify the several species of Oleaceae and Rhododendron to some extent.

It is a group of outer epidermal cells that can be easily distinguished from the typical epidermal cells by their fan like appearance in cross section and larger sizes. The median cell of bulliform cell is the tallest and the size of the other cells, present on the two sides, diminishes gradually. The cells are thin walled, hyaline and have large vacuole.

The cells contain much water and are devoid of chloroplastids. The cell walls are composed of cellulose and pectic substances cutin occurs on the outermost wall that is covered by cuticle. They usually form isolated strips that are situated on parallel between the veins. They are present on the outer epidermis of the leaves of Poaceae and other mono­cotyledons except Helobiae (Fig. 12.3).

Opinion varies regarding the functions of bulliform cell, which are:

(i) The rolled leaf in bud unrolls with the help of bulliform cell.

(ii) The turgidity and flaccidity of bulliform cell due to water uptake and loss respectively cause the closing and opening of mature leaves.

(iii) These cells act as water reservoir.

(iv) These cells are often filled with silica and their outer walls become thick and cuticularized thus providing mechanical rigidity to leaves.

Stoma (pl. stomata, physiologists usually call stomate) that occurs predominantly on leaves and young stems can be defined as a pore enclosed by two specialized cells —the guard cells that move to open and close the pore, and thus control gaseous exchange during transpiration, respiration and photosynthesis.

In Greek the word stoma means mouth. ‘Link and de Candole in 1827 jointly claimed to be the first to have called the pores by that name’. Stoma is reported as early as lower Devonian period (about 390 million years ago) from the extinct genera Rlnjnia, Asteroxylon, Zosterophyllum and Drepattophycus.


20 Questions on Plant Diagnosis

This is the third fact sheet in a series of 10 designed to provide an overview of key concepts in plant pathology. Plant pathology is the study of plant disease including the reasons why plants get sick and how to control or manage healthy plants.

Proper diagnosis of plant problems is a key factor in plant health management. As urban forester Alan Siewert quips: “Treatment without diagnosis, as in medicine, is malpractice.” Despite this, diagnostics is often not given adequate attention.

There are three challenges to consider when embarking on plant problem diagnostics:

  • Some plant problems are very obvious, while other problems are very obscure.
  • Some plant problems will not be diagnosed with your first effort. In fact, some plant problems may never be fully diagnosed.
  • Clients usually want an immediate and clear cut answer which produces great pressure to provide a quick-draw, clear-cut diagnosis.

Typically, diagnostics is a process to come up with the best possible explanation of why a good plant has gone wrong. Unfortunately, it almost always involves unknown variables and uncertainties that make an absolute slam-dunk diagnosis the exception rather than the rule. This reality sometimes conflicts with our desire to provide quick, clear answers. Remember that an incorrect diagnosis will lead to an incorrect treatment. Speed should never supersede accuracy.

However, if the plant problem is an insect pest and the pest is present, a quick and accurate diagnosis can be made by simple pest identification. Diagnosing what is causing leaves on little leaf linden to become skeleton-like with only the veins remaining is relatively easy if Japanese beetles are swarming the tree.

Of course, you should always remain aware that a plant may be suffering from multiple problems, and the most obvious may not be the most significant. The plant problem diagnostic process is not unlike our judicial process and the same dangers associated with “pre-judging” also apply. Starting with a diagnosis, then selectively gathering facts to support the diagnosis is likely to produce an incorrect diagnosis to wrongly convict. Plant problem diagnostics should be guided by the axiom: don’t make the symptoms fit the diagnosis do make the diagnosis fit the symptoms.

Plant problem diagnostics should follow a systematic approach to finding proper treatment, and the place to begin is to consider the questions that must be answered. You do not necessarily need to know the answers to all of the questions, nor do you have to ask them in order. Often, however, failure to accurately answer some of the early basic inquiries at the start is the reason for the faulty diagnosis.

Question 1: What is the plant?

This is the first of three key questions concerning the plant itself. It is one of the reasons why truly useful, comprehensive diagnostic keys are so difficult to create the plant ID key alone would be huge. In diagnosis and treatment, determining whether a plant is a pine or a spruce, determining if it is naturally variegated or deciding if it is supposed to be a dwarf are all crucial.

Be cautious with common names. White ash (Fraxinus americana) and mountainash (Sorbus americana) are good examples. Both have compound leaves however, the arrangement of the leaves on the stems provides the first clue that these trees are not related. Ash trees have compound leaves attached opposite to one another on the stem while the leaves of mountainash are attached in an alternate pattern. How the common names are written also show that these trees are unrelated. Mountainash is not a “true ash,” so the name is written as a contraction. A hyphen may also be used to denote the same thing as in the case of stinking-ash (Ptelea trifoliata), which is not a “true ash.”

Spend time focusing on what plant you are looking at or having described to you. Many diagnoses flounder by initial misidentification. Identifying a plant properly leads to a focused consideration of questions such as the ones that follow.

Question 2: What is normal for the plant?

Plant characteristics are variable enough that what is perfectly healthy for one plant may be a sign of a serious problem for another. A good example can be found in deciduous conifers such as baldcypress, dawn redwood and larch. These three trees bear cones and needles, and neophyte plant lovers may think they are evergreens.

Figure 2. Baldcypress with fall color it is a deciduous conifer. Figure 3. Taxus ‘Helen Corbit’ foliage is variegated with needles trimmed in yellow.

However, they are indeed deciduous, with fall colors ranging from spun gold to reddish brown, followed by leaf drop. Many a baldcypress has felt the bite of the saw from new homeowners who notice a completely brown-leaved tree in their new landscape in late fall. Indeed this total browning of foliage would be a sign of almost certain death on a true evergreen conifer, such as pine. Knowing how to identify these deciduous conifers and understanding that their fall color and leaf drop is normal can be all you need for proper diagnosis.

Similarly, knowing that some yews, such as Taxus ‘Helen Corbit’, naturally have needles trimmed in bright yellow should give a horticulturist pause if someone wonders if the yellowing is due to photosynthetic-inhibitor herbicide injury. Knowing that leaves of Naruto Kaede trident maple (Acer buerganum ‘Naruto Kaede’) naturally curl-up along the edges will reduce the chances that the leaf curl will be diagnosed as being caused by moisture stress, herbicide injury or aphids. Knowing that the needles of dragons-eye pine (Pinus densiflora ‘Oculus Draconis’) naturally have yellow banding will help prevent a recommendation to treat for a needle-caste disease. Knowing that the greenish, strap-like bracts on lindens naturally turn brown after flowering is key to responding to a concern that the browning is associated with some type of fungal disease.

These examples do not prove there is nothing wrong with the plant. After all, the Taxus and trident maple may very well also have herbicide injury, and there may still be diseases on the pine and linden. Nevertheless, knowing what is normal for a particular plant provides a great early perspective in the diagnostic process.

Question 3: What are the common problems with the plant?

Another good diagnostic perspective is to consider a plant’s common problems. All plants have their own set of diseases, insect problems, and cultural dilemmas there are no problem-free plants. Pondering these common quandaries can create somewhat of a bias, especially if you are seeing something new, but it helps rule certain problems out.

Figure 4. A characteristic “shepherd’s crook” on crapapple caused by bacterial fire blight.

For example, fire blight, caused by the bacterium Erwinia amylovora, causes a blighting of shoots that result in discolored leaves and a curling of the shoot often characterized as a “shepherd’s crook.” This symptom is helpful in considering fire blight as a possibility. However, such symptoms can also be caused on many plants by far simpler problems, such as moisture stress, resulting in leaf and shoot wilting. For which plants should fire blight be considered a possibility? As it turns out, fire blight occurs only on plants in the rose family (Rosaceae). So if you see a crabapple, firethorn or mountainash with a shepherd’s crook symptom, fire blight should be considered and investigated. If the plant is a maple, white ash, or pine—not members of the rose family—fire blight is not a possibility.

Some plant problems are specific to a particular genus. Native ash trees (Fraxinus spp.) in North America are under threat from the non-native emerald ash borer (Agrilus planipennis). Ash trees are members of the olive family (Oleaceae) however, other members of this family, such as lilacs and forsythia, are not threatened by the borer. As noted above and in question #1, since mountainash belongs to the rose family, it may suffer from fire blight but it will not be attacked by emerald ash borer. Stinking-ash does not belong to the rose family, nor is it a true ash, so the plant dodges both the fire blight and emerald ash borer bullets.

Be aware that some plant pathogens and plant pests have alternate hosts, so two very different plant groups may be affected by the same problem in different ways. The fungus Gymnosporangium juniperi-virgianae produces large, reddish-brown plant galls that sprout fungal horns on the stems of junipers. On apple leaves, the fungus produces round, lipstick-red leaf spots. The common name for this disease reflects the two hosts: cedar-apple rust. The “cedar” refers to eastern redcedar (Juniperus virginiana) which is a juniper, not a true cedar as indicated with the contraction used in the common name. Once again, beware of common names!

Fungi are not the only gall-makers. There are a number of insects and mites that direct the growth of plant galls, and most are highly specific to their hosts. Indeed, most are so specific the gall-maker can be identified to species based on the gall structure alone without the need to see the actual gall-maker. There are over 800 gall-making insects that are specific to oaks, and over 700 are tiny wasps belonging to the family Cynipidae. The cynipid wasp Amphibolips prunus stimulates plant cells in pin oak acorn caps to grow an unusual ball or plum-shaped structure that surrounds a single wasp larva. The galls are commonly called oak plum galls, or oak acorn galls, and they are only produced by this wasp on pin oaks.

Figure 5. Cedar-apple rust fungal spore horns sprouting from a plant gall caused by the fungus on juniper. Figure 6. These bracket-like fruiting structure of the wood-decay fungus Polyporus squamosus is a disease “sign.”

Knowing your plants, and even what family and genus they are in, is a great starting point for diagnostics. This, of course, helps not just with identifying infectious diseases like fire blight and rust, but with other problems as well. Consider a yew or rhododendron growing in poorly drained soil. Knowing these plants are particularly prone to root decline and root rot in poorly drained sites helps immensely with a proper diagnosis when plant decline is evident. It should not blind you to other possibilities, but it certainly is the type of smoking gun that should be investigated.

Question 4: What do you see that looks abnormal?

Plant abnormalities are categorized in terms of “signs” and “symptoms.” Signs are the actual causal agent some part of the pathogen is visible. A fungal pathogen that causes interior rot in a tree is revealed when it produces bracket-like fruiting structures that grow out of the side of the tree. Powdery mildews reveal themselves when they produce white, powdery mycelia that cover leaf surfaces. Both the bracket-like fruiting structures and the powdery mycelia are “signs” since they are the actual causal agents for the diseases.

Symptoms result from interactions between the plant and pests, pathogens or environmental elements (e.g., high soil pH). In other words, a “sign” is the actual pathogen while a “symptom” is what the pathogen does to the plant. Symptoms include such abnormalities as: off-colored foliage deformed or stunted foliage leaf spots, blotches, blisters or scabby spots stem dieback stem cankers root rot or root loss canopy thinning and overall plant decline.

Remember that the same symptom may be produced by multiple causes. Twisted, deformed leaves can be caused by sucking insects such as aphids, or exposure to plant growth regulator herbicides. Tiny leaf spots can be caused by a leaf-spotting fungus or bacterium, or lace bugs and mites. Yellowed leaves (leaf chlorosis) may be caused by nutrient deficiencies in the soil, or by a soil pH that makes the nutrients unavailable to the plant.

It is important to clearly consider and list what signs and symptoms are present that make you believe there is a problem in the first place. For example, are there signs of insect or mite feeding? If so, is injury from pests with chewing or sucking mouthparts? Similarly, are there signs of fungal diseases, such as the orange fungal growth of rust disease? Are leaves missing off-color, abnormally small or scorched? Is there abnormally peeling bark? Are there girdling roots—or are roots rotted in the pot or in the soil? Are there abnormal growths such as galls or discolored cankered areas on stems?

The list can be extended and extended. It is important to walk around the plant—looking at it up close and from far away—and to catalog every noticed item as you work on your diagnosis of what may possibly be multiple problems.

Figure 7. The “symptom complex” for lace bugs is illustrated by these chrysanthemum lace bugs.

Finally, when considering symptoms, keep in mind that there are often a series of symptoms, known as the “symptom complex,” which together helps fingerprint a particular problem. When questioning if lace bugs are a problem, check not only for flecking and yellowing of leaf tissue, but also for tarlike excrement deposits. When checking for Verticillium wilt on maple, check not only for leaf scorching and stem dieback but also for discolored streaking of the vascular tissue.

Question 5: What is the overall health of the plant?

It is a good reminder to put into perspective overall plant health. Presumably you have found something abnormal or you would not be continuing with the diagnosis, but step back for a moment to consider overall health. This helps later in terms of what you will recommend and how important various problems on the plant might be, but it also helps provide focus relative to how long problems might have been present. Consider, relative to a healthy specimen of the same plant, such questions as whether leaf size and color are normal, if the canopy is full or if the growth rate is normal.

Figure 8. The thinning canopy of this ash tree indicates an overall decline in the health of the tree. Figure 9. Annual growth rate. (Graphic drawn by Joe Boggs.)

For example, the terminal buds on many deciduous trees produce a distinct scar that delineates seasonal growth. If you measure the space between terminal bud scars on the twig, you can tell how much it has grown in recent years. It is a little tricky to know what a normal rate of growth is and whether lower than normal rates necessarily mean the plant is unhealthy. However, declining rates of growth over the past several years can be telling, and they can often even be traced to a particular event, such as installation of new sewer lines or a new driveway. Conversely, pointing out normal annual growth can also help allay fears that something major is wrong with the plant—for example, on maple when all that is found is some tarry spots on the leaves.

Question 6: What exactly do you see?

At this point, it is important to inject a “reality check” in the diagnostic process. Are you on the right track? After stepping back to consider the overall health of the plant, force yourself to step back again to reconsider in more detail Question 4: What do you see that looks abnormal? The key to diagnosis is often in such details, sometimes related to others who help with the diagnosis, such as a diagnostic lab technician or coworker in your company.

It is very important to note the pattern of damage. Is the damage on older leaves, newer leaves or both? Is the damage only on the lower part of the plant, upper part of the plant or throughout the plant? Do symptoms appear to be located on a particular part of the leaf? A good example of this is the difference in symptoms between maple anthracnose and physiological leaf scorch of maple.

Figure 10. Physiological leaf scorch on maple is characterized by leaf-browning that starts along the edges of leaves, and moves inward. Figure 11. Diplodia (Sphaeropsis) tip blight of pine is characterized by browning and stunting of new shoots.

To the casual observer, both problems involve blotchy, scorchy, brown discoloration of the leaves. However, the details are quite different. With anthracnose, caused by a fungus, the blotched areas are more of a reddish brown than a tannish brown, but more importantly, are concentrated along the leaf veins. With physiological leaf scorch, caused by excess evaporation of water from leaves due to a variety of factors, the blotches are not concentrated along the leaf veins and are typically more to the outer margins of the foliage. Knowledge of this difference in symptoms is the sort of fine-tuning that diagnosticians develop as they improve their observational and reporting skills.

As can be seen with this maple example, noticing where symptoms are occurring is critical. Diplodia (Sphaeropsis) tip blight of pine is characterized by browning and stunting of new growth on young Austrian, red, Scots and mugo pine shoots, in addition to dieback of this new growth (the growth farthest out on the branch). This disease typically occurs on the bottom branches of the tree first and works its way upward over the years. Compare this to the normal seasonal loss of inner needles from previous years that occur on pines. Every fall, many people become worried about the yellowing, browning and falling needles on pine, even though loss of older needles is normal. Each evergreen species drops needles of different ages, so good plant identification and knowledge is essential. Careful observation of the details of whether the browning needles are on new or old growth is crucial for good diagnosis.

Diagnosing animal damage often depends on looking for details associated with how the animal feeds. Deer do not have upper front teeth. They have lower front teeth, and a tough pad instead of upper front teeth. When deer feed, they pinch-and-pull plant material between their lower teeth and the pad. This typically produces ragged edges rather than clean cuts on foliage, and tips of twigs may look like toothpicks because the outer bark has been pulled off. Rabbits have very sharp upper and lower front teeth and when a rabbit bites, their incisors cross at a 45 degree angle. So, they produce clean cuts that are angled at 45 degrees.

Figure 12. Deer feeding activity on hosta is often signaled by torn leaves that have tattered edges. Figure 13. Rabbit feeding damage is characterized by cuts made at 45 degree angles.

Question 7: What do you see on other plants?

Now take note of the condition of surrounding plants. Are other specimens similarly affected? What is their general health? If you are looking at a grouping of a particular species, does symptom severity seem to relate to any kind of gradient of drainage or sun exposure? Trying to answer such questions often provides key clues about major environmental factors. If, for example, a number of different vegetables in a garden are all dying, it is unlikely they are deteriorating from an infectious disease since most disease-causing pathogens have limited host ranges. It is more likely that some environmental factor, such as extended flooding or poor soil conditions, is involved.

Figure 14. Black sooty mold on the surface of a magnolia leaf.

Often noticing what is occurring on overhanging plants can prevent embarrassing misdiagnoses. Scale insects, which suck sap from plants, excrete this processed sap out the other end in the form of “honeydew.” Often this clear, sugary, sticky liquid becomes covered with a sooty mold fungus that simply grows on the sugary substance, rather than plant tissue itself. Calico scale is a prolific producer of honeydew. Consider what happens to the leaves of plants underlying a tree that is heavily infested with this scale insect. The underlying plants are not infested however, their leaves become blackened with sooty mold. Of course, an effective treatment must focus on the scale infestation rather than non-infested plants with blackened leaves.

Question 8: What are the plant’s site conditions?

Question 7 leads directly to a more focused examination of the site in which the plant is growing. A few key site characteristics can include everything from soil characteristics and exposure to sun and rain, to construction history and competition from other plants.

The soil type relative to drainage, extent of compaction, amount of organic matter and acidity/alkalinity can tell a great deal about the success and failure of various plants. Poorly drained soils with poor internal aeration sooner or later result in death of Taxus.

Acid-loving plants often develop yellowing between the veins (or to put it more stuffily—interveinal chlorosis) if growing in alkaline soils (pH above 7) due to iron deficiency. This can be diagnostically investigated by using soil tests and even plant tissue analysis, or by simply looking at the plants on-site. If you notice rhododendrons, birches, white pines and other acid-loving plants thriving in a location, then a diagnostician might suspect the yellowing of leaves on the similarly acid-loving pachysandra is due not to iron deficiency, but rather to other factors such as overexposure to sun.

Figure 15. Iron chlorosis on pin oak may signal a high soil pH.

Sun and shade exposure is also critical to the success of many plants. Japanese maples tend to thrive in protected sites, developing physiological leaf scorch in hot, sunny areas. Flowering dogwoods generally do poorly in open, hot sites (and often develop borer problems if stressed) and also in densely shaded sites where diseases, such as dogwood anthracnose, are favored. Partial shade is best for flowering dogwood.

Exposure to wind can result in desiccation of leaf tissue of broad-leaved evergreens such as rhododendron in winter and should be considered while diagnosing these plants and the extent of wind exposure. Even exposure to rain can be an important clue. Diagnosticians often miss the implication of overhangs from houses when wondering why herbaceous ornamentals near structures seem to be languishing despite adequate recent rainfall.

The effects of construction are also a factor that should be investigated relative to the site. How much soil grades were raised, the effects of bulldozers on soil compaction and root destruction, installation of sewer lines, driveways, roads and structures all play a role in plant health, often many years after the fact. Diagnosis would be easy if raising the soil grade 6 inches during construction activity caused trees to fall over within a week or two.

Figure 16. Soil compaction from construction equipment.

The truth, however, is that this kind of stress on root systems, due to reduced oxygen concentrations for the now-buried roots, can have effects for years from the contribution to overall plant stress. Nailing down exactly how much damage is due to various factors is difficult—if not impossible—to pinpoint, but it is the job of the diagnostician to put it into as clear a perspective a possible.

Always keep an eye on plant selection when assessing site conditions. It is very difficult to modify the site once a plant is planted. In cases where it’s “wrong plant, wrong site,” your recommendation may be to replace the plant. Good plant health management means starting with the right plant in the right place.

Question 9: Who knows the most about the plant?

One of the limits of diagnosing plant problems, unlike with human medicine, is that the patients cannot talk. However, asking questions of the person who knows the most about the plant often yields the most important information of all. People who work in a diagnostic laboratory will tell you the information on the sample is often more important than the sample itself. Try to find out from them the answers to the next question.

In some cases, the person who is most familiar with the plant is not the person with the direct responsibility for managing the plant. Consider a landscape around a commercial office building. The building’s owner may not be located in the building, and the company hired to manage the landscaping may only make periodical visits. Who knows most about the plants? It may be the people who work in the building. Do not overlook interviewing the people who actually see the plants on a day-to-day basis.

Question 10: When did symptoms first appear?

Although listed as #10, this is a very important question: when did the symptoms of the problem in question first become evident? Sometimes the answer is unreliable we have all heard “it up and died overnight.” We can check this out, though, by looking at annual growth and symptoms, such as long-term branch decay and peeling bark. Sometimes people do provide crucial information that helps solve the problem, such as noting that foliar collapse occurred soon after a spring frost.

Plant symptoms sometimes progress through a series of different “looks.” It is helpful to consider symptom progression in the context of having a beginning, middle and end. The oak shothole leafmining fly (Agromyza viridula) uses its sharp ovipositor to puncture young expanding oak leaves to cause liquids they feed upon to flow. They often produce holes in a row, and if new leaves are punctured prior to unfolding, the holes will appear as mirror images on different parts of the leaf. In the beginning, the holes are extremely small however, they become larger as the leaves expand. In the end, they may measure over ½ inch in diameter. Of course, by this time, the fly is long gone.

Figure 17. Damage from the oak shothole leafmining fly. Figure 18. “Mulch mounds” or “volcano mulching.”

Plant stress may also produce a progression of symptoms. However, there are two types of plant stress: acute and chronic. Acute stress is caused by an immediate event, such as flooding, drought or defoliation by an insect pest. Symptoms are usually immediately evident and easy to diagnose since the cause is close at hand.

Chronic stress is caused by more subtle conditions such as the site problems listed in Question #8 or horticultural problems that will be covered in the next question. The effects of chronic stress are usually cumulative, meaning that the damage adds up over time to eventually produce dramatic results. A mature oak tree that has had its root system damaged or reduced by construction may take years to show the full effects, with symptoms such as a thinning crown gradually progressing towards the death of the entire tree. The tree does not “up and die overnight”—it began the slow spiral towards death when it was first exposed to chronic stress.

Question 11: What is the horticultural history of the plant?

This inquiry involves a whole series of important questions, some of which can be answered only by others, some of which you can determine from evidence at hand. For example, what is the plant’s transplant history? Looking at a declining 40-foot tree can be a puzzle that is pretty easily put together when you discover the tree was transplanted two years previously. On younger plants, transplant history is often quite evident. A declining rhododendron that has branches growing out of the ground and is planted 6 inches deeper than the root ball grade tells a great deal about the causes of decline. Apply the axiom: “plant them low, never grow plant them high, watch them die plant them right, sleep at night.”

The same combination of questions to ask and clues to look for apply to horticultural practices such as fertilization, mulching, pesticide spray programs, plant hardiness, use of girdling wires and the source of plant material. You can ask about fertilization rates, but you can often find telltale signs that help ask more pointed questions, such as an excessive pile of granular fertilizer on the ground or on mulch. Check the depth of organic mulches. The recommended amount is 2 to 2½ inches, although more commonly 6 to 8 inches (or even more) is applied, or mulch piles up over the years with reapplication exceeding breakdown. Additionally, mulch is often piled up against the trunk of a tree or base of a plant. The result of this overmulching may be the reduction of oxygen availability to feeder roots, especially on young plants, and excessive moisture retention may potentially lead to crown and root rots. Mulch mounded against the base of the crown can also provide a perfect protected location for rodents in winter, which can severely damage or kill young, thin-barked trees and shrubs.

Figure 19. Late-spring freeze damage to trident maple.

Again, consider the always important question of timing. An irrigation system that is present and seemingly functional may not have been working during the hottest portion of the summer, when observed damage really was caused. Conditions may be cool and non-stressful in September, but what if a large tree was transplanted on a 100 degree day in July?

Question 12: What is the environmental history?

In addition to what we do horticulturally, it is important to consider past environmental events. How harsh have recent winters been, and how does this match up to a particular plant’s hardiness range? Also, severe freezes in a given year can result in plant dieback and death well into a growing season.

Often clients think if a plant flowers normally or leafs-out normally, then all is well with regard to surviving winter damage. Sometimes, bud tissue breaks however, early freeze damage to a plant’s cambium prevents that plant from growing beyond that initial bud break, and stems, or the entire plant, may die. These symptoms of delayed winter injury are quite common in cherries, as well as other Prunus selections.

Plants may also bud out and look fairly normal well into late spring and early summer, then hot weather occurs, and the underlying damage to the cambium causes dieback to occur. This type of problem again highlights the separation in time of the cause of damage and the obvious symptoms of this injury that make diagnosis such an art.

If a plant is known to have difficulty under droughty conditions, early hot, dry weather in a given season can have major effects on plants such as turfgrass and tender perennials, including Ligularia and Astilbe. Severe drought in past years should be factored into the current condition of certain drought-sensitive trees, such as beech. How a plant responds to particular additional stress depends upon its entire horticultural and environmental history.

Question 13: What does the client think the problem is?

A careful interview of the client regarding their thoughts on the problem can provide critical pieces of information that contribute to an accurate diagnosis. Remember that you are interviewing the client, not interrogating! Avoid asking “leading” and accusatory questions, such as, “Did you over-fertilize the plant?” or “Did you give the plant too much water?” These questions are not likely to yield useful information they are more likely to yield an angry client.

Figure 20. Leaf scorch on dogwood caused by a summer drought.

Consider phrasing questions in a way that induces dialogue. For example, you may ask, “Tell me about your fertilization program” or “Tell me how you water your plants.” Both of these questions require more than “yes” or “no” answers, and they are not “leading” questions, meaning that you are not influencing the answer. Your goal is to simply gather more information.

Having a two-way conversation with the client will also help you learn the client’s true concerns which will be very helpful in making a recommendation. Asking for their opinion on what they think the problem is makes the client a partner in the diagnostic process. A partner will be more likely to follow-through with your recommendations.

Question 14: What diagnostic tools are available?

Useful tools for diagnosis can obviously be high-tech, ranging from ever more elaborate microscopes and enzyme-linked immunosorbent assay tests for viruses and fungi in diagnostic labs to equipment from the gas company to check for gas leaks on properties where trees and turfgrass along a gas line are dying. However, for horticulturists making a field diagnosis, basic equipment can be far more manageable and less expensive. Here are six basic items:

Soil probe

This tool is useful diagnostically for soil sampling to check soil pH and nutrient levels. It can help explain, for example, foliar chlorosis due to iron deficiency on acid-loving plants like pachysandra, white pine, river birch and rhododendron growing in alkaline soils. Probes can have more immediate diagnostic uses as well, such as determining the soil texture, or checking to see how compacted or dry soils are or the depth of mulches.

Hand lens

A good 10X or 20X magnification hand lens is useful to check for mites and small insects on plant foliage or to look for fungal fruiting bodies on leaf tissue.

Cutting tools

Good, sharp hand pruners are important for cutting small twigs to look more closely at stem and leaf problems. It is also unprofessional, to say the least, to collect a sample by stripping a twig from a plant rather than making a good pruning cut.

For larger stems, a small foldable pruning saw is also easy to carry. A knife is useful for cutting into a stem to check for discoloration of the vascular system (typical of Dutch elm disease or Verticillium wilt disease) or to check stems for the presence of insect borers. Although less portable, pruning poles can also be useful tools to get samples from high in a tree.

Figure 21. A hand lens provides useful magnification for field diagnostics. Figure 22. Hand pruners are useful for collecting samples.

Digging tools

It is often helpful to dig a bit around the base of a plant to check for girdling roots or twine, to check where the pre-transplant root system was located, or to collect a root sample. A collapsible spade is quite handy, but sometimes blunt, wedge-like knife blades can do the trick.

Recording tools

It is important to take good notes of what you observe to later refresh your own memory and to accurately relay relevant information to others. Have a good field notebook, as well as weatherproof pens and markers. A hand-held recorder can also be helpful if you do many field diagnoses. Finally, a camera can help convey symptoms and site characteristics for others and can be a valuable validation of the plant's condition at the time of inspection. This photographic evidence becomes especially useful if post-visit changes are made, such as the cutting down of an affected tree.

Sampling equipment

In addition to soil probes and pruners, it is always a good idea to carry along some large plastic bags for collecting samples. Avoid leaving foliage samples exposed to the heat of the sun, and if collecting soil samples for nematodes, a small cooler can be quite helpful.

Question 15: What additional resources are available?

Of course, the most important diagnostic resource you have is your experience and the collective experience of your cohorts. Also be aware of the number of reliable resources on plant identification and selection problem identification and specific damage by insects, diseases, wildlife and other pests. These sources range from books to great web sites to a wide range of educational programs provided by green industry organizations and university extension services.

Furthermore, recognize that diagnostic observations in the field sometimes need verification at a diagnostic lab. These labs use microscopic examination, fungal culturing and a wide range of tests to help confirm or deny the presence of certain problems. Take advantage of the university, government or private diagnostic labs in your area. In addition, other laboratories specialize in different pieces of the puzzle. Examples are soil test and foliar analysis laboratories used for information on possible nutrient deficiencies or excesses, and analytical laboratories that check for chemical residues in plant tissue.

Question 16: How do I take samples?

At this point in the diagnostic process, you probably have a tentative diagnosis of the problem. You are now focusing on confirming your suspicions. Each type of plant problem can require special techniques to get the best sample back to colleagues or to a diagnostic laboratory. Following are a few hints adapted from Ohio State University Extension bulletins:

Obviously, many times you can only sample a small portion of a plant, but when large numbers of small plants are affected, collect entire plants, including roots. If 500 rhododendrons are going down, do not just send a leaf or two. Dig plants to keep roots intact rather than simply pulling the material out of the ground. Remove excess soil by gently shaking or washing with water. Do not wet leaves or stems. Wrap roots so clinging soil won't be loose in the packaging. Do not ship wet plants let them air-dry first.

If only a portion of a plant is sampled, include the part showing symptoms. Also, when possible, collect about a pint of roots, soil and fine rootlets.

When only localized parts of a plant are affected (leaf spots, stem cankers), ship several examples of the affected parts. Stem and branch sections should include a short section of healthy tissue so the transition area between healthy and diseased tissue is included. For example, if collecting a sample to check for Verticillium wilt disease, select 1-inch diameter stem sections about 6 inches long, ideally from the area where the stem transitions between healthy and diseased tissue, rather than collecting dead stems.

If shipping, press non-woody plants or leaves on small twigs between paper and put them between pieces of stiff cardboard, then place in a padded envelope. For succulent plants, samples packed in airtight plastic often decay before arriving in a lab. Place the leaves of such specimens between paper towels before packing. Use strong containers, filling spaces with shredded paper or other materials to cushion the sample in transit. Use rapid mail delivery for best results.

Question 17: What else needs to be considered?

By now, having asked all kinds of questions and in some cases consulting others or sending in samples for analysis, a good diagnostician asks for the last time, “What else might I be missing?” Question #6 provided the first “reality check” in the diagnostic process this is the second. You should stop and reconsider everything you have learned thus far. Does it all add up to support a good diagnosis?

Figure 23. An emerald ash borer adult beetle. Figure 24. Emerald ash borer “D”-shaped adult emergence holes.

Emerald ash borer provides a good example of the value of stopping to reconsider everything before making a diagnosis. Prior to the discovery in 2002 that this non-native beetle was living in the United States, people were certainly aware that ash trees were dying. However, correctly diagnosing a plant problem that is not known to occur is without doubt the most difficult diagnosis to make. We tend to focus on the “known.”

It was known that a number of tree-killing diseases could occur on ash, including ash yellows and Verticillium wilt. Ash trees were generally considered “tough trees” and they were often planted in challenging sites such as in parking lot planters, or along street curbs. It was no surprise that many died. Finding holes in these trees was also no surprise since it was well known that several native insect borers target stressed ash trees.

Reconsidering Question #4, “What do you see that looks abnormal?,” along with Question #6, “What exactly do you see?” would have been helpful in disclosing the presence of emerald ash borer. Native borers produce round or oblong adult emergence holes emerald ash borer produces distinctly “D”-shaped holes. Sadly, it is now known that emerald ash borer was living in the United States at least 10 to 15 years prior to its discovery prior to a correct diagnosis. “What else?” should always be a nagging question on a diagnostician’s mind.

Question 18: What is the diagnosis?

The last example brings us to several cruel realities of diagnosis. First, sometimes you just won’t have the insight to ask the “What else?” question that starts your light bulb blinking. Second, even when you do ask the question, it may not result in an open-and-shut case. With emerald ash borer, diagnosticians in 2001 simply did not know that the borer should be added to the list of possible problems that could occur on ash trees.

Even if your diagnosis focuses on a well-known problem, the reality is that you are almost always somewhat uncertain as to your diagnosis. A more reasonable goal for diagnosis is to strive to come up with the best diagnosis possible while acknowledging the possibility of other factors. That being said, it is important to be clear about what you did diagnose and also, often just as importantly, about what you did not find. In reporting your diagnosis, remember to do the following:

  1. Describe the symptoms you observed clearly and in detail.
  2. Identify the problem or problems you think these symptoms signify.
  3. Indicate how you made this connection (consulting with colleagues, references and lab tests).
  4. List what you did not find. As indicated above, what you did not find can often be critical. If you do not find Dutch elm disease or other infectious diseases, if there is no evidence of bronze birch borers or Asian long horned beetles, and if the symptoms and/or residue analysis is not suggestive of growth-regulator herbicide injury, this may go directly to the heart of your client's greatest concerns.
  5. Put diagnoses into perspective and provide recommendations.

Question 19: What is the significance of the problem?

After making a diagnosis, it is important to put the suggested problem into proper perspective relative to overall plant health. For example, most pest and disease problems are insignificant relative to plant health. Beech blight aphids have a nasty sounding name, their white woolly bodies may cover beech branches, and their honeydew may rain down to cover underlying sidewalks with sticky goo however, they cause little harm to their host tree. Powdery mildew of lilac occurs every year and seems to cause little effect relative to overall lilac health and survivability. Most of the mite and insect galls on plant leaves are quite fascinating but cause negligible effects on plant health.

However, here you need to be a good communicator, to understand your clients and listen to their concerns. Just because a problem will not affect plant health, or in your opinion, affect aesthetics significantly, does not mean your client agrees. In some sense, plant problems are in the eye of the beholder. While powdery mildew of lilac may be irrelevant to plant health in one landscape, it may matter a great deal to a client who will simply take his or her business elsewhere if you do not do something about the problem. And it certainly matters to a garden center displaying lilacs in its sales area.

Figure 25. Beech blight aphids. Figure 26. Powdery mildew of lilac.

Question 20: What are my recommendations?

We’ve reached the all-important decision of what you recommend to fix the problem. First, remember that sometimes “doing nothing” is the best recommendation. If the problem is trivial and the customer is not concerned about it, then simply letting the client know that the maple bladder gall mites are insignificant and nothing needs to be done is a good recommendation.

Second, sometimes nothing can be done to make the plant recover. In such cases, often the best recommendation relates to considerations for the timely removal and replacement of the plant.

Third, when action recommendations are given, always remember the crucial element of proper timing. If you diagnose Diplodia (Sphaeropsis) tip blight of pine in July, it is important to specify that any chemical to prevent new infections be applied the next spring since fungicides applied at any other time will be of no use for disease control.

Fourth, recommendations should be made within a range of proper expectations. A good example is of pin oak planted in highly alkaline soil at an institutional site. Years later, the root system has grown out beyond the original root ball and amended soil into the alkaline soil. The tree begins to show symptoms of iron chlorosis, starting with interveinal yellowing (chlorosis). After years of this, the problem becomes more severe, with leaf necrosis (browning) and stems dying back. Everyone begins to notice, and it is agreed that something must be done. Experts are called in and asked for diagnosis and recommendations. With reasonable certainty, buttressed with clear-cut symptoms, as well as soil and foliar analysis tests, iron deficiency is diagnosed.

However, recommendations that actually solve the problem are another matter. There are a lot of possible treatments, ranging from trunk implants of iron to the use of chelated iron fertilizers in the soil to injections of iron in the roots. All are problematical relative to a long-term cure of the problem, especially if the situation is severe. If you make it seem like your recommendations are absolute, then you put the grounds maintenance people who have to act on your recommendations in jeopardy of being deemed incompetent once treatments fail.

Finally, the art and science of professional plant diagnostics are often overlooked by those with instant answers to every problem. Beware of those easy answers, especially if the diagnostician did not even ask a question. Diagnostics requires good detective and communication skills, and plant diagnosticians need a thorough knowledge of horticulture, botany, entomology and plant pathology. No one can ever be the perfect diagnostician, and there is always room to improve and grow, to make and correct mistakes.

Always remember that with plant diagnostics, as with human medicine, it is useful to cultivate humility. The first surefire rule of plant diagnostics is nothing is surefire.

For detailed information on each of the IPM strategies, see the fourth fact sheet in this series, Keeping Plants Healthy: An Overview of Integrated Plant Health Management.


Trends

Glandular trichomes produce large amounts of volatile organic compounds (VOCs) and constitute an excellent model system to investigate transport processes of small hydrophobic molecules.

There is increasing evidence for the involvement of active transport in VOC trafficking out of epidermal cells. The contribution of these processes to VOC delivery from secretory cells to the cavity has not yet been studied.

Storage of high VOC concentrations requires biophysical mechanisms to retain them within the cavity.

Electron microscopy studies indicate that the formation of the storage spaces involves highly localized partial cell wall lysis, cell wall expansion, and deposition of cell wall material with low VOC permeability.

Immunostainings with antibodies against cell wall components suggest that pectin demethylation has a role in storage cavity formation.

Trichome-specific transcriptomic along with reverse-genetic approaches will enable the discovery of the biological players involved in VOC release to the trichome cavity and their impact on VOC sequestration.

Plant glandular trichomes are able to secrete and store large amounts of volatile organic compounds (VOCs). VOCs typically accumulate in dedicated extracellular spaces, which can be either subcuticular, as in the Lamiaceae or Asteraceae, or intercellular, as in the Solanaceae. Volatiles are retained at high concentrations in these storage cavities with limited release into the atmosphere and without re-entering the secretory cells, where they would be toxic. This implies the existence of mechanisms allowing transport of VOCs to the cavity but preventing their diffusion out once they have been delivered. The cuticle and cell wall lining the cavity are likely to have key roles in retaining volatiles, but their exact composition and the potential molecular players involved are largely unknown.


Discussion

Based on previous studies, the determination and morphogenesis of the multicellular trichomes of cucumber are regulated by HD-ZIP transcription factors 5,15,16,17,19,20 , which show differences from unicellular trichomes. The known genes related to the cucumber trichome (spine) development are tril, mict, and Tu (tuberculate fruit). Mict mediates the morphogenesis of trichomes, and disruption of Mict makes most trichomes remain in the single-cell bulge state. In this study, evidence from our multi-omic analysis shows that Mict acts as a regulator in the very beginning phase to turn on an array of biological processes related to trichome development, directly or indirectly. Although the functions of Mict target genes need to be tested in further research, our results indicate that Mict can activate genes involved in flavonoid and cuticular lipid biosynthesis. The main purpose of this study was to identify the connection between transcriptional regulation and metabolic pathways in trichome development via multi-omics analysis and provide evidence to support that Mict regulates metabolic pathways (e.g., flavonoids and cuticle) both directly and indirectly.

Mict turns on the regulatory network at the beginning of trichome development

Based on our spatial-temporal expression analysis results, Mict is highly expressed at Stage III and Stage IV and is reduced at Stage V, implying that Mict functions at a particular stage during trichome development in leaf. Moreover, transcription of Mict can hardly be detected in any other organs except fruit spines, which implies that Mict acts as a regulator in the very beginning of trichome initiation, modulating a large number of downstream target genes for a transient period. Then, the regulated genes continually promote processes, such as cell division and secondary metabolite biosynthesis, to promote proper trichome development. Other evidence that Mict acts as a turn-on switch is that Mict is accurately detected in the top cell of the multicellular trichome, inducing successive trichome cells to develop spontaneously after Mict activates the regulatory network in the top cell. The expression of transcription factors (e.g., CsWIN1, CsMYB6, and CsMYB36) is absent in mict mutants, which implies complex interactions between transcription factors are involved in the transcript regulatory network. As a result, the mict mutant shows a defective phenotype in wax biosynthesis. In this study, we narrowed the Mict binding sites into a 500 bp region in the promoter of the four downstream genes. Common cis-element analysis of these four promoters suggested that only light response elements were identified in all four promoters (Fig. S3c). Taken together, Mict might bind novel elements in the promoters that specifically regulate trichome development, which merits further investigations.

Mict regulates cuticle metabolism in epidermal cells

The plant cuticle is an extracellular lipid structure deposited on the aerial surfaces of organs to protect the plant against environmental stresses 21 . Cuticles are complex mixtures of VLCFAs, with chain lengths of more than 26 carbons. In mutant leaf, the wax composition with more than 26 carbons changed significantly compared to that of WT. However, in mutant fruit, similar significant changes in wax components were lacking. Therefore, the change in VLCFA contents might contribute to observed phenotypes between the two organs. Transcriptomic study revealed significant DEGs involved in fatty acid and cuticle biosynthesis pathways, and led us to examine the cuticle’s wax composition. In combination with metabolomics data, we constructed a schematic diagram to explain wax changes, such as VLCFAs, alkanes, and secondary alcohols, in mict mutants (Fig. 5b). Downregulation of CER26 and CER26-like indicated the elongation of 30 carbons or more was blocked in the mict mutant 13 . Downregulated CER8 in the mict mutant caused the lacing of alkanes and redundancy of free fatty acids 22 .

Moreover, low expression of CER1 and CER3, which act together to catalyze the formation of alkanes from VLCFA-CoA, also explains the lack of alkanes in the mict mutant 23 . MAH1, a midchain alkane hydroxylase that catalyzes secondary alcohol formation, was downregulated and related to the absence of 2-C18:OL in the mict mutant 24 . In addition, downregulated ABCG13 and ABCG32, functional ABC transporters involved in wax component transport, may also influence the transport of wax from the plasma membrane (PM) to the exterior of the cell 25,26 . In summary, the downregulated expression of the genes mentioned above could partly explain some of the compound changes in cuticle biosynthesis pathways, which means that Mict positively regulates the downstream genes directly or indirectly to control these pathways in cucumber leaves and fruits. These results are consistent with the mutation of genes related to trichomes, such as GL1, MYB16, MYB106, and CER6, which can also affect cuticle formation in Arabidopsis 4,27,28,29 .

Interestingly, a recent report found that the specific cuticular wax composition varies between different epidermal cell types, i.e., trichome and pavement cells 30 . However, in this context, it is remarkable that Mict has much stronger expression in trichome cells than in other types of cells, leading us to speculate that Mict regulates the specific cuticular wax components. Notably, CER26 has much stronger expression in trichome cells than in other epidermal cells in Arabidopsis 13 , making it reasonable that Mict positively regulates CsCER26 to synthesize the trichome-specific cuticular wax composition. It is technologically challenging for us to isolate multicellular trichomes from cucumber leaves, limiting an accurate examination of the trichome-specific cuticular wax composition. In the future, we believe that identification of the trichome-specific cuticular wax composition could reveal the function of Mict in cooperative regulation of trichome development and material metabolism.

Mict regulates flavonoid metabolism in trichome development

The trichome is regarded as a bio-factory of secondary metabolites in plants. The advantages of trichome-specific secondary metabolite biosynthesis could be maximizing the protective effects while minimizing the harm of their overaccumulation. Flavonoids are frequently used as pigments and involved in response to the environment and developmental processes 31 . Transcriptional control of the structural genes in the flavonoid biosynthetic pathway has been most extensively studied in plants. In our research, Mict was shown to regulate catalytic enzymes, such as CsTT4 and CsFLS1, which participate in flavonoid biosynthetic pathways. The expression of catalytic enzymes usually determines the content of metabolites as they are always located in the downstream regulation area. FLS is a key enzyme in flavonoid biosynthesis 32 , and TRANSPARENT TESTA (TT) family members were found to work as transcription factors or enzymes in flavonoid biosynthesis pathways in Arabidopsis. Among those, CHALCONE SYNTHASE (CHS), which is encoded by the TT4 gene, catalyzes the first step of flavonoid biosynthesis for producing naringenin chalcone, a common precursor of various flavonoids 33 . In addition, IF7MAT (isoflavone 7-O-glucoside-6”-O-malonyltransferase), ANS (anthocyanidin synthase), HCT (O-hydroxycinnamoyltransferase), and IFR (isoflavone reductase) also play key roles in flavone, flavonol, and flavonoid biosynthesis pathways. The aforementioned genes were greatly downregulated in mict mutants, suggesting they may participate in the trichome-specific secondary metabolite biosynthesis pathway via Mict-mediated regulation. Accordingly, together with the genes–metabolites association analysis in this study, Mict may act upstream in a way that activates regulatory networks in trichome morphogenesis and specific metabolic pathways, which ensures that trichomes develop correctly.

Mict activates CsMYB36 to form fruit wax-powders

The cucumber fruit wax-powder is a thin layer of white powder that surrounds the fruit, and it is mainly composed of VLCFAs (and its derivatives), such as alkanes, aldehydes, and primary and secondary alcohols 34 . Several cucumber mutants show no wax-powder on the fruits, such as csmyb36, tril/csgl3, and mict. It is clear that tril exhibits recessive epistasis with mict, as mict does to csmyb36, which suggests that CsMYB36 is the main regulator of wax-powder formation. In this study, we found that Mict could bind to the CsMYB36 promoter and stimulate its transcription. This result explains the absence of wax-powder on mict and tril fruits. Accordingly, the interaction between Mict and CsMYB36 directly bridges trichome development and wax-powder formation, and it provides us with a better understanding of the significance of trichome cell differentiation.

In this study, we identified the metabolic changes related to leaf trichome and fruit spine development in cucumber, and linked flavonoid and cuticle metabolism with transcriptional regulation. However, there are still many metabolites that we could not associate them with corresponding genes due to the limited cucumber molecular pathway databases. Fortunately, in recent years, with the rapid advances in trichome-related gene identification in cucumber, we believe our results will provide important clues to discover biochemical mechanisms in cucumber and reveal the molecular regulatory network involved in trichome development.


Acknowledgements

I wish to thank Frank Syrowatka from the Multidisciplinary Centre of Materials Science, Halle (Saale), Germany, for the environmental SEM pictures of tomato leaves and Ana Simonovic and Sladjana Todorović for providing SEM pictures of Tanacetum parthenium florets and Salvia officinalis leaves and Angelos Kanellis for providing pictures of Salvia trichomes. Work in my laboratory is funded by the EU under the project TERPMED (grant number KBBE-227448) and by the Leibniz Institute of Plant Biochemistry. I would also like to thank Bettina Hause, Thomas Vogt and Michael Walter for critical reading, and both reviewers for suggestions which significantly improved the manuscript. I would like to apologize to authors whose work could not be cited due to space limitations. The author declares no competing financial interests.


Watch the video: Week 8: How To Check Trichomes (September 2022).


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